Structure and Ontogeny of the Pericarp of Six Eupatorieae (Asteraceae) with Ecological and Taxonomic Considerations

“main” — 2010/4/27 — 17:28 — page 279 — #1 Anais da Academia Brasileira de Ciências (2010) 82(2): 279-291 (Annals of the Brazilian Academy of Sciences) ISSN 0001-3765 www.scielo.br/aabc Structure and ontogeny of the pericarp of six Eupatorieae (Asteraceae) with ecological and taxonomic considerations JULIANA MARZINEK1 and DENISE M.T. OLIVEIRA2 1Instituto de Biologia, Universidade Federal de Uberlândia Rua Ceará, s/n, Bloco 2D, sala 28, Umuarama, 38405-315, Uberlândia, MG, Brasil 2Departamento de Botânica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais Avenida Antonio Carlos, 6627, Pampulha, 31270-901 Belo Horizonte, MG, Brasil Manuscript received on October 20, 2008; accepted for publication on June 4, 2009 ABSTRACT The ontogeny of cypselae and their accessory parts were examined using light and scanning electron microscopy for the species Campuloclinium macrocephalum, Chromolaena stachyophylla, Mikania micrantha, Praxelis pauciflora, Symphyopappus reticulatus, and Vittetia orbiculata, some of these being segregated from the genus Eupatorium.A layer of phytomelanin observed in the fruit appears to be secreted by the outer mesocarp into the schizogenous spaces between the outer and inner mesocarp; its thickness was observed to vary among the different species examined. The bristles of the pappus are vascularized, except in M. micrantha, and have cells that are superficially projected and arranged acropetally; in S. reticulatus some of the projections are retrorse and a fracture line on the floral disk that is only seen in this species may indicate a double dispersal process. Numerous differences observed among the cypselae examined here reinforce earlier segregations of the genus Eupatorium sensu lato. Key words: : anatomy, Asteraceae, carpopodium, fruit, pappus, phytomelanin. INTRODUCTION bination of all of the characters cited above (Leszek et al. 1997), have been used to delimit tribes, genera, and The tribe Eupatorieae (Asteraceae) comprises 190 gen- even species of this family. era and 2,000 species (Anderberg et al. 2007) that are In spite of the global occurrence of the family, the encountered primarily in Mexico and Central and South fruits of the Asteraceae have not been intensively stud- America, with some representatives from North Amer- ied, as can be confirmed by the discordance seen in the ica, but with few species in the Old World (King and names attributed to its fruits. Marzinek et al. (2008) Robinson 1987). Eupatorium is a very complex genus took into consideration many anatomical and histori- and has experienced numerous segregations, as com- cal aspects of Asteraceae fruits, especially their com- piled by King and Robinson (1987). plex origin, and reaffirmed their true nature as cypselae. Wagenitz (1976) suggested that anatomical stud- One character that stands out in descriptions of the ies of the fruits of the Asteraceae might aid in eluci- cypselae of the Heliantheae s.l. (which includes Eupato- dating its systematics. Characteristics of the indumen- rieae) is their dark appearance (Anderberg et al. 2007). tum (Ritter and Miotto 2006), pappus (Bean 2001), car- This aspect can be attributed to the deposition of a rigid popodium (Haque and Godward 1984), and the anatomy layer of phytomelanin, an organic material that fills the of the pericarp itself (Bruhl and Quinn 1990), or a com- schizogenous space of the pericarp during the devel- Correspondence to: Denise Maria Trombert Oliveira opment of the cypsela after fertilization (Pandey and E-mail: [email protected] An Acad Bras Cienc (2010) 82 (2) “main” — 2010/4/27 — 17:28 — page 280 — #2 280 JULIANA MARZINEK and DENISE M.T. OLIVEIRA Dhakal 2001). Sometimes this phytomelanin layer is gitudinal sections 6 to 10μm thick were prepared us- referred to as a “carbon layer” or black pigment layer ing a rotary microtome, stained with 0.05% toluidine (see Roth 1977). Panero (2007) indicates the occurrence blue at pH 4.7 (O’Brien et al. 1964), and subsequently of phytomelanin as a synapomorphy of the Asteraceae. mounted in synthetic resin. The permanent slides were The Phytomelanin Cypsela Clade (PCC) is composed of observed under an Olympus BX41 optical microscope more than 5,000 species that produce this substance. and the images captured digitally. Ontogenetic studies of the reproductive organs of The following histochemical tests were performed: Eupatorieae are generally scarce, and are almost exclu- ruthenium red to indicate the presence of polysaccha- sively limited to the publications of Pandey and Singh rides and pectins (Jensen 1962); phloroglucinol with (1983, 1994). In spite of the importance of the Eupato- hydrochloric acid to stain lignified cell walls (Sass rieae within the Asteraceae, no single study has focused 1951); Sudan IV to stain lipidic substances; Lugol so- simultaneously on the ontogeny, indumentum, pappus, lution for starch; ferric chloride with sodium carbon- and carpopodium of the species within this taxon. ate to indicate the presence of phenolic compounds (Jo- The present work analyzed the development of the hansen 1940); Dragendorff reagent to detect alkaloids pericarp and the accessory parts of the cypselae of six (Yoder and Mahlberg 1976); and bromophenol blue for species of Eupatorieae from Brazil (some of them segre- proteins (Mazia et al. 1953). gated from Eupatorium s.l.), emphasizing the most rel- The results are described in ontogenetic terms. Con- evant structures to taxonomic and ecological questions sidering the inferior origin of the ovary, the lato sensu related to this tribe. definition of the pericarp was adopted, in which theex- ocarp is produced by the outer epidermis of the infe- MATERIALS AND METHODS rior ovary, the endocarp by the inner epidermis, and the Specimens of Campuloclinium macrocephalum (Less.) mesocarp by the ground region where the vascular tis- DC., Chromolaena stachyophylla (Spreng.) R. King and sues are inserted. H. Robinson, Mikania micrantha H.B.K., Praxelis pau- Trichomes distribution was analyzed using both ciflora (H.B.K.) R. King and H. Robinson, Symphyopap- scanning electron and optical microscopy. Ten cypse- pus reticulatus Baker, and Vittetia orbiculata (DC.) R. lae (n=10) of each species mounted in glycerin were King and H. Robinson were collected in cerrado veg- also observed. The terms scarce (when trichomes were etation fragments in the municipality of Botucatu, São present on up to 10% of the cypsela surface), frequent > Paulo State, Brazil. Reference plant material was pre- (between 11% and 50%), and abundant ( 50%) were pared and deposited in the BOTU Herbarium (Holmgren used. et al. 1990) as collections 25,552 to 25,557. The terminology used to designate the outward pro- Micromorphological analyses of the surfaces of jected portions of the pappus fibers was adapted from the cypselae were performed with material fixed in glu- Hickey (1979), considering the angle of divergence be- taraldehyde (2.5% in 0.1M phosphate buffer, pH 7.3, tween the distal portion of the projected cells and the maintained at 4◦C). Samples were processed and, then, apex of the bristles. These angles were classified as nar- > mounted on aluminum stubbs, gold coated, and subse- row ( 45◦) or moderate (between 45◦ and 65◦) acute. quently examined using a scanning electron microscopy The nomenclature proposed by Barthlott et al. (1998) (Quanta 200, FEI Company) to generate digital images. was used to describe the bristle surfaces. Anatomical studies were performed on ovaries and cypselae in different phases of development that had RESULTS been fixed in FAA 50 for 48 hours (Johansen 1940) and, The ovaries of the floral buds of the species examined then, conserved in 70% ethanol (Jensen 1962). The fixed were all inferior (Fig. 1), bicarpelar, and unilocular material was dehydrated in an ethanol series and em- (Fig. 2), oblong shaped, and had variable numbers of bedded in methacrylate (LeicaTM) following the manu- longitudinal ridges that were visible during the develop- facturer’s recommendations. Both transversal and lon- ment of the pericarp. An Acad Bras Cienc (2010) 82 (2) “main” — 2010/4/27 — 17:28 — page 281 — #3 PERICARP OF EUPATORIEAE (ASTERACEAE) 281 Fig. 1 – Ovary of the floral bud. Figs. 1, 5, 7, 9 – Longitudinal sections. Figs. 2-4, 6, 8, 10 – Transversal sections. Fig.1– Campuloclinium macrocephalum; note lignified basal epidermal cells (arrow). Fig. 2– Mikania micrantha. Figs. 3-4 – Detail of the ovarian walls of Campuloclinium macrocephalum and Symphyopappus reticulates, respectively. Fig. 5 – Campuloclinium macrocephalum; note fusiform nuclei of the inner mesophyll cells (arrowhead). Fig. 6 – Detail of the ovarian wall of Mikania micrantha showing the transmission tissue (*). Fig. 7 – Detail of the floral disk of Praxelis pauciflora; note salient external cells of the pappus with evident nuclei (arrow). Fig. 8 – Detail of part of the floral disk of Mikania micrantha demonstrating secretory cavities near the vascular bundles. Figs. 9-10 – Carpopodium of Symphyopappus reticulatus. (ca – carpopodium; ie – inner epidermis; im – inner mesophyll; lo – locule; oe – outer epidermis; om – outer mesophyll; ov – ovule; ow – ovarian wall; pa – pappus; pe – petal; vb – vascular bundle). Bars – 250μm (1); 150μm (2, 10); 20μm (3, 5); 50μm (4,6-7,9); 100μm (8). The outer ovarian epidermis is uniseriate (Fig. 3) The ovarian mesophyll is composed of two re- with cubical cells, slightly longitudinally elongated (Fig. gions. The outer mesophyll region demonstrates juxta- 5) and vacuolated,

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