Review of Palaeobotany and Palynology 221 (2015) 192–203 Contents lists available at ScienceDirect Review of Palaeobotany and Palynology journal homepage: www.elsevier.com/locate/revpalbo Palynological study of heterostylous species of Melochia L. (Byttinerioideae-Malvaceae) occurring in Bahia, Brazil Cristiano Eduardo Amaral Silveira Júnior a,⁎, Luciene Cristina Lima e Lima b,MarileideDiasSabac a Universidade do Estado da Bahia, Programa de Pós-Graduação em Biodiversidade Vegetal, Campus VII, Senhor do Bonfim-BA, Brazil b Universidade do Estado da Bahia, Laboratório de Estudos Palinológicos, Campus II, Alagoinhas-BA, Brazil c Universidade do Estado da Bahia, Laboratório de Estudos Palinológicos, Campus VII, Senhor do Bonfim-BA, Brazil article info abstract Article history: Melochia L. species are morphologically characterized by a pentacarpelar gynoecium with free papillose styles at Received 11 September 2014 the apex, whereas some are connate. The genus comprises about 60 species, with 22 occurring in Brazil. We an- Received in revised form 7 July 2015 alyzed pollen grains from 12 heterostylous species occurring in the State of Bahia to contribute to the palynology Accepted 10 July 2015 of the Melochia Group, including Melochia betonicifolia A. St.-Hil., M. caracasana Jacq., M. chamaedrys A. St.-Hil., Available online 31 July 2015 M. hirsuta Cav., M. illicoides K. Schum., M. parvifolia Kunth, M. pilosa (Mill.) Fawc. & Rendle, M. pyramidata L., M. spicata (L.) Fryxell, M. splendens A. St.-Hil. & Naudin, M. tomentosa L., and M. villosa Fawc. & Rendle. Pollen Keywords: fl Palynology grains were provided from specimens deposited at the CEPEC, HUEFS, and SP herbaria. The oral morphs of all Heterostyly specimens had been previously studied with a stereomicroscope. Acetolyzed pollen grains were investigated Taxonomy by light microscopy, and non-acetolyzed pollen grains were observed by scanning and transmission electron Melochia microscopy. It was confirmed in two pollen types of the two floral morphs: (1) the longistylous morph was char- Malvaceae acterized as medium sized; prolate spheroidal; 3-colporate, with ectoapertures comparatively larger, endo- aperture lalongate; reticulate-perforate; exine ultrastructure with nexine differentiated into endexine and a foot layer (M. pyramidata) or undifferentiated (M. betonicifolia); a 4-stratified sexine may also occur; (2) the brevistylous morph showed medium to large-sized pollen grains; oblate spheroidal; 3(4)-colporate, ectoaperture short, endoaperture lalongate; microechinate and echinate; semitectum perforate and microreticulate-rugulate (M. betonicifolia); M. pyramidata wall ultrastructure with nexine showing “inter- nal foramina.” Pollen dimorphism was confirmedinthegenusduetoheterostyly.Thetypeandnumberof apertures were not as useful as exine ornamentation to characterize and distinguish the pollen grains. © 2015 Elsevier B.V. All rights reserved. 1. Introduction that continent. For example, Brizicky (1966) and Dorr and Barnett (1989) studied North American species; Robyns and Cuatrecasas The genus Melochia L. consists of approximately 60 species that are (1964) and Faife-Cabrera et al. (2014) investigated Central American widespread in tropical and subtropical regions but most species are species; and Fernández and Grande (2007), Cristóbal (2007),and confined to the Americas. Twenty-two species are found in Brazil, Rondón (2007a, 2007b, 2009) collected South American species. To three of them are endemics (Goldberg, 1967; Dorr and Barnett, 1989; date, the most well-known floristic survey in Brazil was of the tradition- Esteves, 2010). al Sterculiaceae family, such as those of Cristóbal (1983) for the Flora of Melochia is included within the Malvaceae family and the Santa Catarina State; Cristóbal and Tressens (1986) and Cristóbal et al. Byttnerioideae subfamily, because their flowers have five stamens (1995) for the Flora of Bahia State; Esteves (1986) for the Flora of and no or reduced staminodes (Bayer et al., 1999). The genus be- Minas Gerais State; Vicentini and Silva (1999) for the Amazon Forest; longs to the Hermannieae tribe, which is morphologically recog- and Cruz (2007) for the Flora of São Paulo State. nized by five antipetal stamens and plane petals (Dorr and Barnett, Heterostyly in Melochia was studied by Martin (1966),who 1989). According to Goldberg (1967), Melochia is mainly character- observed a self-incompatibility system related to non-germination of ized by a pentacarpelar gynoecium and by the five free styles that pollen grains and restricted pollen tube growth to the stigma and are partially connate and papillose at the apex. style. Heterostyly is a genetically established floral heteromorphism The Americas hold most species within this genus; therefore, some that occurs in at least 24 flowering plant families. Heterostyly is related of the samples of the relevant taxonomic studies were extracted from to pollen dimorphism within species, mainly regarding size and or- namentation of the exine. This trait is remarkably characteristic of ⁎ Corresponding author. Melochia and is shared only by the genus Waltheria in the Malvaceae E-mail address: [email protected] (C.E.A. Silveira Júnior). family (Ganders, 1979). http://dx.doi.org/10.1016/j.revpalbo.2015.07.005 0034-6667/© 2015 Elsevier B.V. All rights reserved. C.E.A. Silveira Júnior et al. / Review of Palaeobotany and Palynology 221 (2015) 192–203 193 Palynological studies that considered the influence of heterostyly on M. caracasana, M. chamaedrys, M. hirsuta, M. illicoides,andM. splendens, pollen grain morphological characters have been included to Melochia were studied due to an insufficient number of longistylous flower sam- taxonomy. Pollen grains with different aperture length dimensions ples in the herbaria. and exine ornamentation can be found within the same species, but it LM were performed under a Zeiss Axioskop 2 microscope (Jena, depends on the floral morphs analyzed (Dorr and Barnet, 1989; Saba Germany) in the Palynology Laboratory of the Universidade do Estado et al., 2004; Faife-Cabrera et al., 2014). Several authors, such as da Bahia, Campus VII. The pollen grains were prepared using an Erdtman (1952), Sharma (1970), Melhem et al. (1976), Palácios- acetolysis protocol (Erdtman, 1960) from anthers belonging to more Chávez et al. (1990), Roubik and Moreno (1991), Miranda et al. than one flower of each specimen. The acetolyzed material was (1992), and Saba and Santos (2000; 2003) described palynologically mounted between a slide and a coverslip, immersed in glycerin jelly, species of Melochia; however, they did not mention that pollen dimor- and sealed with hot paraffin. phism is derived from heterostyly. Moreover, these studies were pre- The main morphometric parameters (equatorial and polar diame- dominantly performed using light microscopy (LM), making it difficult ters) were measured in 25 pollen grains, when possible, and the re- to clearly visualize all of the morphological characters of the pollen. A maining parameters (aperture diameter and exine, sexine, and nexine few authors have used scanning electron microscopy (SEM), and some thicknesses) were measured in 10 randomly selected pollen grains studies have used transmission electron microscopy (TEM) to study using an ocular micrometer. Malvaceae. TEM allows detailed visualization of the pollen grain surface Quantitative results were statistically analyzed by calculating the ar- and stratification of the exine. ithmetic average, standard deviation, standard deviation of the average, Thus, in this study, we characterized pollen morphology from coefficient of variation, and the 95% confidence intervals for the pollen heterostylous Melochia spp. occurring in the State of Bahia, Brazil. parameter measurements with a sample size of 25. The other measure- ments had a sample size of 10, and only the arithmetic average was 2. Material and methods calculated. The pollen grains were acetolyzed, washed, and dehydrated for Based on a literature review (Saba and Santos, 2003; Cruz, 2007; 10 min each in an ascending ethanol series (50, 70, 90, and 100%) for Esteves, 2010) and the CEPEC (Centro de Pesquisa do Cacau), HUEFS the SEM pollen surface analysis. Absolute alcohol containing the pollen (Universidade Estadual de Feira de Santana), and SP (Instituto de Botânica grains was dripped directly over a SEM stub (specimen holder), dried de São Paulo) herbaria databases (acronyms according to Thiers, 2014), completely, coated using gold evaporation under high vacuum, and we selected 26 specimens belonging to 12 Melochia heterostylous species the pollen grains were qualitatively analyzed using a JEOL 6390LV mi- occurring in the State of Bahia. We used additional specimens from other croscope (Tokyo, Japan) at the Electron Microscopy Platform of Centro states when necessary and selected specimens identified by Malvaceae de Pesquisas Gonçalo Moniz-FIOCRUZ. taxonomic experts when possible. Pollen grains were collected from at The TEM analysis was also performed at FIOCRUZ. All non-acetolyzed least four different specimens of each species whenever possible. Mature material was fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate floral buds of all specimens were studied under LM to determine the buffer solution for 2 h. The samples were washed in sodium cacodylate floral morphs. Only the brevistylous flowers of some species, such as buffer and post-fixedin1%osmiumtetroxide,0.8%potassium Plate I. Pollen grains of Melochia L. (see on page 194) 1-4 Melochia betonicifolia