Abstracts

ORIGINAL PAPERS

Molecular phylogeny, morphology and their implications for the taxonomy of Eriocaulaceae

Filogenia molecular, morfologia e suas implicações para a taxonomia de Eriocaulaceae

Ana M. Giulietti^I,VII,* * Author for correspondence: anagiulietti@hotmail.com ; Maria José G. Andrade^I,IV; Vera L. Scatena^II; Marcelo Trovó^VI; Alessandra I. Coan^II; Paulo T. Sano^III; Francisco A.R. Santos^I; Ricardo L.B. Borges^I,V; Cássio van den Berg^I

^IUniversidade Estadual de Feira de Santana, Depto. Ciências Biológicas, Av. Transnordestina s.n., 44036-900, Feira de Santana, BA, Brazil

^II Universidade Estadual Paulista, Instituto de Biociências, Depto. Botânica, C.P. 199, 13506-900 Rio Claro, SP, Brazil

^III Universidade de São Paulo, Instituto de Biociências, Depto. Botânica, R. Matão 277, Cidade Universitária, 05508-900, Butantã, SP, Brazil

^IVUniversidade do Estado da Bahia, Depto. Educação – DEDC/VIII, R. Gangorra 503, 48608-240, Paulo Afonso, BA, Brazil

^VUniversidade do Estado da Bahia, Depto. Ciências Humanas – DCH/VI, Av. Contorno s.n., 46400-000, Caetité, BA, Brazil

^VIUniversidade Federal do Rio de Janeiro, Instituto de Biologia, Depto. Botânica, CCS, bl. A1, s. 82, 21941-590, Rio de Janeiro, RJ, Brasil

^VIIRoyal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, England

ABSTRACT

The pantropical family Eriocaulaceae includes ten genera and c. 1,400 species, with diversity concentrated in the New World. The last complete revision of the family was published more than 100 years ago, and until recently the generic and infrageneric relationships were poorly resolved. However, a multi-disciplinary approach over the last 30 years, using morphological and anatomical characters, has been supplemented with additional data from palynology, chemistry, embryology, population genetics, cytology and, more recently, molecular phylogenetic studies. This led to a reassessment of phylogenetic relationships within the family. In this paper we present new data for the ITS and trnL-F regions, analysed separately and in combination, using maximum parsimony and Bayesian inference. The data confirm previous results, and show that many characters traditionally used for differentiating and circumscribing the genera within the family are homoplasious. A new generic key with characters from various sources and reflecting the current taxonomic changes is presented.

Key words: anatomy, ITS, phylogenetics, pollen, trnL-trnF.

RESUMO

Eriocaulaceae é uma família pantropical com dez gêneros e cerca de 1.400 espécies, com centro de diversidade no Novo Mundo, especialmente no Brasil. A última revisão da família foi publicada há mais de 100 anos, e até recentemente, as relações genéricas e infra-genéricas ainda eram pouco resolvidas. Entretanto, tem havido nos últimos 30 anos, um grande esforço por parte de pesquisadores brasileiros para preencher as lacunas existentes, utilizando caracteres morfológicos e anatômicos, complementados por dados adicionais de diferentes fontes, como palinologia, química, embriologia, genética de populações, citologia e, mais recentemente, estudos de filogenia molecular. Tal conjunto de dados tem levado a uma re-avaliação do relacionamento filogenético dentro da familia. Neste trabalho são apresentados novos dados para as regiões de ITS e trnL-F, analisadas separadamente e em combinação, usando máxima parcimônia e inferência Bayesiana. Os dados obtidos confirmam resultados já publicados, e mostram que muitos caracteres tradicionalmente usados para diferenciação e circunscrição dos gêneros dentro da família são homoplásicos. Uma nova descrição e chave genérica para a família, utilizando caracteres de várias fontes são apresentadas, refletindo a taxonomia atual das Eriocaulaceae.

Palavras-chave: anatomia, ITS, filogenia, palinologia, trnL-trnF.

Introduction

The Eriocaulaceae are easily distinguished from other monocot families because most of the species have short stems and leaves in a rosette, long scapes with small unisexual flowers grouped in dense heads (capitula), a 3- or 2-locular ovary with a single pendulous ovule per locule, and spiraperturate pollen grains (Giulietti et al. 1995, 2000).

Some species have considerable economic importance in Brazil, especially the genera Syngonanthus Ruhland and the recently re-established Comanthera L.B. Sm. (Parra et al. 2010). These are collected when their flowers are still at anthesis and dried in the sun, to be sold as ornamental objects and are often exported to different countries as "everlasting plants" (Giulietti et al. 1988, 1996). Some species such as Comanthera elegans (Bong.) L.R. Parra & Giul. have been marketed at the rate of 40,000 kg/year (Giulietti et al. 1988,1996) and some such as C. magnifica (Giul.) L.R. Parra & Giul. and C. mucugensis (Giul.) L.R. Parra & Giul.. are critically endangered due to over-exploitation (Pereira et al. 2007). Currently, the scapes of Syngonanthusnitens (Bong.) Ruhland, popularly known as "golden grass", are widely commercialized and used for the production of various local handcrafts (Schmidt et al. 2007). In the regions where these plants occur naturally, they are one of the main sources of income for local inhabitants, especially in the "campos rupestres" vegetation of Espinhaço Range (states of Minas Gerais and Bahia, Brazil), Serra Geral (state of Goiás, Brazil), Cerrado in Jalapão (state of Tocantins, Brazil), and western Bahia.

The Eriocaulaceae is a sub- to Pantropical family, and Eriocaulon L. includes 400-800 species occurring on five continents. Paepalanthus Mart. (>500 species) and Syngonanthus (c. 130 species) occur disjunctly in the Americas and Africa. Mesanthemum Körn. is restricted to the African continent, and Lachnocaulon Kunth to North America. All other genera: Actinocephalus (Körn.) Sano, Comanthera, Leiothrix Ruhland, Rondonanthus Herzog, and Tonina Aubl., are practically endemic to South America, whereas Tonina extends to Central America. The main diversity centre of the family is in Brazil with 629 species, 482 in the Cerrado, so that Eriocaulaceae is the fourth family in number of species in this biome (Giulietti et al. 2010; Forzza et al. 2010).The greatest diversity is in the "campos rupestres" (high-altitude rocky savannas) of the Espinhaço Range in the states of Minas Gerais and Bahia, where about 500 species occur. Eriocaulaceae was a very important family for the definition of endemism areas in Minas Gerais (Echternacht et al. 2011a).

Judd et al. (2002) and APG III (2009) included Eriocaulaceae within Poales, but the family was previously placed in its own order Eriocaulales (Cronquist 1981), within the Commeliniflorae (Dahlgren et al. 1985) or Commelinales (Judd et al. 1999). Although it is a morphologically well-delimited family (Dahlgren et al. 1985) and considered a monophyletic group (Giulietti et al. 2000; Davis et al. 2004), its inter- and infra-generic relationships are still not well resolved and the traditional generic circumscriptions were mostly based on few floral characters (Stützel 1998; Giulietti et al. 1995, 2000).

The last full revision of Eriocaulaceae was done by Ruhland (1903) who established the taxonomic basis of the family, which is still in use. This author recognized about 560 species and two subfamilies: Eriocauloideae with diplostemonous flowers and glandular petals, including Eriocaulon and Mesanthemum; and Paepalanthoideae with isostemonous flowers and eglandular petals, including: Paepalanthus, Tonina, Lachnocaulon, Philodice Mart., Syngonanthus, Leiothrix and Blastocaulon. Since then, more than twice the original number of species have been described especially in the last five years in Comanthera, Eriocaulon, Paepalanthus and Syngonanthus. Also, four new genera have been proposed: Actinocephalus, Carptotepala Moldenke, Comanthera, Moldenkeanthus P. Morat. Of all the genera described in Eriocaulaceae, six are already considered to be synonymous to existing genera: Moldenkeanthus in Paepalanthus (Stützel 1987), Wurdackia in Rondonanthus (Hensold & Giulietti 1991), Carptotepala and Comanthera both in Syngonanthus (Giulietti & Hensold 1991), Philodice in Syngonanthus (Giulietti et al. 2009) and Blastocaulon in Paepalanthus (Andrade et al. 2011). Rondonanthus and Actinocephalus are currently accepted and Comanthera was recently reestablished (Parra et al. 2010). Currently the genera accepted in Eriocaulaceae are: Actinocephalus, Comanthera, Lachnocaulon, Leiothrix,Paepalanthus, Rondonanthus, Syngonanthus and Tonina within Paepalanthoideae, and Eriocaulon and Mesanthemum within Eriocauloideae.

The circumscription of genera in Eriocaulaceae has been based primarily on a few floral characters including the following: level of petal union in pistillate flowers; presence of glands on petals; number of stamens; and number of microsporangia per anther (Ruhland 1903; Stützel 1985; Giulietti et al. 1995, 2000).

Recent studies involving more than 30 researchers from six institutions in Brazil based on morphology, anatomy, floristics, taxonomy, chemistry, population biology and genetics, economic botany, physiology and molecular phylogenetics have provided fresh data for a better understanding of the family. These studies have also shown that, besides the characters used by Ruhland (1903), vegetative and floral parts also display many other characters, especially those relating to form and anatomy of the floral parts, embryology, type of hairs, seed testa and apertures and sculpturing of the pollen grains. The aims of this paper are to summarize the state of knowledge for the family and to present a reassessment of the relationships within Eriocaulaceae, especially in Paepalanthus, which is the largest genus in the Brazilian flora, with 357 species (Forzza et al. 2010).

Material and Methods

The material examined in the different studies is listed in ^{Appendix 1} Appendix 1 - Species and collections used in the morphological and anatomical studies of vegetative and reproductive organs. (morphological, and anatomical data) and ^{Appendix 2} Appendix 2 - Names, vouchers and Genbank accessions for DNA samples used for molecular phylogenetic analyses. The voucher specimens are deposited in the following Herbaria: HUEFS (molecular data). Voucher material has been deposited in the herbaria BH, C, FLAS, HRCB, HUEFS, K, MU, NY, PH and SPF.

For molecular phylogenetic analyses 71 species were sequenced. The ingroup consisted of 67 species, representing all five genera of Paepalanthoideae, including all main infrageneric divisions in Paepalanthus, proposed by Ruhland (1903) (^{Appendix 2} Appendix 2 - Names, vouchers and Genbank accessions for DNA samples used for molecular phylogenetic analyses. The voucher specimens are deposited in the following Herbaria: HUEFS ). Four species of Eriocaulon were defined as the outgroup (E. linearifolium Körn., E. ligulatum (Vell.) L.B. Sm., E. modestum Kunth and E. cinereum R. Br.). The DNA was extracted mostly from fresh or silica-gel leaves using a modified version on the CTAB procedure of Doyle & Doyle (1987). The programs of PCR and methods for amplification and sequencing of DNA are described in detail by Andrade (2007) and Andrade et al. (2010). For amplification and sequencing of ITS, we used the primers 75 and 92 of Desfeaux et al. (1996) and for some samples we used the primers 17SE and 26SE (Sun et al. 1994). For amplification and sequencing of trnL-F, we used two universal primers (C, F) of Taberlet et al. (1991). The PCR fragments were purified by enzymatic treatment with Exonuclease I (EXO) and Shrimp Alkaline Phosphatase (SAP) (Amersham Biosciences). The cycle-sequencing reactions were performed with the Big Dye Terminator version 3.1 (Applied Biosystems). Samples were sequenced in both directions using the Spectrumedix SCI SCE9624 automated sequencer at Universidade Estadual de Feira de Santana (UEFS).

Electropherograms were edited and assembled using Staden Package (Staden et al. 1998) and aligned using CLUSTAL X (Thompson et al. 1997). The resulting alignment was corrected manually following the guidelines in Kelchner (2000). Maximum Parsimony (MP) analyses were performed using PAUP* version 4.0b10 (Swofford 2002) with Fitch parsimony (equal weights, unordered; Fitch 1971) as the optimality criterion. We performed two separate searches for the ITS and trnL-F datasets. A third analysis included the combined data from both DNA regions. Each search consisted of 1,000 random taxon-addition replicates, with TBR algorithm, and limited swapping on up to 15 trees per replicate to prevent extensive swapping on islands with many trees. The resulting trees were then used as starting trees for TBR swapping with an upper limit of 30,000 trees. Nonparametric bootstrap support was estimated from 1,000 bootstrap (BP) replicates incorporating heuristic parsimony searches using addition sequence and branch-swapping options as in our MP analyses (Felsenstein 1985).

The model-based analysis was performed with Bayesian inference (Larget & Simon 1999; Lewis 2001), using MrBayes version 3.01 (Ronquist & Huelsenbeck 2003). The model used for trnL-F and ITS was GTR+I+G, as indicated by nested likehood-ratio tests using MrModeltest version 2.2 (Nylander 2004). MrBayes was run for 1,300,000 generations for trnL-F and 1,000,000 for ITS and combined analyses; with two separate analysis with four chains each, sampling trees every 100 generations. The burn-in stage needed to reach a stable state was determined by plotting the likelihood scores against the number of generations. The trees sampled from within the burn-in stage were excluded (480 for trnL-F, 260 for ITS and 400 for combined analyses), and the remaining trees were assumed to be representative of the posterior probability distribution. The majority rule consensus tree was calculated in PAUP* and the resulting group frequencies estimated the posterior probabilities (PP).

For light microscopy (LM), the median portion of adult roots, stems, leaves and scapes were sectioned by hand, stained with basic fuchsin and astra blue (Roeser 1962) and mounted in glycerine jelly (Kaiser 1880). For floral anatomy and embryology, inflorescences were dehydrated, embedded in historesin, sectioned, and the sections were stained with periodic acid – Schiff's Reagent (PAS reaction) and toluidine blue (Feder & O'Brien 1968). Material for scanning electron microscopy (SEM) was dehydrated through an ethanol series; critical-point dried; coated with gold; and examined using a JEOL JSM-5410 scanning electron microscope.

For pollen analysis, chemically (acetolysis) treated and untreated pollen grains were observed using LM and SEM (LEO 1430 VP – Carl Zeiss) respectively.

Results and Discussion

Morphology and anatomy

As mentioned above, the Eriocaulaceae are easy to recognize and considered monophyletic, but different patterns in the morphology and anatomy can occur in the group, and what may help to define one subgenus or section can vary at the species or population level in another.

Vegetative organs

The roots of Eriocaulaceae either store air in the cortex, with exodermis and arm cells, as in Eriocaulon and a few species of Leiothrix and Comanthera (Fig. 1a), or else lack aerenchyma and exodermis, possessing instead isodiametric cortical cells, especially in Paepalanthus and Actinocephalus (Fig. 1b). The stems are erect or rhizomatous, either with primary thickening, as in various species of Comanthera (Fig. 1c), or without as in Toninafluviatilis Aubl. (Fig. 1d). These anatomical characters of root and stem appear to depend closely on environmental factors. Because of this, they are not considered to be appropriate for establishing taxonomic groups within the family (Scatena et al. 2005).

The characters of leaves and scapes are also particularly useful for infrageneric taxa, species and populations (Scatena & Giulietti 1996; Scatena & Menezes 1996). The leaves (Figs. 1e-i) and scapes (Figs. 1j-l) possess epidermis with thin- (Figs. 1e- f, j) or thick-walled cells (Figs. 1i, l); stomata with (Figs. 1i, l) or without special substomatal chambers (Figs. 1e-h, j-k); presence (Figs. 1h-i, l) or absence of hypodermis (Figs. 1e-g, j-k); mesophyll formed by compact (Figs. 1g-i, k-l) or loosely aggregated chlorenchyma (Figs. 1e-f, j). These characters are closely associated with environmental factors and can be important for defining species clusters as in Leiothrix, Comanthera and Paepalanthus subg. Platycaulon from dry environments, and in Eriocaulon, Tonina, Syngonanthus sect. Carphocephalus and S. sect. Syngonanthus from wet environments (Scatena et al. 2005).

Except for the paraclades that define Actinocephalus (Fig. 2a) (Sano 2004; Costa & Sano 2006), characters related to the morphological architecture of the plants in Eriocaulaceae (Figs. 2a-i) have been restricted largely to infra-generic categories, especially in Paepalanthus (see Ruhland 1903). In Paepalanthus, the branching patterns of the stems and the position of the inflorescences might be important tools to develop a more natural classification of the genus associated with the flowers. For example, the fused scapes that define P. subg. Platycaulon Körn. are a good example of a feature that can be used to help resolve problems seen in clades PATL and P1 of the molecular analysis (Fig. 5).

Floral organs

Floral morphology plays a central role in Eriocaulaceae classification, with subfamilies and genera defined primarily by floral features. These are mainly: number of androecial parts; presence of glands in the petals; absence or reduction of petals in the pistillate flower; form of the pistillodes in the staminate flower; fusion of the petals in the pistillate flower; and the presence or absence of stylar appendages (Körnicke 1863; Ruhland 1903; Stützel 1998). Recent re-evaluation of these characters with SEM and other anatomical tools has revealed new information (Rosa & Scatena 2003, 2007; Coan & Scatena 2004; Borges 2008; Borges et al. 2009) and the importance of these characters has also been evaluated here.

The staminate and pistillate flowers of Eriocaulaceae are dichlamydeous and heterochlamydeous, with free petals, united from the base or united in the middle and free at the base and apex (syngonanthoid fusion), as in Mesanthemum (Figs. 3a-b), Syngonanthus (Figs. 3c-d) and Comanthera (Figs. 3e-f). Lachnocaulon and Tonina have monochlamydeous flowers, and the petals are reduced to hairs, especially in the pistillate flowers (Figs. 3g-j). Most of the family has trimerous flowers (Figs. 3b, k), but in Eriocaulon, Paepalanthus and Syngonanthus dimerous flowers also occur (Figs. 3l). Staminate flowers are diplostemonous in Mesanthemum and Eriocaulom (Figs. 3b, m), and are isostemonous in the other genera which present antesepalous scale-like staminodes (Fig. 3n), but not in Rondonanthusroraimae (Oliv.) Herzog with fertile stamens and nectariferous pistillodes (Fig. 3o). The staminodes in Paepalanthoideae probably indicate a reduction of the outer stamen whorl seen in Eriocauloideae (Rosa & Scatena 2003). Pistillate flowers mostly possess scale-like staminodes, except for Rondonanthus, which has elongated, vascularized staminodes (Fig. 3p) and includes R. flabelliformis (Moldenke) Hensold & Giul., observed to have dehisced anthers, but pollen grains have not been seen inside. The gynoecium has an unbranched style with a simple stigma in Eriocauloideae (Figs. 3a, q), or a branched style with simple or bifid stigmas and the stigmatic portions alternating with nectariferous portions in Paepalanthoideae (Fig. 3r-s). In Actinocephalus, Paepalanthus and Syngonanthus, both portions of the style are free at the same height (Figs. 3c, t-u), but in Leiothrix (a monophyletic group in all morphological and molecular analyses) the two style portions diverge at different heights in the column (Fig. 3s).

In all Eriocauloideae and the majority of Paepalanthoideae the anthers are dithecous and tetrasporangiate (Fig. 4a), but bisporangiate anthers occur in few species of Paepalanthus (Andrade et al. 2011) and Lachnocaulon (Fig. 4b), as well as in the species of Philodice considered to be synonymous of Syngonanthus. Variation in the number of anther microsporangia in Toninafluviatilis Aubl. was previously reported by Stützel (1985) using SEM, and is here confirmed by anatomical sections (Fig. 4c). The significance of the number of microsporangia as a useful character in delimiting genera has been stressed throughout the taxonomic history of Eriocaulaceae. This character has been used to delimit genera such as Blastocaulon from Paepalanthus (Giulietti 1978) and Philodice from Syngonanthus (Ruhland 1903), both now in synonymy. Molecular analysis shows that bisporangiate anthers are homoplasious in the family (Fig. 6).

Pollen grains in Eriocaulaceae are generally spherical, small- to medium-sized (22–37 µm), spiraperturate (Fig. 4d) or 2-zonasulcate in Comanthera subg. Comanthera. This is an important taxonomic character for the group (Fig. 4e). The exine surface is echinate to microechinate, sometimes with granules. Paepalanthus is a stenopalinogical genus. A remarkable feature among pollen grains in Eriocaulaceae is presented by T. fluviatilis with large grooved spines, interspersed with smaller ones (Santos et al. 2000, Borges 2008; Borges et al. 2009) (Fig. 4f).

Molecular data

The earliest phylogenetic studies in Eriocaulaceae were carried out based on morphological data (Giulietti et al. 1995, 2000), until the theses of Unwin (2004) and Andrade (2007) presented the first molecular analyses. Andrade et al. (2010) produced a broad molecular analysis of Eriocaulaceae, with 82 species representative of all recognized genera, using individual and combined analyses of sequences of the Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA and regions of plastid DNA (trnH-psbA and trnL-trnF). All combined analyses show higher bootstrap values than the individual analyses. The results of those analyses suggest the need for a new delimitation of taxa within Eriocaulaceae as follows: a) recognition of Eriocaulaceae subfam. Eriocauloideae and Paepalanthoideae as well-supported monophyletic groups; b) confirmation of Eriocaulon and Leiothrix as well-supported monophyletic genera; c) synonymy of Philodice under Syngonanthus and the need for conservation of the latter name (proposed by Giulietti et al. 2009); d) reestablishment of Comanthera (Parra et al. 2010); e) synonymy of Blastocaulon under Paepalanthus; f) transformation of the currently polyphyletic genus Paepalanthus in a monoplyletic group, by the inclusion of Actinocephalus, Lachnocaulon and Tonina. On account of the huge morphological diversity which would be encompassed in such a group, at present we prefer to await further data before embarking on such a step.

Of the five subgenera of Paepalanthus previously recognized by Ruhland (1903), three were sampled in the study mentioned above (Andrade et al. 2010) and were supported as monophyletic in at least some of the analyses: P. subg. Thelxinoë Ruhland, P. subg. Platycaulon Mart. and P. subg. Xeractis Körn. Also Actinocephalus (sensu Sano 2004) was a monophyletic group as associated with P. subsect. Aphorocaulon Ruhland.

In the present study, an additional 13 taxa of Paepalanthoideae were included and sequenced for ITS and trnL-F. Of these, Paepalanthus subg. Xeractis (with six new species) and P. subg. Platycaulon (with five new species), have been emphasized since both are monophyletic (Andrade et al. 2010) and both were recently revised (Hensold 1988; Tissot-Squalli 1997).

A total of 65 taxa were sequenced for trnL-F, including the ingroup of Paepalanthoideae and the outgroup constituted by four species of Eriocaulon (Eriocauloideae). Eriocaulon was chosen as outgroup as shown by Andrade (2007) and Andrade et al. (2010) to possess an insert of about 250 bp (between positions 302 and 508) in relation to all genera of Paepalanthoideae. Maximum parsimony analysis (MP) reached a pre-established limit of 30,000 of the shortest trees of 906 steps (CI 0.7064, RI 0.8525). The ITS region was sampled for 70 species. The MP analysis resulted in 39 trees with 1,938 steps, CI 0.5542, RI 0.8555. Both MP and Bayesian analyses of trnL-F and ITS were congruent, and the strict consensus trees of MP (with the support of bootstrap and posterior probabilities of Bayesian analyses) are plotted in Fig. 5. Analysis of the two regions resulted in similar topologies for the major groups, but differing in the internal resolution of the clades, especially within Paepalanthus. This appears to be due to ITS being more variable than trnL-F.

These MP combined analyses resulted in 4,128 trees with 2,949 steps, CI=0.5894, RI=0.8452. The topology observed in the MP analysis was very similar to that found in the Bayesian analysis except in minor details (Fig. 6). For the clades, the relationships in the combined analysis were more strongly supported than the individual analyses, in both bootstrap (PB) support values and Bayesian posterior probability (PP) (Figs. 5-6).

Using one of the most parsimonious trees of the combined analysis, the clades were named with letters that represent monophyletic groups. The bootstrap (BP) and Bayesian subsequent probability (PP) presented in the text is the result of the combined analyses (Fig. 6). However the BP obtained from individual analyses are presented in Fig. 5 for comparison.

All of the analyses support the monophyly of Paepalanthoideae (Figs. 5, 6, clade A, BP/PP 100). This group is characterized by: isostemonous flowers; eglandular petals and pistillate flowers with branched styles; stigmatic portions alternating with nectariferous portions; and simple or bifid stigmas. Except in Rondonanthus, which has linear and vascularized staminodes (Fig. 3p), all other genera have scale-like staminodes (Figs. 3t-u).

In Paepalanthoideae (Figs. 5, 6; Clade A), two main clades were recognized as having moderate to high bootstrap support and Bayesian posterior probability.

The Clade S (BP 93, PP 100) comprises the species of the monophyletic genus Syngonanthus (including S. sect. Syngonanthus, S. sect. Carphocephalus and Philodice sensu Ruhland). Syngonanthus sensu Parra et al. (2010) is characterized by having: staminate flowers with stamen filaments adnate to the corolla; 3 (or rarely 2) stamens; tetrasporangiate or bisporangiate anthers; and pistillate flowers with syngonanthoid fusion petals smaller than the sepals (Figs. 3c, d). The flavonoids have 6-hydroxy-luteoline derivatives (Ricci et al. 1996). Syngonanthus is strongly positioned as sister to the remainder of Paepalanthoideae in all analyses (Figs. 5, 6).

The clade B includes two main clades C and CM. Clade CM (BP 100, PP 100) is formed by the monophyletic Comanthera (C. subg. Comanthera and C. subg. Thysanocephalus) sensu Andrade et al. (2010) and Parra et al. (2010). This is corroborated here by the inclusion of C. cipoensis (Ruhland) L.R. Parra & Giul., which increases the taxa number in C. subg. Thysanocephalus. Comanthera has pistillate flowers with petals having syngonanthoid fusion that are longer than the sepals (Fig. 3e); staminate flowers (Fig. 3f) with 3 stamens and tetrasporangiate anthers, filaments free from the corolla, pistillodes with apical papillose trichomes; and seeds with rugose surfaces (Parra 2000; Parra et al. 2010). Comanthera has leaves and scapes with an epidermis having thickened cell walls, compact chlorenchyma (Figs. 1h-i), and scapes with malpighiaceous trichomes. It also has different flavonoids from Syngonanthus (Ricci et al. 1996; Coelho et al. 2006). Pollination is entomophilous, especially Diptera, Coleoptera, and Hymenoptera, and nectar is present (Ramos et al. 2005; Oriani et al. 2009). Two subgenera were recognized for Comanthera: Thysanocephalus, which has spiraperturate pollen grains; and Comanthera which has 2-zonasulcate pollen grains that have not been found in other representatives of the family (Figs. 4h-k) (Borges et al. 2009; Parra et al. 2010).

The Clade C includes two main clades: Clade RL including Rondonanthus and Leiothrix and Clade P with Paepalanthus. The Clade RL includes two subclades: L (Leiothrix) and R (Rondonanthus). Clade L (Figs. 5, 6; BP/PP 100) is formed by the monophyletic genus Leiothrix. The monophyly of Leiothrix confirms an earlier phylogenetic analysis based on morphology and anatomy (Giulietti et al. 1995). This genus includes approximately 45 species and is characterized by having pistillate flowers with styles forming a column, and with stigmatic and nectariferous portions free at different levels, basifixed anthers (Fig. 3n,s), and striate seeds. As in Andrade et al. (2010), the sister group of Leiothrix is not still clear. Leiothrix (Clade L) has Rondonanthus capillaceus (Körn.) Hensold & Giul. (Clade R) as the sister group in both ITS (BP 69, PP 86) and combined analyses (BP 53, PP 63). However, in trnL-F analysis, Clade CM appears more related to Clade L (BP >50%, not shown), and in polytomy with Clades P and R, which are, on the other hand, slightly sustained. Comanthera (Clade CM) and Leiothrix (Clade L) are shown to be monophyletic and highly sustained in all of the analyses. The inclusion of more species of Rondonanthus in further studies might lead to a better explanation of the phylogenetic position of this genus.

Clade P in the trnL-F analyses (Fig. 5; BP 91, PP 74), ITS (Fig. 5; BP 100, PP 100), and combined analyses (Fig. 6; BP 100, PP 100), is formed by the genera Paepalanthus,Actinocephalus, Lachnocaulon and Tonina, and divided into two subclades, corroborating the previous data in Andrade et al. (2010). The Clade PATL (PB 80, PP 100) presented better resolution in combined analyses and includes a polytomy. Toninafluviatilis and Lachnocaulonanceps Morong form a monophyletic group. They are morphologically characterized by the reduced perianth, the coalescence of the spathe with the scape and the pollen grains with grooved spines in Tonina (Figs. 2d, i, 3g-j, 4f), and by the geographic distribution in Lachnocaulon (T. Stützel pers. comm.). Paepalanthus lamarckii, with bisporangiate anthers, and P. obtusifolius Körn. and P. tortilis (Bong.) Mart., with tetrasporangiate anthers, form a well supported group (BP 100, PP 100). The other subclade includes P. almasensis Moldenke (dimerous flowers) as a sister group of a trichotomy. The first group is formed by species with dimerous flowers, included in P. sect. Diphyomene Ruhland and in P. series Dimeri Ruhland. The second monophyletic group includes P. leucocephalus Ruhland, P. scleranthus Ruhland (the unique species of P. subgen. Thelxinoë Ruhland), and P. exiguus (Bong.) Körn. All have dimerous flowers and are a sister group of P. stannardii Giul. & L.R. Parra and P. distichophyllus Mart. with elongate stems and trimerous flowers. The last group is formed by the sampled species of Actinocephalus and the species of Paepalanthus sect. Aphorocaulon. A new circumscription of Actinocephalus is being proposed by Costa & Sano (2006) for including the species of P. subsect. Aphorocaulon into Actinocephalus also corroborated by morphologic data. The genus is characterized by: the presence of paraclades; the capitula arrangement; anatomical data; and chromosome size, the largest in the family (Figs. 1b, 2a, 3k,t).

The subclade P1 presented better resolution in combined analyses and includes two subclades (Fig. 6). The first is formed by many species of P. subgen. Paepalanthus (including P. erigeron Mart. ex Körn., the type species). The other is formed by two other groups. The first includes several sections, subsections and series of Paepalanthus subgen. Paepalanthus (sensu Ruhland 1903). The second includes Paepalanthus (Blastocaulon) albidus Gardner and Paepalanthus (Blastocaulon) rupestris Gardner with bisporangiate anthers as a sister group of a monophyletic, well-supported clade, formed by Paepalanthus subgen. Platycaulon and P. subgen. Xeractis. The internal resolution of the clade is a polytomy. Paepalanthus subgen. Platycaulon is a monophyletic group, but P. sect. Divisi (P. tuberosus (Bong.) Kunth, P. bromelioides Silveira and P. vellozioides Körn.) and P. sect. Conferti (P. macropodus Ruhland and P. planifolius (Bong.) Körn.) are not sustained. Likewise, P. subgen. Xeractis is paraphyletic and the two sections analysed (P. sect. Xeractis and P. sect. Chrysostegis) are not sustained. Paepalanthus sect. Chrysostegis (P. argenteus (Bong.) Körn., the only species sampled) appeared collapsed with the two other series included in P. sect. Xeractis. Paepalanthus series Albidi (P. nigrescens Silveira, the only species studied) appear collapsed with those of the P. series Fuscati. Hensold (1988) considered P. ser. Albidi the most primitive group in P. subg. Xeractis and also considered that P. uncinatus Gardner was the most primitive species in the group. The reason is that involucral bracts are all green and leaf-like. On the other hand, P. nigrescens was the most specialized member in this series, mainly due to the acaulescent, thickened stem, and the leaves having a well-developed hypodermis. However, P. ser. Fuscati, including P. comans Silveira, P. augustus Silveira, P. mollis Kunth, and P. superbus Ruhland, appeared as a monophyletic group, characterized morphologically by the acaulescent habit, semi-succulent leaves with reduced buttresses and radially arranged mesophyll (Fig. 1g), and floral trichomes with rounded apices. Hensold (1988) proposed four alliances for P. ser. Fuscati, but the detection of different evolutionary lines in the series could not be determined in our study. We consider that more extensive sampling within the series should help to resolve the issue. Echternacht et al. (2011b) present a morphologic phylogeny of the group and the phylogenetic tree obtained agrees with many of Hensold's (1988) propositions. Firstly, all sections and series previously proposed appear to be monophyletic and the published hierarchy is also confirmed.

In spite of only c. 5% of the genus Paepalanthus being sampled, some lineages could be considered as subgenera: P. subgen. Leptocephali; P. subgen. Paepalanthus; P. subgen. Telxinoë, and P. subgen. Platycaulon.

Key to the genera of Eriocaulaceae

Flowers diplostemonous, stamens 4 or 6, petals glandular (Figs. 3b, m) ............................... 2

Flowers isostemonous, stamens 2 or 3, petals eglandular .................................................. 3

Pistillate flowers with petals free (Fig. 3q) ..................................................... Eriocaulon

Pistillate flowers with pistillate petals united in the middle and free at the base and apex (Fig. 3a) .................................................................................................. Mesanthemum

Pistillate flowers, petals reduced to small lobes covered with hairs, or lacking ............... 4

Pistillate flowers with petals never reduced or lacking .............................................. 5

Plants rosulate, scape subtended by a closed spathe, free from the main axis. Pistillate and staminate flowers with sepals free and petals usually lacking, anthers dithecous, bisporangiate (Figs. 2d; 3g-h) ......................................................... Lachnocaulon

Plants long-stemmed, leaves spirally arranged, scape subtended by an open spathe, fused with the main axis. Pistillate flowers with petals reduced to small hairy lobes, staminate flowers with sepals and petals united, anthers dithecous, tetrasporangiate, rarely bisporangiate, pollen exine with grooved spines (Figs. 2i; 3i-j) ................ Tonina

Pistillate flowers with linear staminodes or stamens present, staminate flowers with petals united or free (Figs. 2g; 3o-p,r) ........................................ Rondonanthus

Styles with the stigmatic and nectariferous portions (sensu Rosa & Scatena 2007) separating at different levels, seed surface striate (Fig. 3s) ....................................................... Leiothrix

Styles with stigmatic and nectariferous portions (sensu Rosa & Scatena 2007) separating at the same level, seed surface never striate .......................................................................... 7

Pistillate flowers with petals free .............................................................................. 8

Pistillate flowers with petals united in the middle and free at the base and apex ................ 9

Flowers 3-merous; staminate flowers with corolla base conspicuously fleshy, anthers dithecous, tetrasporangiate, capitula in leafy paraclades, pistillate flowers with stigmatic portions completely united (sensu Sano 2004; Costa & Sano 2006, including Paepalanthus sect. Aphorocaulon) (Figs. 2a; 3k,t) .................................................................................................... Actinocephalus

Flowers 2 or 3-merous; staminate flowers with corolla base membranous, anthers dithecous, bior tetrasporangiate, capitula never in leafy paraclades, pistillate flowers with stigmatic portions completely united or not (including Blastocaulon) ....................................................................................................... Paepalanthus

Scapes without malpighiaceous trichomes. Flowers 3-merous rarely 2-merous, unisexual rarely bisexual; staminate flowers with petals united to above the middle, anthers bi- or tetrasporangiate, filaments adnate to the corolla, pollen grains spiraperturate, apex of pistillodes without papillate trichomes; pistillate flowers with petals elliptic to obovate and short lobed, style with nectariferous portion without papillate trichomes; seed surface reticulate (including Philodice)(Figs. 3c-d) ...................................................................................................Syngonanthus

Conclusion

Considerable advances in our knowledge of Eriocaulaceae have been made since Monocots II (Giulietti et al. 2000). At present we recognized ten genera in Eriocaulaceae, with Philodice being synonymized under Syngonanthus and Blastocaulon under Paepalanthus, as well as the re-establishment of the genus Comanthera. Despite this, much still remains to be done to establish a phylogenetic classification within the family, especially in Paepalanthuss.l. This will require a considerable expansion of molecular information, as well as an elucidation of the nature, ontogeny and function of a wide range of recently discovered morphological features within the family. In addition, studies exploring species evolution and phylogenetic significance within Eriocaulaceae are needed.

Based on the data presented here, we recognize four monophyletic genera in Paepalanthoideae: Syngonanthus, Comanthera, Leiothrix and Rondonanthus. As regards the polyphyletic genus Paepalanthus, some molecular and morphological analyses are now in progress, with a view to producing a new classification of Paepalanthus.

Acknowledgements

We thank the Organizing Committee of the Fourth International Conference on the Comparative Biology of the Monocotyledons (Copenhagen 2008) and especially Dave Simpson and Paula Rudall the coordinators of the Poales Symposium where this paper was presented. We also thank John Conran and two anonymous referees for providing valuable comments on the manuscript and linguistic assistance, and we also thank Raymond Mervyn Harley and Richard Lindsay for revision of the English. We thank the Brazilian Research Council (CNPq) for the productivity grant to A.M.Giulietti, C. van den Berg, F.A.R.Santos, P.T.Sano and V.L.Scatena, and for the postdoc scholarship to M.J.G.Andrade, and for the PhD grant to A.I.Coan; the Brazilian Council for Graduate Studies (CAPES) for the Master grant to R.Borges and Ph.D to M.Trovó; to CNPq, FAPESP, CAPES and DAAD to support the participation of AMG, MT and CS in the Monocot IV Meeting; to the Brazilian Ministry of Science and Technology (MCT)/CNPq for funding through IMSEAR and PPBIO Projects. We also thank all herbarium curators for facilitating the use of the collections, and Ricardo Secco and Anna Luiza Ilkiu-Borges for the invitation to publish this paper in this special number of the Rodriguesia.

Artigo recebido em 05/05/2011.

Aceito para publicação em 21/10/2011.

Actinocephalus bongardii (A.St.-Hil.) Sano: Sano CFSC13023 (SPF), Santana do Riacho-MG; Scatena et al. 203 (HRCB). A. polyanthus (Bong.) Sano: Scatena et al. 214 (HRCB), Santana do Riacho-MG; CFCR 6349 (SPF). A. ramosus (Wikstr.) Sano: Scatena & Giulietti 165, Rio de Contas-BA; (HRCB); L.P. Felix 9525, Rio de Contas-BA (HUEFS). A. robustus (Silveira) Sano: Scatena et al. 224 (HRCB), Rio de Contas-BA. Comanthera aurifibrata (Silveira) L.R. Parra & Giul.: Rossi et al. CFCR 1079 (SPF), Joaquim Felício-MG. C. cipoensis (Ruhland) L.R. Parra & Giul.: Scatena et al. CFSC 10421, Santana do Riacho-MG; 10466, 10913 (SPF); Scatena et al. 195 (HRCB). Comanthera aff. circinnata (Bong.) L.R. Parra & Giul.: Zappi et al. CFCR 11292 (SPF), Diamantina-MG. C. elegans (Bong.) L.R. Parra & Giul.: Giulietti CFCR 3789 (SPF), Diamantina-MG. C. imbricata (Körn.) L.R. Parra & Giul.: Giulietti et al. 1047 (SPF), Guarapari-ES; Andrade Lima s/n (SPF). C. nitida (Bong.) L.R. Parra & Giul.: Magalhães 4335 (SPF), Santana do Riacho- MG. C. squarrosa (Ruhland) L.R. Parra & Giul.: Scatena CFCR 11309 (SPF), Santa Bárbara-MG. C. vernonioides (Kunth) L.R. Parra & Giul.: Giulietti CFSC 4968, Scatena, Santana do Riacho-MG; CFSC 10912, Scatena et al.; CFSC 10387 (SPF). C. xeranthemoides (Bong.) L.R. Parra & Giul.: Zappi et al. CFCR 11290 (SPF), Diamantina-MG. Eriocaulon aquatile Körn.: Giulietti CFSC 5055 (SPF), Santana do Riacho-MG. E. cipoense Silveira: Giulietti CFSC 5053 (SPF), Santana do Riacho-MG. E. crassiscapum Bong.:Giulietti CFSC 5138 (SPF), Santana do Riacho-MG. E. elichrysoides Bong.: Giulietti 5050 (SPF), Santana do Riacho-MG; Scatena et al. 183 (HRCB). E. ligulatus (Vell.) L. B. Sm.: Scatena et al. 199 (HRCB), Santana do Riacho-MG.; Teixeira s.n. SPF 97479 (SPF), Itabirito-MG; CFCR 1522 (SPF). E. linearifolium Körn. : Giulietti et al. CFCR 6294 (SPF), Joaquim Felício-MG. E. melanolepis Silveira: Hensold 582 (SPF), Santana do Riacho-MG. E. modestum Kunth: Harley et al. 26224 (SPF), Rio de Contas-BA. E. vaginatum Körn.: Scatena s.n. SPF 76169 (SPF), Santana do Riacho-MG. Lachnocaulon anceps Morong: Unwin 201 (MU), Alabama, USA; Harper 443 (PH), Georgia, USA. L. engleri Ruhland: Unwin 237 (MU), Alabama, USA. L. minus Small: Unwin 230 (MU), Alabama, USA. Leiothrix crassifolia (Bong.) Ruhland: Giulietti et al. CFCR 4672 (SPF), Couto Magalhães-MG. L. flavescens (Bong.) Ruhland var. distichophylla (Silveira) Giul. & Hensold: Cerati et al. CFCR 4287 (SPF), Datas-MG. L. flavescens (Bong.) Ruhland var. flavescens: Scatena et al. 209 (HRCB), Rio de Contas-BA. L. fluitans (Mart.) Ruhland: Giulietti CFSC 5051 (SPF), Santana do Riacho-MG; Rosa et al. 10 (HRCB); CFCR 8317 (SPF). Mesanthemum auratum Lecomte: Chillon 1835 (C), Senegal, Africa. Paepalanthus applanatus Ruhland: Silva et al. CFCR 8018 (SPF), Datas-MG. P. (Blastocaulon) albidus Gardner: Arbo et al. 4362 (SPF), Diamantina-MG; Hensold 234 (HUEFS). P. bromelioides Silveira: Sano et al. CFSC 12859 (SPF), Santana do Riacho-MG. P. bryoides Kunth: Giulietti et al. s.n. (SPF), Santana do Riacho-MG. P. chlorocephalus Silveira: Coan et al. 4 (HRCB), Santana do Riacho-MG. P. flaccidus (Bong.) Kunth: Scatena et al. 235 (HRCB), Santana do Riacho-MG. P. geniculatus (Bong.) Kunth: Hensold CFCR 4219 (SPF), Serro-MG. P. gibbosus Silveira: Giulietti CFCR 57 (SPF), Diamantina-MG. P. incanus (Bong.) Körn.: Zappi et al. CFCR 10527 (SPF), Diamantina-MG. P. latipes Silveira: Tissot & Scatena 29 (SPF), Santana do Riacho-MG. P. leucocephalus Ruhland: M.J.G. Andrade 488 (HUEFS), Grão Mogol-MG. P. longicaulis Silveira: Silveira 692 (SPF), Santana do Riacho-MG. P. macrocaulon Silveira: L.P. Felix 9539 (HUEFS), Rio de Contas-BA. P. macrocephalus (Bong.) Körn.: Silva et al. CFCR 11126 (SPF), Serro-MG. P. macropodus Ruhland: Benko-Iseppon s.n. (SPF), Santana do Riacho-MG. P. neglectus Körn.: L.P. Felix 9543 (HUEFS), Rio de Contas-BA. P. obtusifolius Körn.: Harley et al. 19812 (HUEFS), Rio de Contas-BA. P. phaeocephalus Ruhland: Giulietti CFCR 1073 (SPF), Alto Paraíso de Goiás-GO. P. planifolius (Bong.) Körn.: Mello-Silva et al. CFCR 8023 (SPF), Santana do Riacho-MG. P. prostratus (Benth. & Hook.) Koern: Hensold 496 (SPF), Santo Antonio do Itambé-MG. P. (Blastocaulon) rupestris Gardner: M.J.G. Andrade 543 (HUEFS), Diamantina-MG. P. scleranthus Ruhland: Scatena et al. 220 (HRCB), Santana do Riacho-MG. Scatena et al. 247 (HRCB). P. (Blastocaulon)scirpeus Mart. ex Körn.: Hensold 767 (SPF), Barão de Cocais-MG, Scatena et al. 246 (HRCB). P. subtilis Miq.: Scatena & Giulietti s.n. (HUEFS), Rio de Contas-BA; F. Juchum 16 (HUEFS). P. tortilis (Bong.) Mart.: M.J.G. Andrade 465 (HUEFS), Subaúma-BA; M.J.G. Andrade 472 (HUEFS), Grão Mogol-MG. P. vellozioides Körn.: Martens s.n. (SPF), Brumadinho-MG. Rondonanthus roraimae (Oliver) Herzog: Huber & Alarcon 10526 (SPF), Bolívar, Venezuela. Syngonanthus anthemidiflorus var. anthemidiflorus (Bong.) Ruhland: Hensold CFSC 5190 (SPF), Santana do Riacho-MG. S. appressus (Körn.) Ruhland: Giulietti et al. CFCR 4572 (SPF), Couto Magalhães-MG. S. caulescens (Poir.) Ruhland: Pereira CFCR 11310 (SPF), Serra Azul de Minas-MG; Scatena et al. 142 (HRCB). S. fuscescens Ruhland: Semir et al. CFSC 5191 (SPF), Santana do Riacho-MG. S. (Philodice) hoffmannseggii (Mart.) Giul & M.J.G. Andrade: Giulietti 1381 (HUEFS), Mato Grosso-MT S. nitens (Bong.) Ruhland: CFSC 12980 (SPF), Santana do Riacho-MG. S. verticillatus Ruhland: Giulietti CFCR 3785 (SPF). Tonina fluviatilis Aubl.: Harley et al. 17980 (SPF), Alcobaça-BA; Alves & Pinto s.n. (HRCB); Andrade 616 (HUEFS), Recife-PE.

¹, SPF², BH³, NY⁴ and FLAS⁵. For additional information on the geographic origin of taxa see Andrade et al. (2010). Asterisks indicate registry number of the sample in the DNA bank (FSA) at the Universidade Estadual de Feira de Santana, Brazil. GenBank accession numbers are given in the following sequence ITS and trnL-T, replaced with # where not sampled.

Actinocephalus bongardii (A.St.-Hil.) Sano¹: MJG Andrade 501; *1927; EU924282, EU924434. A.brachypus (Bong.) Sano¹: MJG Andrade 521; *1938; EU924281, EU924433. A. ciliatus (Bong.) Sano¹: MJG Andrade 544; *1956; EU924283, EU924435. A. ramosus (Wikstr.) Sano¹: BRN Araújo 78; *1798; EU924284, EU924436. A. stereophyllus (Ruhland) Sano¹: MJG Andrade 514; *1933; EU924285, EU924437. Comanthera aciphylla (Bong.) L.R. Parra & Giul.¹: MJG Andrade 532; *1948; EU924339, EU924491. C. cipoensis (Ruhland) L.R. Parra & Giul.²: Trovó 203; *6945, GQ861448, #. C. curralensis (Moldenke) L.R. Parra & Giul.¹: MJG Andrade 595; *6890; EU924340, EU924492. C. elegans (Bong.) L.R. Parra & Giul.^1,2: Trovó 340; *6941; GQ861447, #. C. hatschbachii (Moldenke) L.R. Parra & Giul.¹: AC Pereira 122; *6891; EU924341, EU924493. C. vernonioides (Kunth) L.R. Parra & Giul.¹: AM Giulietti 2185; *220; EU924343, EU924499. Eriocaulon cinereum R. Br.¹: AM Giulietti 2582; *6875; EU924280, EU924432. E. ligulatum (Vell.) L.B. Sm¹: AM Giulietti 2368; *298; EU924278, EU924430. E. linearifolium Körn.¹: AM Giulietti 2366; *297; EU924277, EU924429. E. modestum Kunth¹: MJG Andrade 445; *780; EU924279, EU924431. Lachnocaulon anceps Morong³: D. Goldman s/n; *6877; #, EU924442. Leiothrix arrecta Ruhland¹: AM Giulietti 2496; *6872; EU924296, EU924449. L. curvifolia (Bong.) Ruhland¹: MJG Andrade 553; *1964; EU924293, EU924446. L. distichoclada Herzog¹: MJG Andrade 458; *792; EU924294, EU924447. L. flagellaris (Guill.) Ruhland¹: MJG Andrade 485; *1920; EU924297, EU924450. L. flavescens (Bong.) Ruhland¹: MJG Andrade 440; *775; EU924291, EU924444. L.vivipara (Bong.) Ruhand¹: AM Giulietti 2503; *7415; EU924298, EU924451. P. acantholimon Ruhland²: Trovó 250; *6924; GQ475232, #. P. cf. acantholimon Ruhland¹: MJG Andrade 522; *1939; #, EU924466. P. acuminatus Ruhland²: Trovó 175; *6928; GQ475234, GQ475204. P. (Blastocaulon) albidus Gardner¹: MJG Andrade 541; *1953; EU924287, EU924439. P. almasensis Moldenke¹: MJG Andrade 429, *768; EU924315, EU924468. P. argenteus (Bong.) Körn.¹: MJG Andrade 539; *1952; EU924331, EU924484. P. augustus Silveira²: Borges 178; *6963; GQ475236, #. P. bromelioides Silveira²: Trovó 219; *6905; GQ475238, GQ475207. P. canescens Körn.¹: MJG Andrade 536; *1950; EU924324, EU924477. P. cinereus Giul & L.R. Parra¹: AM Giulietti s/n; *1293; EU924316, EU924469. P. comans Silveira¹: MJG Andrade 540; *6820; EU924329, EU924482. P. distichophyllus Mart.²: Trovó 218; *6907; GQ475245, #. P. elongatus (Bong.) Körn.¹: MJG Andrade 572; *6884; EU924314, EU924467. P. erigeron Mart. ex Körn.¹: A.A Ribeiro-Filho 107; *6882; EU924306, #. P. eriophaeus Ruhland¹: MJG Andrade 504; *6811; EU924307, EU924459. P. exiguus (Bong.) Körn.^1,2: E Guarçoni 710; *6885; EU924328, EU924481. P. giganteus Sano¹: MJG Andrade 527; *1943; EU924325, EU924478. P. glareosus Kunth¹: MJG Andrade 548; *1959; EU924322, EU924475. P. implicatus Silveira¹: MJG Andrade 550; *1961; EU924319, EU924472. P. lamarckii Kunth¹: DS Carneio-Torres 461; *6880; EU924303, EU924456. P. leucocephalus Ruhland¹; MJG Andrade 620; *6886; EU924334, EU924487. P. macrocaulon Silveira¹: MJG Andrade 431; *766; EU924317, EU924470. P. macropodus Ruhland²: Trovó 214; * 6922; GQ475251, GQ475220. P. mollis Kunth²: Trovó 376; *6961; GQ475253, GQ475222. P. neglectus Körn.¹: BRN Araújo 85; *1805; EU924308, EU924460. P. nigrescens Silveira²: Trovó 204; *6932; GQ475254, GQ475223. P. obtusifolius Körn.¹: R Harley 54802; *1321; EU924304, EU924457. P. planifolius (Bong.) Körn.¹: MJG Andrade 526; *1942; EU924332, EU924485. P. pulchellus Herzog¹: AM Giulietti 2423; *1409; EU924309, EU924461. P. pulvinatus N.E. Br.¹: R Harley 54634; *638; EU924313, EU924465. P. regalis Mart.¹: R Harley 54640; *660; EU924310, EU924462. P. (Blastocaulon) rupestris Gardner¹: MJG Andrade 542; *1954; EU924288, EU924440. P. (Blastocaulon) scirpeus Mart. ex Körn.^1,2: JR Pirani 4162; *6876; EU924289, EU924441.P. scleranthus Ruhland¹: MJG Andrade 537; *1951; EU924335, EU924488. P. silveirae Ruhland¹: MJG Andrade 568; *1976; EU924311, EU924463. P. spathulatus Körn.¹, R Harley 55476, *6883; EU924312, EU924464. P. sphaerocephalus Ruhland¹: MJG Andrade 456; *790; EU924327, EU924480. P. stannardii Giul. & L.R. Parra¹: MJG Andrade 438; *773; EU924320, EU924473. P. strictus Körn.¹: MJG Andrade 491; *1926; EU924318, EU924471. P. superbus Ruhland¹: AM Giulietti 2504; *6858; EU924330, EU924483. P.tortilis (Bong.) Mart.¹: MJG Andrade 479; *1914; EU924302, EU924455. P. trichophyllus (Bong.) Körn.¹: MJG Andrade 439; *774; EU924326, EU924479. P. tuberosus (Bong.) Kunth¹: C Van den Berg 1364; *1641; EU924333, EU924486. P. vellozioides Körn.²: Trovó 198; *6906; GQ475261, GQ475229. Rondonanthus capillaceus (Körn.) Hensold & Giul.¹: C Van den Berg 1792; *6889; EU924338, GQ478282. Syngonanthus arenarius (Gardner) Ruhland¹: MJG Andrade 493, *6802; EU924342, EU924498. S. caulescens (Poir.) Ruhland¹: MJG Andrade 452; *786; EU924344, EU924500. S. (Phil odice) hoffmannseggii (Mart.) Giul & M.J.G.Andrade¹: AM Giulietti 2483; *6887; EU924336, EU924489. S. verticillatus Ruhland²: Trovó 222; * 6944; GQ861446, #. Tonina fluviatilis Aubl.¹: MJG Andrade 616; *6895; EU924345, EU924501

Appendix 1  - Species and collections used in the morphological and anatomical studies of vegetative and reproductive organs.

Appendix 2  - Names, vouchers and Genbank accessions for DNA samples used for molecular phylogenetic analyses. The voucher specimens are deposited in the following Herbaria: HUEFS

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