bs_bs_banner Botanical Journal of the Linnean Society, 2015, 179, 255–265. With figures Fruits of Heterocoma (Vernonieae-Lychnophorinae): taxonomic significance and a new pattern of phytomelanin deposition in Asteraceae 1 Universidade Federal de Minas Gerais – Departamento de Botânica, Belo Horizonte 31270-901, Minas Gerais, Brazil 2 Universidade Federal de Uberlândia – Instituto de Biologia, Uberlândia 38400-902, Minas Gerais, Brazil Received 2 April 2015; revised 29 May 2015; accepted for publication 22 July 2015 Heterocoma is a Brazilian endemic genus resulting from the dismemberment of Sipolisiinae, in which only representatives with fruit containing phytomelanin were included in the genus. As the fruits of Asteraceae are known to be systematically important at various taxonomic levels and Heterocoma fruit has not been described previously, we studied the morphology and anatomy of the cypselas of all species of the genus, comparing them with other fruits in the family containing phytomelanin and evaluating the systematic potential at the specific and tribal levels. The fruits were analysed by scanning electron microscopy (SEM) and light microscopy. The morphological features of the fruit, including the carpopodium, ribs and pappi, varied in the genus and demonstrated potential for species discrimination. The anatomy showed a pattern for the genus with a uniseriate exocarp, the outer mesocarp composed of fibres arranged in several layers, the inner mesocarp composed of several layers of parenchyma, the endocarp, and phytomelanin deposited between the inner and outer mesocarp. This anatomical pattern of phytomelanin deposition differs from that of other Asteraceae with phytomelanin in their fruit. Heterocoma is also the only genus in Vernonieae that has phytomelanin deposition in the cypselas. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179, 255–265. ADDITIONAL KEYWORDS: anatomy – Bishopalea – cypsela – Sipolisia – Xerxes. INTRODUCTION Heterocoma DC. is an endemic genus of the Brazilian central plateau grasslands, comprising six species, characterized by foliose subinvolucral bracts, fimbrillate or paleaceous receptacles, glabrous cypselas, caducous biseriate pappi and cypselas with phytomelanin (Loeuille et al., 2013). [Two terms are found in use to describe the fruit of Asteraceae: achene and cypsela. Although both types have a single seed attached to the pericarp by a single point (funiculus), the fruits of Asteraceae come from the inferior ovary and have an outer layer of origin, not the carpel, whereas an achene fruit originates only *Corresponding author. E-mail: jmarzinek@gmail.com from carpel tissue. Thus, cypselas and achenes are not homologous, and the fruit of Asteraceae, by this definition, is a cypsela (Marzinek, De-Paula & Oliveira, 2008)]. Heterocoma in its current circumscription is the result of the dismemberment of Sipolisiinae, at which point Hololepis DC. was excluded and the other genera, Heterocoma DC., Bishopalea H.Rob., Sipolisia Glaz. ex Oliv. and Xerxes J.R.Grant, were included in the single genus, Heterocoma (Loeuille et al., 2013). This change was first made on the basis of morphological studies in which Hololepis was considered not to be related to the other genera of Sipolisiinae, mostly based on it lacking cypselas with phytomelanin, but also as a result of its petiolate leaves with a glabrous adaxial surface and its persistent pappus, characteristics not © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179, 255–265 255 Downloaded from https://academic.oup.com/botlinnean/article/179/2/255/2416526 by guest on 21 June 2022 FERNANDA S. FREITAS1, ORLANDO C. DE-PAULA2, JIMI N. NAKAJIMA2 and JULIANA MARZINEK2* 256 F. FREITAS ET AL. MATERIAL AND METHODS For this study, all species of Heterocoma were sampled. Details, including voucher information, are provided in Table 1. For scanning electron microscopy (SEM), the samples were mounted on aluminium stubs and coated with gold using a sputter coating device (Leica EM SCD050). The samples were analysed under a Zeiss EVO MA 100 and the images were digitally acquired. For anatomical studies, dried ovaries and cypselas were rehydrated with 5 M NaOH solution for 36 h (Anderson, 1963), dehydrated in an ethanol series and embedded in methacrylate resin, following the manufacturer’s protocol. The samples were sectioned using a rotary microtome at 10–12 μm thickness. The material was stained with 0.05% toluidine blue in acetate buffer, pH 4.7 (O’Brien, Feder & McCully, 1964, modified) and mounted in synthetic resin. The slides were observed under a light microscope (Olympus BX41) and the images were digitally acquired. The results are described considering the origin of the inferior ovary. Accordingly, we adopted the definition of the pericarp sensu Roth (1977), in which the exocarp originates from the outer epidermis of the ovary, the endocarp originates from the inner epidermis and the mesocarp originates from the ground and vascular tissues among both the inner and outer epidermis. The terminology for pappus morphology follows Hickey (1979), with the angle of divergence of the bristle being classified as narrow (< 45°) or moderate Table 1. Voucher information for species of Heterocoma used in this study Species Voucher Heterocoma albida (DC. ex Pers.) DC. & Toledo Nakajima 3074 T. Alves et al. 283 H.S. Irwin 28979 Loeuille 450 Bringel 91 R. Romero et al. 5550 Martinelli et al. 16417 Roque 2047 Roque et al. 2705 Heterocoma ekmaniana (Philipson) Loeuille, J.N.Nakaj. & Semir Heterocoma erecta (H.Rob.) Loeuille, J.N.Nakaj. & Semir Heterocoma gracilis Loeuille, J.N.Nakaj. & Semir Heterocoma lanuginosa (Glaz. ex Oliv.) Loeuille, J.N.Nakaj. & Semir Heterocoma robinsoniana Loeuille, J.N.Nakaj. & Semir Loeuille et al. 520 Glaziou 8969 L.S. Kinoshita 08-116 G. Hatschbach 72231 R. Romero 5029 R. Romero 5075 R. Romero et al. 1708 © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179, 255–265 Downloaded from https://academic.oup.com/botlinnean/article/179/2/255/2416526 by guest on 21 June 2022 found in other members of Sipolisiinae. Therefore, the authors synonymized Bishopalea, Sipolisia and Xerxes with Heterocoma. Phylogenetic analyses based on morphological and molecular data (Loeuille, Keeley & Pirani, 2015a; Loeuille et al., 2015b) have shown that Hololepis is not closely related to the monophyletic Heterocoma, and both genera belong to Lychnophorinae (Sipolisiinae being a synonym), still included in Vernonieae as proposed by Robinson (1996, 1999). The presence of phytomelanin in Heterocoma cypselas, first observed by Robinson (1996), is now a morphological synapomorphy for the new circumscription of the genus (Loeuille et al., 2013). The structural peculiarities of the cypselas of Asteraceae include the pappus and the carpopodium, both structures with great taxonomic significance. The pappus is a deeply modified calyx that can be used in the determination of species, as in Cardueae (Bean, 2001) and Solidago L. (Hood & Semple, 2003). The carpopodium is the abscission zone of the cypsela and is related to its dispersal. Its morphology (symmetrical, asymmetrical or undifferentiated) is used in the delimitation of taxa (Haque & Godward, 1984). The internal structure (anatomy) also has taxonomic importance at different taxonomic levels, as demonstrated by, for example, Roth (1977), Pandey & Singh (1982), Källersjö (1985), Bruhl & Quinn (1990), Pak, Park & Whang (2001), Marzinek, De-Paula & Oliveira (2010), Pandey, Stuessy & Mathur (2014) and Tadesse & Crawford (2014). Some cypselas contain a black and insoluble substance, called phytomelanin, which may be secreted by the fibres comprising the pericarp (De-Paula et al., 2013). It is involved in the protection of the embryo against light damage and insect attack (Johnson & Beard, 1977). The presence of phytomelanin in the cypsela is a common feature of most genera of the Heliantheae alliance (Anderberg et al., 2007), one of the later diverging clades of the family, comprising 13 tribes (Pandey et al., 2014). Some authors, including Tadesse & Crawford (2014) and Pandey et al. (2014), have used the way in which phytomelanin accumulates to establish deposition patterns of the pericarp for related taxa. This study describes and compares the morphology and anatomy of the cypselas in all six Heterocoma spp., searching for morphological patterns in the genus and species. As the genus is the only member of Vernonieae with phytomelanin in the cypsela wall, we compared the pattern of deposition of this compound in Heterocoma with the patterns previously described in the pericarp of members of the Heliantheae alliance, also evaluating the potential of phytomelanin for classification at higher taxonomic levels. PHYTOMELANIN IN HETEROCOMA (ASTERACEAE) (45–65°). The nomenclature proposed by Barthlott et al. (1998) was used to describe the bristle surfaces. RESULTS MORPHOLOGY All species have cylindrical and glabrous cypselas (Fig. 1A–F). They are erect in H. ekmaniana (Philip- 257 son) Loeuille, J.N.Nakaj. & Semir, H. erecta (H.Rob.) Loeuille, J.N.Nakaj. & Semir and H. gracilis Loeuille, J.N.Nakaj. & Semir (Fig. 1B–D) or slightly curved at the base in H. albida (DC. ex Pers.) DC. & Toledo, H. lanuginosa (Glaz. ex Oliv.) Loeuille, J.N.Nakaj. & Semir and H. robinsoniana Loeuille, J.N.Nakaj. & Semir (Fig. 1A, E, F). The ribs are conspicuous in H. albida, H. ekmaniana, H. erecta and H. lanuginosa Downloaded from https://academic.oup.com/botlinnean/article/179/2/255/2416526 by guest on 21 June 2022 Figure 1. Fruit morphology of Heterocoma. A–F, General view. A, H. albida. B, H. ekmaniana. C, H. erecta. D, H. gracilis. E, H. lanuginosa. F, H. robinsoniana. G–L, Carpopodium. G, H. albida. H, H. ekmaniana. I, H. erecta. J, H. gracilis. K, H. lanuginosa. L, H. robinsoniana. M–R, Nectary. M, H. albida. N, H. ekmaniana. O, H. erecta. P, H. gracilis. Q, H. lanuginosa. R, H. robinsoniana. Arrow, rib; arrowhead, pappus scar; ca, carpopodium; pa, pappus; ne, nectary. Scale bars: A–F, 400 μm; G–N, P–R, 200 μm; O, 100 μm. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179, 255–265 258 F. FREITAS ET AL. (Fig. 1A–C, E) and only weakly evident in H. gracilis and H. robinsoniana (Fig. 1D, F). All species have an apparent nectary (Fig. 1N–R). In the basal region of the fruit, the carpopodium (abscission zone) is inconspicuous in H. albida and H. robinsoniana (Fig. 1G, L) and conspicuous in H. ekmaniana, H. erecta, H. gracilis and H. lanuginosa (Fig. 1H–K). The carpopodium is symmetrical in H. ekmaniana (Fig. 1H) and asymmetrical in H. erecta (Fig. 1I), H. gracilis (Fig. 1J) and H. lanuginosa (Fig. 1K). The pappus is biseriate in all species (Fig. 1M–R). Both series, internal and external, are morphologically similar in H. ekmaniana, H. gracilis, H. lanuginosa and H. robinsoniana (Fig. 1N, P–R). In H. albida (Fig. 1M), the external series is short, with each unit having a rounded edge format (Fig. 1M), and remnants of this short cylindrical outer series were observed in H. erecta (Fig. 1C). The pappus has a laminar form at the base (Fig. 2A, C, E, G, J, L). The distal extremities of the pappus cell projections (bristles) occur only at the margin in H. ekmaniana, H. gracilis and H. robinsoniana (Fig. 2C, G, L). In H. albida, H. erecta and H. lanuginosa, the bristles are located around the pappus (Fig. 2A, E, J). The angle of divergence of the bristles is narrow in all species studied. The bristle surface is reticulate in H. ekmaniana and H. gracilis (Fig. 2H) and smooth in H. albida, H. erecta, H. lanuginosa and H. robinsoniana (Fig. 2B, F, K, M). With regard to the apex of the pappus, H. albida (Fig. 2B) and H. erecta (Fig. 2F) have more congested bristles, and H. ekmaniana, H. gracilis, H. lanuginosa and H. robinsoniana have more lax bristles (Fig. 2C, G–M). ANATOMY The exocarp is uniseriate, with flattened cells in the periclinal sense. The mesocarp is divided into two regions: the outer mesocarp consists of several layers of sclerenchyma and the inner mesocarp comprises several parenchymatic layers (Figs 3A–F, 4A–L). A collateral vascular bundle inserted into the mesocarp was observed to supply the pericarp. In H. albida and H. gracilis, all of the ribs have vascular bundles. In H. ekmaniana, H. erecta, H. lanuginosa and H. robinsoniana, some of the ribs lack vascular bundles (Fig. 3B, C, E, F). Phytomelanin is deposited inside the intercellular spaces, first between the parenchyma and sclerenchyma (Fig. 4A, C, E, G, I). Subsequently, an outer portion of the parenchyma becomes lignified in the mature cypselas (Fig. 4B, D, F, H, J–L). The endocarp and the inner layers of the mesocarp are consumed during the development of the cypsela (Fig. 3A–F). © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179, 255–265 Downloaded from https://academic.oup.com/botlinnean/article/179/2/255/2416526 by guest on 21 June 2022 Figure 2. Pappus morphology of Heterocoma. A, B, H. albida. A, Base. B, Apex. C, D, H. ekmaniana. C, Base. D, Apex. E, F, H. erecta. E, Base. F, Apex. G–I, H. gracilis. G, Base. H, Detail of setae; note the wall ornamentation (arrow). I, Apex. J, K, H. lanuginosa. J, Base. K, Apex. L, M, H. robinsoniana. L, Base. M, Apex. Arrow, wall ornamentation. Scale bars: A–D, F, I, K, M, 200 μm; E, G, H, J, L, 100 μm. PHYTOMELANIN IN HETEROCOMA (ASTERACEAE) 259 DISCUSSION According to Robinson (1999) and Keeley & Robinson (2009), the cypselas of Vernonieae generally have biseriate trichomes (Zwillingshaare) and, according to Roth (1977) and Bremer (1994), this type of trichome is common in Asteraceae and is highly characteristic of the pericarp of many members of the family. The lack of trichomes on the pericarp of Heterocoma distinguishes the genus from most other Vernonieae. In addition, in Vernonieae, the cypselas mostly have a biseriate pappus with or without a reduced outer series (Robinson, 1999). Among the species studied, H. albida has a reduced series and a short and flat pappus, whereas H. erecta has a short and cylindrical series. According to Ramiah & Sayeeduddin (1958), the pappus is a deeply modified © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179, 255–265 Downloaded from https://academic.oup.com/botlinnean/article/179/2/255/2416526 by guest on 21 June 2022 Figure 3. Transversal sections of Heterocoma fruit. A, H. albida. B, H. ekmaniana. C, H. erecta. D, H. gracilis. E, H. lanuginosa. F, H. robinsoniana. Arrow, rib; arrowhead, phytomelanin; pe, pericarp; vb, vascular bundle; sc, seminal chamber; se, seed. Scale bars: A–F, 400 μm. 260 F. FREITAS ET AL. Downloaded from https://academic.oup.com/botlinnean/article/179/2/255/2416526 by guest on 21 June 2022 Figure 4. Transversal sections of Heterocoma cypselae pericarp. A, B, H. albida. A, Immature fruit. B, Mature fruit. C, D, H. ekmaniana. C, Immature fruit. D, Mature fruit. E, F, H. erecta. E, Immature fruit. F, Mature fruit. G, H, H. gracilis. G, Immature fruit. H, Mature fruit. I, J, H. lanuginosa. I, Immature fruit. J, Mature fruit. K, L, H. robinsoniana. K, Immature fruit. L, Mature fruit. Arrowhead, phytomelanin; ec, exocarp; mc, mesocarp; vb, vascular bundle. Scale bars: A–L, 100 μm. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179, 255–265 Curved Inconspicuous Inconspicuous With and without vascular bundles Similar to inner series Marginal Smooth Lax Curved Symmetric Conspicuous With and without vascular bundles Similar to inner series Scattered Smooth Lax Outer series of the pappus Projections of pappus cells (bristles) Ornamentation of the bristles Distribution of bristles on the apex of the pappus Curved Inconspicuous Conspicuous All with vascular bundles Rounded edge Scattered Smooth Congested Base of cypsela Carpopodium Ribs Straight Symmetric Conspicuous With and without vascular bundles Similar to inner series Marginal Reticulate Lax Straight Asymmetric Conspicuous With and without vascular bundles Cylindrical Scattered Smooth Congested Straight Asymmetric Inconspicuous All with vascular bundles Similar to inner series Marginal Reticulate Lax H. robinsoniana H. gracilis H. erecta H. ekmaniana H. albida Features Species studied Table 2. Fruit morphological features of the Heterocoma species studied © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179, 255–265 261 Downloaded from https://academic.oup.com/botlinnean/article/179/2/255/2416526 by guest on 21 June 2022 calyx and, according to Roth (1977), the sepals of Asteraceae are initiated, together forming a ringshaped structure (‘Ringwall’), which probably refers to H. albida and H. erecta having the outer series of the pappus that interrupts their growth in the early stages. In addition to the pappus, the base of the cypsela, ribs, carpopodium and the organization of the pericarp tissues proved to be important features for the segregation of Heterocoma spp. (Table 2). The abscission zone of the cypsela (carpopodium) may or may not be conspicuously developed (Haque & Godward, 1984). In the case of the studied species, the taxonomic value of this structure is confirmed, as it is inconspicuous in H. albida and H. robinsoniana, symmetric in H. ekmaniana and H. lanuginose, and asymmetric in H. erecta and H. gracilis. Heterocoma ekmaniana, H. erecta and H. lanuginosa have some ribs lacking vascular bundles. According to Marzinek et al. (2010), the formation of ribs on cypsela might be more related to the position occupied by the flower in the inflorescence axis than the presence of the vascular bundles, also resulting in ribs without bundles, as in Heterocoma. The anatomy of cypselas of Vernonieae is poorly understood. Studies examining species of Vernonia (Pandey & Singh, 1980; Basak & Mukherjee, 2003; Martins & Oliveira, 2007; Galastri & Oliveira, 2010) and genera (Mukherjee & Sarkar, 2001) have generally shown that the pericarp of all species contains a wide variety of crystals. The exocarp is uniseriate at maturity, the outer mesocarp consists of fibres arranged in several layers, the inner mesocarp consists of several layers of parenchyma and the endocarp is consumed during development. Heterocoma spp. share this arrangement of the pericarp, and it corroborates the pattern already established for Vernonieae. However, crystals have not been observed in the pericarp of Heterocoma. Phytomelanin was observed between the fibrous outer mesocarp and the inner parenchyma. According to King & Robinson (1987), cypselas containing phytomelanin do not contain crystals, and vice versa, with the exception of rare representatives of Heliantheae, in which phytomelanin and crystals occur simultaneously. In Asteraceae, in addition to the Heterocoma phytomelanin deposition pattern, two other patterns are found in Eupatorieae and Heliantheae (Table 3). The Eupatorieae pattern of phytomelanin deposition is found only in Eupatorieae. In these species, the pericarp possesses a uniseriate exocarp, an outer mesocarp consisting of parenchyma, an inner mesocarp consisting of sclerenchyma and parenchyma cells and an endocarp consumed during cypsela development (Pandey et al., 1989; Pandey & Singh, 1994; Pandey, H. lanuginosa PHYTOMELANIN IN HETEROCOMA (ASTERACEAE) 262 F. FREITAS ET AL. Table 3. Comparative data for phytomelanin pattern deposition in the fruit of the Asteraceae family (black areas, phytomelanin; ex, exocarp; pa, parenchyma; sc, sclerenchyma) Tribes Species References Eupatorieae Eupatorieae Adenostema viscosum J.R.Forst. & G.Forst Ageratina adenophora (Spreng.) R.M.King & H.Rob. Ageratina pseudochilca (Benth.) R.M.King & H.Rob. Ageratum conyzoides L. Pandey & Singh (1983) Pandey & Singh (1983) Pandey & Singh (1994) Pandey & Singh (1983); Franca et al. (2015) Franca et al. (2015) Pandey & Singh (1994) Marzinek & Oliveira (2010) Pandey & Singh (1994) Marzinek & Oliveira (2010) Heliantheae Coreopsideae Heliantheae Madieae Millerieae Neurolaeneae Senecioneae Tageteae Heterocoma Vernonieae Ageratum fastigiatum (Gardner) R.M.King & H.Rob. Bartlettina platyphylla (B.L.Rob.) R.M.King & H.Rob. Campuloclinium macrocephalum DC. Chromolaena odorata (L.) R.M.King & H.Rob. Chromolaena stachyophylla (Spreng.) R.M.King & H.Rob. Eupatorium serotinum Michx. Liatris aspera Michx. Liatris scariosa (L.) Willd. Mikania micrantha Kunth Mikania scandens (L.) Willd. Neomirandea araliaefolia (Less.) R.M.King & H.Rob. Praxelis clematidea (Griseb.) R.M.King & H.Rob. Praxelis diffusa (Rich.) Pruski Praxelis pauciflora (Kunth) R.M.King & H.Rob. Symphyopappus reticulatus Baker Vittetia orbiculata (DC.) R.M.King & H.Rob. Coreopsis tinctoria Nutt. Bidens gardneri Baker Bidens pilosa L. Coreopsis auriculata L. Coreopsis stillmannii (A.Gray) S.F.Blake Cosmos sulphureus Cav. Clibadium F.Allam. ex L. Eclipta prostrata (L.) L. Heliopsis helianthoides ssp. scabra (Dunal) T.R.Fisher Lagascea mollis Cav. Parthenium hysterophorus L. Parthenium hysterophorus L. Sphagneticola calendulacea (L.) Pruski Spilanthes acmella (L.) L. Verbesina encelioides (Cav.) Benth. & Hook.f. ex A.Gray Zinnia angustifolia Kunth Achyrachaena mollis Schauer Desmanthodium Benth. Icthyothere Baker Sigesbeckia orientalis L. Tridax procumbens (L.) L. Galinsoga quadriradiata Ruiz & Pav. Erechites valerianifolius (Wolf) DC. Tagetes erecta L. Flaveria trinervia (Spreng.) C.Mohr Porophyllum ruderale (Jacq.) Cass. Heterocoma albida (DC. ex Pers.) DC. & Toledo Heterocoma ekmaniana (Philipson) Loeuille, J.N.Nakaj. & Semir Heterocoma erecta (H.Rob.) Loeuille, J.N.Nakaj. & Semir Heterocoma gracilis Loeuille, J.N.Nakaj. & Semir Heterocoma lanuginosa (Glaz. ex Oliv.) Loeuille, J.N.Nakaj. & Semir Heterocoma robinsoniana Loeuille, J.N.Nakaj. & Semir Pandey & Singh (1994) Pandey & Singh (1994) Pandey & Singh (1994) Marzinek & Oliveira (2010) Pandey & Singh (1994) Pandey & Singh (1994) Batista (2014) De-Paula et al. (2013) Marzinek & Oliveira (2010) Marzinek & Oliveira (2010) Marzinek & Oliveira (2010) Pandey & Singh (1982) Julio & Oliveira (2009) Julio & Oliveira (2009) Pandey & Singh (1982) Pandey & Singh (1982) Batista (2014) Stuessy & Liu (1983) Batista (2014) Pandey & Singh (1994) Pandey & Singh (1994) Pandey & Singh (1994) Batista (2014) Pandey & Singh (1994) Pandey & Singh (1994) Misra (1972) Pandey & Singh (1994) Pandey & Singh (1994) Stuessy & Liu (1983) Stuessy & Liu (1983) Batista (2014) Frangiote-Pallone & Souza (2014) Batista (2014) Batista (2014) Pandey (1998) Misra (1964) Frangiote-Pallone & Souza (2014) This work This work This work This work This work This work © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179, 255–265 Downloaded from https://academic.oup.com/botlinnean/article/179/2/255/2416526 by guest on 21 June 2022 Phytomelanin pattern of deposition PHYTOMELANIN IN HETEROCOMA (ASTERACEAE) cypselas containing phytomelanin in Asteraceae fall into three groups according to their pattern of deposition: the pericarp consists of outer parenchymatic mesocarp and inner sclerenchymatic mesocarp with the development of schizogenous space; the pericarp consists of outer parenchymatic mesocarp and inner sclerenchymatic mesocarp without the development of schizogenous space; and the pericarp consists of outer sclerenchymatic mesocarp and internal parenchymatic mesocarp without forming a schizogenous space (Table 3). The comparison showed that the pattern of cypsela phytomelanin deposition in Heterocoma has not been described previously for the family, reaffirming the taxonomic potential and the importance of anatomical studies in Asteraceae. ACKNOWLEDGEMENTS The authors thank Benoît Loeuille and Nádia Roque for providing mature cypselas, and Francielle Batista da Silva [Laboratório Multiusuário de Microscopia Eletrônica, Faculdade de Engenharia Química (UFU)] for support with SEM. They also thank Benoît Loeuille for critical and helpful reading of the manuscript. REFERENCES Anderberg AA, Baldwin BG, Bayer RG, Breitwieser J, Jeffrey C, Dillon MO, Eldenas P, Funk V, Garcia-Jacas N, Hind DJN, Karis PO, Lack HW, Nesom G, Nordenstam B, Oberprieler CH, Panero JL, Puttock C, Robinson H, Stuessy TF, Susanna A, Urtubey E, Vogt R, Ward J, Watson LE. 2007. Compositae. In: Kubitzki K, Kadereit JW, Jeffrey C, eds. The families and genera of vascular plants. Berlin, Heidelberg: Springer Verlag, 61–576. Anderson LC. 1963. Studies on Petradoria (Compositae): anatomy, cytology, taxonomy. Transactions of the Kansas Academy of Science 66: 632–684. Barthlott W, Neinhuis C, Cutler D, Ditsch F, Meusel I, Theisen I, Wilhelmi H. 1998. Classification and terminology of plant epicuticular waxes. Botanical Journal of the Linnean Society 126: 237–260. Basak N, Mukherjee SK. 2003. Taxonomic significance of cypselar features in some species of Vernonia (Vernonieae – Asteraceae). Journal of Hill Research 16: 9–15. Batista MF. 2014. Morfoanatomia comparada da flor e do fruto (pericarpo) em desenvolvimento de espécies de Asteraceae. Centro de Ciências Biológicas, Maringá: Universidade Estadual de Maringá. Bean AR. 2001. Pappus morphology and terminology in Australian and New Zealand thistles (Asteraceae, tribe Cardueae). Austrobaileya 6: 139–152. Bremer K. 1994. Asteraceae: cladistics and classification. Portland, OR: Timber Press. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179, 255–265 Downloaded from https://academic.oup.com/botlinnean/article/179/2/255/2416526 by guest on 21 June 2022 1998; Marzinek & Oliveira, 2010). Between the outer and inner mesocarp, there is a separation of tissues, forming a schizogenous space in which phytomelanin is deposited. The projections of sclerenchyma that connect with the parenchyma under the epidermis (hypodermis) were described as peg-like by Pandey et al. (2014). They developed a technique of diaphanization of the outer mesocarp (in the case of other species of the Heliantheae alliance, where it is parenchymatous). The remaining structure after treatment is phytomelanin, which was deposited previously. The phytomelanin is deposited, acquiring different formats, and, from this starting point, the authors were able to establish phytomelanin distribution patterns. In Heterocoma, the technique of Pandey et al. (2014) could not be used because, unlike the other species of the Heliantheae alliance, the outer layer of the pericarp is sclerenchymatous and resistant to the action of the diaphanization agent. The Heliantheae phytomelanin deposition pattern occurs in Coreopsideae, Heliantheae, Madieae, Millerieae, Neurolaeneae and Tageteae. In such cases, the pericarp is similar to the pericarp of Eupatorieae (Table 3) with a uniseriate exocarp, an external mesocarp composed of parenchyma, an inner mesocarp composed of sclerenchyma and parenchyma cells, and an endocarp consumed during cypsela development (Misra, 1964, 1972; Pandey & Singh, 1982, 1994; Stuessy & Liu, 1983; Pandey, 1998). However, there is no formation of a schizogenous space, and phytomelanin is deposited in the intercellular spaces between the parenchyma and sclerenchyma mesocarp. In all three patterns described so far for the family, phytomelanin is always associated with fibres. According to De-Paula et al. (2013), fibres are responsible for the secretion of phytomelanin, re-using the arrangement of organelles employed in the secretion of the wall carbohydrates. Furthermore, phytomelanin has been found recently in the pericarp of Erechtites valerianifolius (Link ex Spreng.) DC. (Senecioneae) (Batista, 2014). This new finding only reinforces the assertion of De-Paula et al. (2013), who stated that the presence of phytomelanin in cypselas of Asteraceae is underestimated in the family, and that its occurrence has been reported in the roots and rhizomes of some species of Cardueae (Fritz & Saukel, 2011) and in the stems and leaves of species of Lychnophorinae (Lusa, 2014). In this article, we provide the first description of the structure of the cypselas of Heterocoma, the only genus in Vernonieae with cypselas containing phytomelanin, and evaluate its interspecific taxonomic value (Table 2). In addition, the anatomical pattern in Heterocoma was compared with other patterns found in members of Asteraceae with cypselas containing phytomelanin. Our study suggests that the 263 264 F. FREITAS ET AL. Loeuille B, Semir J, Lohmann LG, Pirani JR. 2015b. A phylogenetic analysis of Lychnophorinae (Asteraceae: Vernonieae) based on molecular and morphological data. Systematic Botany 40: 299–315. Lusa MG. 2014. Morfoanatomia e fitoquímica de espécies da subtribo Lychnophorinae (Asteraceae: Vernonieae) como subsídios para as análises filogenéticas do grupo. Instituto de Biologia, Campinas: Universidade Estadual de Campinas. Martins MA, Oliveira DMT. 2007. Morfoanatomia comparada dos frutos em desenvolvimento de Vernonia brevifolia Less. e V. herbacea (Vell.) Rusby (Asteraceae). Revista Brasileira de Botânica 30: 99–110. Marzinek J, De-Paula OC, Oliveira DMT. 2008. Cypsela or achene? Refining terminology by considering anatomical and historical factors. Revista Brasileira de Botânica 31: 549–553. Marzinek J, De-Paula OC, Oliveira DMT. 2010. The ribs of Eupatorieae (Asteraceae): of wide taxonomic value or reliable characters only among certain groups? Plant Systematics and Evolution 285: 127–130. Marzinek J, Oliveira DMT. 2010. Structure and ontogeny of the pericarp of six Eupatorieae (Asteraceae) with ecological and taxonomic considerations. Anais da Academia Brasileira de Ciências 82: 279–291. Misra S. 1964. Floral morphology of the family Compositae. 2. Development of the seed and fruit in Flaveria repanda. Botanical Magazine Tokyo 77: 290–296. Misra S. 1972. Floral morphology of the family Compositae. IV. Tribe Vernonieae – Vernonia anthelmintica. Botanical Magazine Tokyo 85: 187–199. Mukherjee SK, Sarkar AK. 2001. Study of macromorphological and anatomical structures of cypselas of eighteen taxa of the tribe Vernonieae (Asteraceae). Journal of the National Botanical Society 55: 85–104. O’Brien TP, Feder N, McCully ME. 1964. Polychromatic staining of plant cell walls by toluidine blue O. Protoplasma 59: 368–373. Pak IH, Park IK, Whang SS. 2001. Systematic implications of fruit wall anatomy and surface sculpturing of Microseris (Asteraceae, Lactuceae) and relatives. International Journal of Plant Sciences 162: 209–220. Pandey AK. 1998. Development of phytomelanin layer in fruit wall of Tagetes patula L. (Asteraceae). Journal of the Indian Botanical Society 77: 35–38. Pandey AK, Lee WW, Sack FD, Stuessy TF. 1989. Development of the phytomelanin layer in fruits of Ageratum conyzoides (Compositae). American Journal of Botany 75: 739–746. Pandey AK, Singh RP. 1980. Development and structure of seeds and fruits in tribe Vernonieae – some Vernonia and Elephantopus species. Flora 169: 443–452. Pandey AK, Singh RP. 1982. Development and structure of seeds and fruits in the Compositae, tribe Senecioneae. Botanische Jahrbucher fur Systematik, Pflanzengeschichte und Pflanzengeographie 103: 413–422. Pandey AK, Singh RP. 1983. Development and structure of seeds and fruits in Compositae: tribe Eupatorieae. Journal of the Indian Botanical Society 62: 276–281. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179, 255–265 Downloaded from https://academic.oup.com/botlinnean/article/179/2/255/2416526 by guest on 21 June 2022 Bruhl JJ, Quinn CJ. 1990. Cypsela anatomy in the ‘Cotuleae’ (Asteraceae-Anthemideae). Botanical Journal of the Linnean Society 102: 37–59. De-Paula OC, Marzinek J, Oliveira DMT, Machado SRM. 2013. The role of fibers and the hypodermis in Compositae melanin secretion. Micron (Oxford, England: 1993) 44: 312–316. Franca RO, De-Paula OC, Carmo-Oliveira R, Marzinek J. 2015. Embryology of Ageratum conyzoides L. and A. fastigiatum R. M. King & H. Rob. (Asteraceae). Acta Botanica Brasilica 29: 8–15. Frangiote-Pallone S, Souza LA. 2014. Ontogenia del papus y cipsela en Asteraceae: las consideraciones structurales de la categoría tribal. Revista Mexicana de Biodiversidad 85: 62–77. Fritz E, Saukel J. 2011. Secretory structures of subterranean organs of some species of the Cardueae, and their diagnostic value. Acta Biologica Cracoviensia Series Botanica 53: 62–72. Galastri NA, Oliveira DMT. 2010. Morfoanatomia e ontogênese do fruto e semente de Vernonia platensis (Spreng.) Less. (Asteraceae). Acta Botanica Brasilica 24: 73–83. Haque MZ, Godward MBE. 1984. New records of the carpopodium in Compositae and its taxonomic use. Botanical Journal of the Linnean Society 89: 321–340. Hickey LJ. 1979. A revised classification of the architecture of dicotyledonous leaves. In: Metcalf CR, Chalk L, eds. Anatomy of the dicotyledons. Oxford: Clarendon Press, 25–39. Hood JLA, Semple JC. 2003. Pappus variation in Solidago. Sida 20: 1617–1630. Johnson AL, Beard BH. 1977. Sunflower moth damage and inheritance of the phytomelanin layer in sunflower achenes. Crop Science 17: 369–372. Julio PGS, Oliveira DMT. 2009. Morfoanatomia comparada e ontogênese do pericarpo de Bidens gardneri Baker e B. pilosa L. (Asteraceae). Revista Brasileira de Botânica 32: 109–116. Källersjö M. 1985. Fruit structure and generic delimitation of Athanasia (Asteraceae-Anthemideae) and related South African genera. Nordic Journal of Botany 5: 527– 542. Keeley SC, Robinson H. 2009. Vernonieae. In: Funk VA, Susanna A, Stuessy TF, Bayer RG, eds. Systematics, evolution, and biogeography of Compositae. Vienna: IAPT, 439–470. King RM, Robinson H. 1987. The genera of the Eupatorieae (Asteraceae). Lawrence, MO: Missouri Botanical Garden. Loeuille B, Keeley SC, Pirani JR. 2015a. Systematics and evolution of syncephaly in American Vernonieae (Asteraceae) with emphasis on the Brazilian subtribe Lychnophorinae. Systematic Botany 40: 286–298. Loeuille B, Nakajima JN, Oliveira DMT, Semir J, Pirani JR. 2013. Two new species of Heterocoma (Asteraceae: Vernonieae) and a broadened concept of the genus. Systematic Botany 38: 242–252. PHYTOMELANIN IN HETEROCOMA (ASTERACEAE) Robinson H. 1999. Generic and subtribal classification of American Vernonieae. Smithsonian Contributions to Botany 89: 1–116. Roth I. 1977. Fruits of angiosperms. Berlin: Gebrüder Borntraeger. Stuessy TF, Liu H. 1983. Anatomy of the pericarp of Clibadium, Desmanthodium and Ichthyothere (Compositae, Heliantheae) and systematics implications. Rhodora 85: 213– 227. Tadesse M, Crawford DJ. 2014. The phytomelanin layer in traditional members of Bidens and Coreopsis and phylogeny of the Coreopsideae (Compositae). Nordic Journal of Botany 32: 80–91. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179, 255–265 Downloaded from https://academic.oup.com/botlinnean/article/179/2/255/2416526 by guest on 21 June 2022 Pandey AK, Singh RP. 1994. Development and structure of seed and fruit in Eupatorieae and Heliantheae (Compositae). Proceedings of the National Academy of Sciences 64: 115–126. Pandey AK, Stuessy TF, Mathur RR. 2014. Phytomelanin and systematics of the Heliantheae Alliance (Compositae). Plant Diversity and Evolution 131: 1–21. Ramiah N, Sayeeduddin M. 1958. Homology of the pappus in the light of trichome distribution. Current Science 10: 402–403. Robinson H. 1996. The status of generic and subtribal revisions in the Vernonieae. In: Hind DJN, Beentje HJ, eds. Compositae: systematics. Proceedings of the International Compositae Conference. Kew: Royal Botanic Gardens, 511– 529. 265