Published online: 30 Jul 2015.

Transcription

1 This article was downloaded by: [ ] On: 03 August 2015, At: 09:27 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: Registered office: 5 Howick Place, London, SW1P 1WG Palynology Publication details, including instructions for authors and subscription information: Pollen morphology in Athenaea Sendtn. and Aureliana Sendtn. (Solanaceae) Izabella Martins da Costa Rodrigues a, Bruno Fernandes Falcão a, João Renato Stehmann a & Soraia Girardi Bauermann b a Laboratório de Sistemática Vegetal, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil b Laboratório de Palinologia, Universidade Luterana do Brasil, Canoas, Brazil Published online: 30 Jul Click for updates To cite this article: Izabella Martins da Costa Rodrigues, Bruno Fernandes Falcão, João Renato Stehmann & Soraia Girardi Bauermann (2015): Pollen morphology in Athenaea Sendtn. and Aureliana Sendtn. (Solanaceae), Palynology, DOI: / To link to this article: PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the Content ) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at

2 Palynology, Pollen morphology in Athenaea Sendtn. and Aureliana Sendtn. (Solanaceae) Izabella Martins da Costa Rodrigues a *, Bruno Fernandes Falc~ao a,jo~ao Renato Stehmann a and Soraia Girardi Bauermann b a Laboratorio de Sistematica Vegetal, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil; b Laboratorio de Palinologia, Universidade Luterana do Brasil, Canoas, Brazil Aureliana and Athenaea (Withaniinae, Solanaceae) are two genera of shrubs or small trees with centres of diversity in the Brazilian Atlantic Rain Forest. They are difficult to distinguish using gross morphology, and are traditionally segregated based on differences in fruiting calyx size. Pollen grains of all taxa were acetolysed, treated and examined with light and scanning electron microscopy, with the aim of identifying diagnostic characters. Microphotographs and illustrations of pollen grains are presented. The species analysed present small- to medium-sized monad pollen grains of varying morphology with long to extremely long colpi, and lalongate endoapertures. The analysis showed that the pollen grains of both genera are very similar, differing in size-related characters. These data contributed to the synonymisation of Athenaea within Aureliana. Keywords: Brazilian Withaniinae; systematics; Atlantic Rain Forest; pollen morphology 1. Introduction The Solanales is a monophyletic order based on both morphological and molecular analysis. This order comprises five families of flowering plants: Convolvulaceae, Hydroleaceae, Montiniaceae, Sphenocleaceae and Solanaceae (APG III 2009), of which Solanaceae is the largest and most diverse at both the generic and specific levels. Solanaceae is an economically important family with worldwide distribution, with about 90 genera and approximately 3000 species, diversity is high in the New World, and particularly in South America (S arkinen et al. 2013). It contains food crops of global economic importance such as the cultivated potato (Solanum tuberosum L.), tomato (S. lycopersicum L.), eggplant (S. melongena L.) and pepper (Capsicum L.), and important species for the pharmaceutical industry, such as the tobaccos (Nicotiana spp.), deadly nightshade (Atropa belladonna L.) and jimsonweeds (Datura L.) (Hunziker 2001; Olmstead et al. 2008; S arkinen et al. 2013). Brazil is a centre of Solanaceae diversity, with an estimated 470 species in 34 genera, many of them endemic (Hunziker 2001; Stehmannetal. 2014). Among the Brazilian genera that are endemic or near endemic are Aureliana Sendtn. (eight species) and Athenaea Sendtn. (seven species), both of which have their centres of diversity in the Brazilian Atlantic rainforest (Mata Atl^antica s.l.). Species of these two genera are concentrated in southeastern and southern Brazil, with only Aureliana fasciculata (Vell.) Sendtn. reaching southeastern Paraguay and Argentina (eastern Misiones), with a few from Bolivia andperu,asshowninthemap(figure 1). Aureliana and Athenaea have traditionally been distinguished by differences in their calyx morphology; Athenaea has a highly inflated and accrescent calyx at fruit maturity (Figure 2). Synopses of the genera have been published (Athenaea, Barboza & Hunziker 1989; Aureliana, Hunziker & Barboza 1990), as well as two new species: Aureliana darcyi and Aureliana angustifolia (Carvalho & Bovini 1995; Almeida-Lafeta 2000) and one change in taxonomic rank: Aureliana sellowiana (Barboza et al. 2010). Recent phylogenetic studies have shown that it is not only sister relationships that are unclear but also the circumscription of both genera; thus the generic status of Athenaea and Aureliana has been rigorously examined, and a new circumscription is about to be published (Zamberlam et al. 2015). Olmstead et al. (2008) placed both genera in the subtribe Withaniinae within the large tribe Physaleae; later results using a larger data set (S arkinen et al. 2013) confirmed this placement. Subtribe Withaniinae comprises, in addition to Aureliana and Athenaea, the genera Withania Pauq. (Old World), Tubocapsicum (Wettst.) Makino (China and Japan), Mellissia Hook. f. (St. Helena), Nothocestrum A. Gray (Hawaii) and Discopodium Hochst. (tropical African mountains). Within Withaniinae, Aureliana and Athenaea are sisters to one another (S arkinen et al. 2013). Olmstead et al. (2008) suggest that the Aureliana/Athenaea clade *Corresponding author. izabellamcr@yahoo.com.br Ó 2015 AASP The Palynological Society

3 2 I. M. da Costa Rodrigues et al. Figure 1. Occurrence map of Aureliana fasciculata. represents a relictual group from the common ancestor of Withaniinae, since all other genera in Physaleae have a distribution restricted to the New World. No morphological synapomorphies have been identified for Withaniinae (Olmstead et al. 2008), but inflated calyces are common amongst the genera. Fundamental aspects of classification involving the origin of characters have been evaluated and discussed in the Aureliana/Athenaea clade. Barboza et al. (2010) suggested that studies with more species and different techniques are necessary for assessing the generic distinctness of Aureliana and Athenaea. Palynological studies have been a rich source of taxonomic information in the Solanaceae in a variety of genera, such as Solanum (Murray & Eshbaugh 1971; Sharma 1974; Anderson & Gensel 1976; Anderson 1977; Srivatstava 1977; Edmonds 1984; Knapp et al. 1998), Brunfelsia L. (Plowman 1998; Batista-Franklim & Gonçalves- Esteves 2002) and non-amazonic Cestrum L. (Vignoli- Silva et al. 2014) and the tribes Cestreae (Gentry 1986), Datureae (Persson et al. 1999) and Salpiglossideae (Stafford & Knapp 2006). In this study, we aimed to carry out a palynological study with Brazilian genera of the Withaniinae subtribe, the traditional Aureliana and Athenaea, describing pollen morphology, and evaluating its taxonomic significance, enlightening through a narrow focus on the available information. 2. Materials and methods Pollinic material was obtained from fertile anthers of flowers from herbarium specimens deposited in the BHCB Herbarium of Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Brazil. The pollen grains were acetolysed according to Erdtman (1960), in the palynology laboratory of Centro de Pesquisa Manoel Teixeira da Costa, (CPMTC) Instituto de Geoci^encias, UFMG. Slides (three for each specimen) were mounted in glycerin jelly and examined by light

4 Palynology 3 Figure 2. A. Aureliana tomentosa fruit. B. Athenaea cuspidata fruit. microscopy (LM) analysis under a Zeiss Primo Star microscope (Zeiss, Germany). Pollen grains were measured and microphotographed up to 7 days after acetolysis (Wanderley & Melhem 1991). Observations and measurements were taken on one standard specimen and two comparison specimens for each taxon. The standard material was chosen based on the collection site of the type material for the species, and, in these, we took 25 readings of polar axis and equatorial diameter in equatorial view of pollen grains. Ten measurements were noted for the comparison specimens, as well as for colpus length, apertures (length and width), exine thickness, distance between the apices of two ectocolpi (d), equatorial diameter in polar view (D) and polar area index (PAI). The terminology used herein is based on Barth and Melhem (1988), Faegri and Iversen (1989) and Punt et al. (2007). Size classes of pollen follow Barth and Melhem (1988), and of apertures, Faegri and Iversen s (1989) definitions. Statistical analysis was conducted to obtain means and standard deviations; coefficients of variation were calculated using the confidence interval of 95%. All images were taken with a Canon Power Shot A650 IS (Canon, Japan) camera and all measurements were performed in the images using the AxioVision v. 4.7 (Zeiss, Germany) computer program. Further details of pollen surface and aperture were revealed through analysis of non-acetolysed pollen grains by scanning electron microscopy (SEM) in the Quanta 200 microscope at Centro de Microscopia Eletr^onica of UFMG. Here, pollen grains were directly transferred to a metallic stub using double-sided sellotape and coated with gold (thickness 10 nm) in a sputtering chamber (Melhem et al. 2003). Illustrative microphotographs were taken. 3. Results The examined species have monad pollen grains, radial or isopolar (Plates 1 5). The pollen grains of Aureliana fasciculata var. tomentella, A. fasciculata var. longifolia and Athenaea (with the exception of Athenaea micrantha) were medium sized, with all other species small- (X < 25 mm) to medium- (25 mm < X < 50 mm) sized with a range of variation between 20.5 and 36.6 mm in polar diameter. Athenaea pogogena exhibited higher mean values, mm and mm, while Aureliana tomentosa presented the lowest averages, mm and mm, respectively, for the polar axis and equatorial diameters (Tables 1 2; supplemental online material). Evaluating the shape, all species showed sub pollen grains, with the majority prolate

5 4 I. M. da Costa Rodrigues et al. Plate 1. Detail of endoaperture and colpus in equatorial and polar view of mature pollen grains of Athenaea and Aureliana (light microscopy). 1. Athenaea pogogena. 2. Aureliana brasiliana. 3. Aureliana fasciculata var. tomentella. 4. Athenaea anonacea. 5. Athenaea martiana. 6. Aureliana wettsteiniana: Inaperturate pollen grain. 7. Athenaea picta: detail of colpus. 8. Aureliana darcyi: detail of endoaperture ends. 9. Aureliana angustifolia: Tetracolporate pollen grains in polar view. Scale bars: 10 mm. ; Aureliana tomentosa was the exception and showed only oblate pollen grains (Table 2 3; supplemental online material). Pollen grains of all species were typically tricolporate, with very long and wide colpus and tuberculate membrane (Plate 1, figures 4,5 7; Plate 2; Plate 3; Plate 4, figure 3; Plate 5, figure 2), lalongate endoaperture with acute ends, geniculum (non-acetolysed grains; Plate 1, figure 8; Plate 3; Plate 4; Plate 5, figures3,5)and prominent fastigium (acetolysed grains; Table 5; Plate 1, figure 5). Tetracolporate (maximum of 10%) grains (Plate 1, figure 9; Plate 2, figure 5) were observed in Athenaea cuspidata, Athenaea martiana, Athenaea anonacea, Aureliana angustifolia, Aureliana fasciculata var. longifolia and Aureliana tomentosa. One of the three Aureliana wettsteiniana specimens presented only inaperturate pollen grains; otherwise, 25% of these grains were found in one specimen of Athenaea martiana and Athenaea anonacea (Plate 1, figure 6; Plate 5, figure 6). A long colpus was found in pollen grains of Athenaea anonacea, Aureliana fasciculata var. tomentella and Aureliana fasciculata var. longifolia. In Athenaea

6 Palynology 5 Plate 2. Mature pollen grains of Athenaea and Aureliana (scanning electron microscopy): 1. Athenaea martiana: polar view. 2. Athenaea micrantha: equatorial view. 3. Athenaea picta: equatorial view. 4. Athenaea pogogena: polar view. 5. Aureliana angustifolia: tetracolporate pollen grain in polar view. 6. Aureliana brasiliana: polar view. Scale bars: 10 mm.

7 6 I. M. da Costa Rodrigues et al. Plate 3. Mature tricolporate pollen grains of Aureliana (scanning electron microscopy): 1, 2. Aureliana fasciculata var. fasciculata: 1. polar view; 2. equatorial view. 3. Aureliana fasciculata var. longifolia: polar view. 4. Aureliana sellowiana: equatorial view. 5. Aureliana velutina: polar view. 6. Aureliana wettsteiniana: equatorial view. Scale bars: 10 mm.

8 Palynology 7 Plate 4. Mature pollen grains of Athenaea and Aureliana, detail of ornamentation (scanning electron microscopy): 1. Athenaea picta. 2. Aureliana angustifolia. 3. Aureliana fasciculata var. fasciculata: geniculum. 4, 5. Aureliana brasiliana: 4. exine; 5. geniculum. 6. Aureliana sellowiana. Scale bars: 1 mm.

9 8 I. M. da Costa Rodrigues et al. Plate Mature pollen grains of Athenaea and Aureliana, detail of ornamentation (scanning electron microscopy): 1, 2. Aureliana wettsteiniana: 1. exine; 2. colpus membrane. 3. Aureliana wettsteiniana: inaperturate pollen grain exine. 4. Athenaea martiana. 5, 6. Anthers: 5. Aureliana tomentosa. 6. Aureliana wettsteiniana: anther with inaperturate pollen grains. Scale bars: 1 mm(1 4); 1 mm (5 6).

10 Palynology 9 Table 1. Polar diameter (P) and exine thickness (EX) measurements (in mm) of pollen grains of the Brazilian species of Athenaea and Aureliana (standard specimens). Taxa Range X SD CV (%) IC 95 (%) P/E EX (mm) Athenaea anonacea Athenaea cuspidata Athenaea martiana Athenaea micrantha Athenaea picta Athenaea pogogena Aureliana angustifolia Aureliana brasiliana Aureliana darcyi Aureliana fasciculata var. fasciculata Aureliana fasciculata var. longifolia Aureliana fasciculata var. tomentella Aureliana tomentosa Aureliana velutina Aureliana wettsteiniana Note: Arithmetic average (X ), standard deviation (SD), variability coefficient (CV), confidence interval (IC) and exine thickness (EX), (n D 25). anonacea, we observed some parasyncolporate pollen grains in which the endoapertures were so wide that the ends of two adjacent endoapertures crossed without connecting, resembling an endocingulum (Table 4; Plate 1, figure 8; supplemental online material). Endoapertures with bifurcated ends were observed in Athenaea martiana, Aureliana fasciculata var. fasciculata, Aureliana brasiliana, Aureliana tomentosa and Aureliana velutina. Aureliana fasciculata var. tomentella presented pollen grains with circular endoapertures (Plate 1, figure 3). A geniculum was observed in the equatorial region of endoapertures in non-acetolysed pollen grains in several species (Plate 2; Plate 3; Plate 4, figures 3, 5). This geniculum was fragile and easily disrupted by acetolysis, creating a cavity, often called a fastigium. Athenaea martiana and Aureliana fasciculata var. longifolia had the most prominently developed fastigium and the least prominent (flattest) were found in Aureliana brasiliana, A. tomentosa and A. wettsteiniana (Plate 1, figure 5). Table 2. Equatorial diameter (P) measurements (in mm) and shape of pollen grains of the Brazilian species of Athenaea and Aureliana (standard specimens). Taxa Range X SD CV (%) IC 95 (%) Shape Athenaea anonacea prolate Athenaea cuspidata prolate Athenaea martiana prolate Athenaea micrantha prolate Athenaea anonacea prolate Athenaea picta prolate Athenaea pogogena prolate Aureliana angustifolia prolate Aureliana brasiliana prolate Aureliana darcyi prolate Aureliana fasciculata var. fasciculata prolate Aureliana fasciculata var. longifolia prolate Aureliana fasciculata var. tomentella prolate Aureliana tomentosa oblate Aureliana velutina prolate Aureliana wettsteiniana oblate Note: Arithmetic average (X ), standard deviation (SD), variability coefficient (CV), confidence interval (IC) (n D 25).

11 10 I. M. da Costa Rodrigues et al. Table 3. Pollen grains of Athenaea and Aureliana: polar (P) and equatorial diameter (E) measurements (in mm) and shape (comparison specimens, n D 10). Taxa Registry P E P/E Shape Athenaea anonacea BHCB BHCB prolate prolate Athenaea cuspidata BHCB BHCB prolate oblate Athenaea martiana BHCB BHCB oblate prolate Athenaea micrantha BHCB BHCB prolate prolate Athenaea picta BHCB BHCB prolate prolate Athenaea pogogena BHCB BHCB oblate oblate Aureliana angustifolia BHCB prolate Aureliana brasiliana BHCB BHCB prolate prolate Aureliana darcyi BHCB BHCB prolate prolate Aureliana fasciculata var. fasciculata BHCB BHCB prolate prolate Aureliana fasciculata var. longifolia BHCB BHCB prolate prolate Aureliana fasciculata var. tomentella BHCB BHCB oblate prolate Aureliana tomentosa BHCB BHCB oblate oblate Aureliana velutina BHCB BHCB prolate prolate Aureliana wettsteiniana BHCB prolate Table 4. Arithmetic average (in mm) of aperture measurements of pollen grains of the Brazilian species of Athenaea and Aureliana (n D 10). Colpus Endoaperture Taxa Length Width Width Athenaea anonacea Athenaea cuspidata Athenaea martiana Athenaea micrantha Athenaea picta Athenaea pogogena Aureliana angustifolia Aureliana brasiliana Aureliana darcyi Aureliana fasciculata var. fasciculata Aureliana fasciculata var. longifolia Aureliana fasciculata var. tomentella Aureliana tomentosa Aureliana velutina Aureliana wettsteiniana

12 Palynology 11 Table 5. Arithmetic average (in mm) of distance between the apices of two ectocolpi (d), equatorial diameter in polar view (D) and polar area index (PAI), of pollen grains of the Brazilian species of Athenaea and Aureliana (n D 10). Taxa D d PAI Athenaea anonacea Athenaea cuspidata Athenaea martiana Athenaea micrantha Athenaea picta Athenaea pogogena Aureliana angustifolia Aureliana brasiliana Aureliana darcyi Aureliana fasciculata var. fasciculata Aureliana fasciculata var. longifolia Aureliana fasciculata var. tomentella Aureliana tomentosa Aureliana velutina Aureliana wettsteiniana PAI varied from 0.22 to A very small PAI (< 0.25) was observed in pollen grains of Athenaea micrantha, Aureliana angustifolia, Aureliana brasiliana, Aureliana tomentosa and A. velutina. In contrast, the pollen grains of Aureliana fasciculata var. tomentella and Aureliana wettsteiniana showed high PAI. All others species showed small PAI (Table 5; supplemental online material). Exine sculpturing varied from scabrate (Plate 3,figures 1, 2, 4; Plate 4, figures 3, 6) in Aureliana fasciculata var. fasciculata and Aureliana sellowiana to scabrate punctate in all other species (Plate 4, figures 1, 2, 4). Some irregularities were seen (Plate 4, figure 2; Plate 5, figures 1, 4), but further studies are needed to determine if such elements are artifacts of the technique of scanning microscopy or particular exine structures. Exine thickness ranged from 1.5 mm inaureliana fasciculata var. tomentella, Aureliana fasciculata var. fasciculata and A. brasiliana to 1.9 mm in Athenaea anonacea, Athenaea picta and Aureliana velutina (Table 1; supplemental online material). In general, the sexine was columellate and as thick as, or slightly thicker than, the nexine. 4. Discussion Solanaceae is a eurypalynous family (Erdtman 1952) that is, pollen morphological features are shared between distantly related groups, while on the other hand pollen grains can vary widely between closely related genera. Aureliana and Athenaea pollen grains are very similar to those found in Nothocestrum, Tubocapsicum and Withania, as much as in some species of Capsicum L., Cestrum L, Physalis and Solanum L. Thus, the distinction of the treated genera is complex upon palynological study, providing only a slight separation through pollen grain measurements. However, these data confirm the position of the Athenaea/Aureliana clade in the subtribe Withaniinae. Overall, the pollen character was not diagnostic at the generic limit, and, together with macromorphological and molecular (DNA) details, led Rodrigues and Stehmann to circumscribe Athenaea as a synonym of Aureliana (Zamberlan et al. 2015). Considering infrageneric taxonomic clues, pollen grain analysis showed that varieties of the Aureliana fasciculata complex are consistently different from other species, eventually recognized as new (Rodrigues and Stehmann, in prep). Few studies of pollen morphology in Withaniinae have been conducted. When dealing specifically with the focus of this work, Cabrera and Cuadrado (2001) analysed pollen morphology of Aureliana fasciculata in northeastern Argentina; Barth and Duarte (2008) examined A. fasciculata var. longifolia and Cruz-Barros and colleagues (2011) investigated Athenaea picta. The description of the pollen grains of the studied species under LM analysis does not differ from the data collected in the present work, except in the exine described as rugulate in A. fasciculata and finely undulated in A. fasciculata var. longifolia, probably because of the lack of SEM analysis. Small- to medium-sized, prolate to oblate, tri- or tetracolporate pollen grains with scabrate, granulate, reticulate and striate ornamentation were observed in Withania somnifera (L.) Dunal, W. coagulans (Stocks) Dunal, W. aristata Pauq., Nothocestrum longifolium A. Gray and N. subcordatum H. Mann (Murray & Eshbaugh 1971; Alwadie 2002; Al-Quran 2004; Perveen & Qaiser 2007). Tubocapsicum anomalum pollen grains have been described as medium-sized, sub and tricolporate, with a tuberculate membrane in the colpus (D Arcy et al. 2001). Pollen grains of Aureliana and Athenaea share a perforate (or punctate) scabrate exine with Acnistus arborescens (L.) Schltdl., Cestrum martii Sendtn., C. megalophyllum Dunal, C. parqui L Her., C. sclhechtendahlii G.Don., Lycium makranicum Schoenbeck-Temesy, L. ruthenicum Murray, Sessea brasiliensis Toledo, Solanum americanum Mill., S. anguivi Lam, S. argenteum ex Dunal Poir, S. cernuum Vell., S. diploconos (Mart.) Bohs, S. hoehnei Morton, S. inaequale Vell., S. indigoferum A.St.-Hil., S. nigrum L., S. pseudoquina A. St.-Hil., S. seaforthianum Andrews, S. surattense Burm.f., S. swartzianum Roem. & Schult., S.vaillantii Dun. and S. viarum Dun. (Roubik & Moreno 1991; Silva et al. 2003; Perveen & Qaiser 2007; Barth &

13 12 I. M. da Costa Rodrigues et al. Duarte 2008; Batista-Franklin & Gonçalves-Esteves 2008), and a punctate exine with at least seven species of Salpiglossideae (Stafford & Knapp 2006). Exine ornamentation is the most variable pollen character in Solanaceae palynology; however,stafford and Knapp (2006) suggest caution in using it to determine relationships due to its homoplastic nature in the family. We observed a structure covering the endoaperture of the non-acetolysed pollen grains, in all species, which we here term geniculum. The presence of a geniculum in genera of about 13 families of angiosperms has been described by Erdtman (1952). The term refers to the similarity of a knee (Potonie 1934) and is described by Punt et al. (2007) as a bulge in the equatorial exine of the colpus, often associated with separation of the sexine from the nexine and the rupturing of the later. If the separation forms a cavity, the term fastigium should be applied. A geniculate aperture has been described only rarely in Solanaceae. Stafford and Knapp (2006) found a central transversely oriented ridge of uplifted exine in Heteranthia decipiens Nees & Mart. and Schwenckia D.Royen ex L. A thin disrupted area of the exine adjacent to the side of the endoaperture was described in Schwenckia curviflora Benth. Ribeiro dos Santos and Melhem (1998). The presence of a fastigium (in the sense of Erdtman 1952) on the endoaperture seems to be common in Solanaceae, and has been observed in pollen grains from species of the genera Capsicum, Cestrum, Lycianthes, Physalis and Solanum, the tribe Lycieae, and Salpichroa origanifolia (Lam) Thell. and Vassobia breviflora (Sendtn). Hunz. (Bernardello & Lujan 1997; Cabrera & Cuadrado 2001; Silva et al. 2003; Batista-Franklin & Gonçalves- Esteves 2008; Cruz-Barros et al. 2011). More insightful discussion should take place about the use of the term geniculum in Solanaceae palynology. We suggest a detailed analysis of non-acetolysed pollen grains for applying the correct term, since after acetolysis, the geniculum cannot be noted and turned to a fastigium. Another character that needs to be carefully analysed is the presence of an endocingulum, which sometimes, can be an optical artefact. We did not find an endocingulum in any species of Aureliana or Athenaea, although some species had an endoaperture so wide that the ends crossed each other without fusing, giving the impression of an endocingulum. This trait has been seen in some species of Solanum (Batista-Franklin & Gonçalves-Esteves 2008), although, in fact, it is correctly applied in Brugmansia and Datura (Persson et al. 1999). Some species in this study had inaperturate pollen grains. These are usually associated with derived breeding systems such as dioecy (Anderson & Gensel 1976; Anderson & Levine 1982; Anderson & Symon 1989). According to Knapp et al. (1998), dioecy has evolved numerous times independently in the genus Solanum. In all of these cases, functionally female flowers have apparently normal anthers and stigmas, but inaperaturate pollen grains. This pollen has living cytoplasm, but apparently never germinates. The retention of nutrient-rich pollen in male plants is likely to be related to the buzz-pollination of Solanum flowers, where pollen is the only reward (Knapp et al. 1998). Aureliana and Athenaea flowers are not buzz pollinated, and the gynoecium presents style heteromorphies that is, flowers with short and long styles in the same individual relatively common in Solanum, Deprea, Withania, etc. (Sawyer & Anderson, 2000; Hunziker 2001; Anderson et al. 2006). So, the biology behind these inaperaturate grains needs detailed study. Male sterility in other Solanaceae genera appears to involve viable pollen grains which, for mechanical reasons, indehiscent anthers or morphological abnormalities, fail to reach the stigma (Atanasova et al. 2001). In Aureliana and Athenaea, anthers with inaperturate pollen grains were markedly smaller than normal ones (Plate 5, figures 5, 6, 8), and in addition, the exine was tuberculate (Plate 5, figure 3, 8c). Further study of floral morphology and reproductive biology in Aureliana and Athenaea will help to clarify whether the presence of inaperturate pollen grains is linked with possible functional dioecy in these species. 5. Conclusions The morphology of pollen grains in Aureliana and Athenaea is similar to that described from other members of the Solanaceae. Only pollen grain characters related to size were sufficient for the identification of species within the group. Our data corroborate the position of the Athenaea/Aureliana clade in the subtribe Withaniinae. The pollen data were not diagnostic at the generic limit and, added to macromorphological and molecular (DNA) details, guided Rodrigues and Stehmann to circumscribe Athenaea as a synonym of Aureliana (Zamberlan et al. 2015). Further studies with standardised protocols are necessary to establish the usefulness of pollen variation in Withaniinae systematics and possible synapomorphies in these characters. Supplemental data Supplemental data for this article can be accessed here. Acknowledgements We thank Karin Elise Bohns Meyer and Raquel Franco Cassino for the support and helpful guidance on the acetolysis process; Andreia Cardoso Pacheco Evaldt and Andre Ramos for their valuable assistance, and for receiving the first author

14 Palynology 13 in the Palynology Laboratory of Universidade Luterana do Brasil; and Sandy Knapp for reviewing the English. Funding This work was supported by the Fundaç~ao de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG) under Grant [APQ ] and Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico (CNPq) under doctoral scholarship to the first author [143004/2009-3]. Author biographies IZABELLA MARTINS DA COSTA RODRIGUES graduated in biology (emphasis on plant biology) from the Universidade Federal de Viçosa, and has a Master s degree in plant science (agronomy) from the same institution (2008). She has a PhD in plant biology from the Universidade Federal de Minas Gerais (2013), and part of this study was performed in London at the Natural History Museum. Izabella has experience in plant biology (systematics and taxonomy), Solanaceae taxonomy, palynology, plant morphology, reproductive biology, allelopathy and weed management. BRUNO FERRNANDES FALCAO e graduated in biology from the Universidade Federal de Minas Gerais. He is currently a Master s student at the same institution. Bruno has experience in plant systematics and taxonomy, Solanaceae reproductive biology and pollination biology. JOAO e RENATO STEHMANN has a PhD in plant biology from the Universidade Estadual de Campinas. He is a specialist on the Solanaceae family and is advisor for the postgraduate Program in Plant Biology at the Universidade Federal de Minas Gerais. Jo~ao researches flowering plant taxonomy, systematics in Solanaceae and floristic studies. Currently, he is curator of the collection of dicotyledons and the BHCB herbarium database, contributes to the management committee of the Institutos Nacionais de Ci^encia e Tecnologia (INCT) Virtual Herbarium of Flora and Fungi of Brazil, and operates in coordination of the Species List of the flora of Brazil. SORAIA GIRARDI BAUERMANN graduated in biology from the Pontifıcia Universidade Catolica do Rio Grande do Sul, and has a Master s degree in botany from the Universidade Federal do Rio Grande do Sul (1989) and a PhD in geosciences from the same institution (2003). Currently, she is a professor at the Universidade Luterana do Brasil, Ulbra Canoas, coordinator of the Palynology Laboratory and research coordinator. Soaraia has experience in botany and palaeontology, acting on the following topics: pollen morphology, surface samples and palaeoenvironmental reconstitution of Quaternary palynology. References Almeida-Lafeta RC A New Species of Aureliana (Solanaceae) from Minas Gerais, Brazil. Novon. 10: Al-Quran S Pollen morphology of Solanaceae in Jordan. Pakistan J Biol Sci. 7: Alwadie HM Ultrastructure of Withania somnifera (L.) Dunal pollen grains. Arab Gulf J Sci Res. 20: Anderson GJ, Bernardello G, Opel MR, Santos-Guerra A, Anderson M Reproductive biology of the dioecious Canary Islands endemic Withania aristata (Solanaceae). Amer J Bot. 93: Anderson GJ, Gensel PG Pollen morphology and the systematics of Solanum section Basarthrum. Pollen Spores 18: Anderson GJ, Levine DA Three taxa constitute the sexes of a single dioecious species of Solanum. Taxon 31: Anderson GJ, Symon DE Functional dioecy and andromonoecy in Solanum. Evolution 43: Anderson RJF Notes on Solanum (Solanceae) in Australia. Austrobaileya 1: Angiosperm Phylogeny Group An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants APG III. Bot J Linn Soc. 161: Atanasova B, Daskalov S, Nikova V Male sterility in three Solanaceae genera (Capsicum, Lycopersicon, Nicotiana) and its application in breeding and hybrid seed production. In: Van der Berg RG, Barendse GWM, Van der Weerden GM editors. Solanaceae V Advances in Taxonomy and Utilization. Nijmehen: Univ. Press; p Barboza G, Hunziker AT Estudios sobre Solanaceae XXIX. Sinopsis Taxonomica de Athenaea. Boletin de la Sociedade Argentina de Botanica 26: Barboza GE, Chiarini FE, Stehmann JR Real identity of Witheringia sellowiana (Solanaceae) typification and chromosome number. Syst Bot. 35: Barth OM, Duarte SG Morfologia polınica de especies arboreas de Solanaceae do Estado de Santa Catarina Brasil. Hoehnea 35: Barth OM, Melhem TS Glossario ilustrado de palinologia. Campinas: Editora da Universidade Estadual de Campinas. Batista-Franklim CPR, Gonçalves-Esteves V Morfologia polınica de Brunfelsia (Solanaceae) do Rio de Janeiro. Rev Bras Bot. 25: Batista-Franklim CPR, Gonçalves-Esteves V Palynology of species of Solanum L. (Solanaceae A. Juss.) from the restingas of Rio de Janeiro State Brazil Acta Botanica Brasilica 22: Bernardello L, Lujan MC Pollen morphology of the tribe Lycieae: Grabowskia, Lycium, Phrodus (Solanaceae). Rev Palaeobot Palynol. 96: Cabrera MM, Cuadrado GA Solanaceae subfamilia Solanoideae Tribus Datureae Jaboroseae y Lycieae In: Pire SM, Anzotegui LM, Cuadrado GA, editors. Flora polınica del Nordeste argentino. Corrientes: EUDENE UNNE. Carvalho LAF, Bovini MG Aureliana darcyi a New Species of Solanaceae from Brazil. Novon 5: Cruz-Barros MAV, Silva EL, Gasparino EC, Souza LN, Oliveira AC Flora Polınica da Reserva do Parque Estadual das Fontes do Ipiranga (S~ao Paulo, Brasil) Famılia:136- Solanaceae. Hoehnea 38(4): D Arcy WG, Keating RC, Zhang ZY, Peng CI The genus Tubocapsicum (Solanaceae). Bot Bull Acad Sinica 42: Edmonds JM Pollen morphology of Solanum L. section Solanum. Bot J Linn Soc. 88: Erdtman G Pollen Morphology and Plant Taxonomy. Angiosperms. Stockholm: Almqvist and Wiksell. Erdtman G The acetolysis technique, a revised description. Svensk Botanisk Tidskrift 54: Faegri K, Iversen J Textbook of pollen analysis. 4.ed. New York: John Wiley.

15 14 I. M. da Costa Rodrigues et al. Gentry JL Jr Pollen studies in the Cestreae (Solanaceae). In: D Arcy WG, editor. Solanaceae: Biology and Systematics. New York: Colombia University Press; p Hunziker AT, Barboza GE Estudios sobre Solanaceae XXX. Revision de Aureliana. Darwiniana 30: Hunziker AT The Genera of Solanaceae. Ruggell: ARG Gantner Verlag K.G. Knapp S, Persson V, Blackmore S Pollen morphology and functional dioecy in Solanum (Solanaceae). Plant Syst Evol. 210: Melhem TS, Cruz-Barros MAV, Corr^ea MAS, Makino- Watanabe H, Silvestre-Capelato MSF, Golçalves-Esteves VL Variabilidade polınica em plantas de Campos do Jord~ao (S~ao Paulo Brasil). Boletim do Instituto de Bot^anica de S~ao Paulo 16: Murray LE, Eshbaug WH A palynological study of Solaninae (Solanaceae). Grana 11: Olmstead RG, Bohs L, Migid HAl, Santiago-Valentin E, Garcia VF, Collier SM A molecular phylogeny of the Solanaceae. Taxon 57: Persson V, Knapp S, Blackmore S Pollen morphology and the phylogenetic analysis of Datura L. and Brugmansia Pers. In: Nee M, Symon DE, Lester RN, Jessop JP, editors. Solanaceae IV. Kew Richmond Surrey: Royal Botanic Gardens; p Perveen A, Qaiser M Pollen morphology of family Solanaceae from Pakistan. Pakistan J Bot. 39: Plowman TC A revision of the South American species of Brunfelsia (Solanaceae). Fieldiana 39: Potonie R I. Zur Morphologie der fossilen Pollen und Sporen. Arb. Inst. Pal aobotanik Petrographie Brennsteine 4:5 24. Punt W, Hoen PP, Blackmore S, Nilsson S, Le Thomas A Glossary of pollen and spore terminology. Rev Paleobotany Palyno. 143:1 81. Ribeiro dos Santos FA, Melhem TD Palinologia de Heteranthia decipiens Nees & Mart. (Scrophulariaceae ou Solanaceae?). Polibotanica 8: Roubik DW, Moreno PJE Pollen and spores of Barro Colorado Island. Monogr Syst Bot. 36: S arkinen T, Bohs L, Olmstead RG, Knapp S A phylogenetic framework for evolutionary study of the nightshades (Solanaceae): a dated 1000-tip-tree. BMC Evolutionary Biology 13:214. Sawyer NW, Anderson GJ Dioecy in South American Deprea (Solanaceae). Biotropica 32: Sharma BD Contribution to the Palynotaxonomy of genus Solanum Linn. Journal of Palynology 10: Silva SN, Carvalho AMV, Ribeiro dos Santos FA Morfologia polınica de doze especies de Cestrum L. (Solanaceae) da Mata higrofila na Bahia Brasil. Acta Sci Biol Sci. 25: Stafford P, Knapp S Pollen morphology and systematics of the zygomorphic-flowered nightshades (Solanaceae; Salpiglossideae sensu D Arcy, 1978 and Cestroideae sensu D Arcy, 1991, pro parte): a review. Systematics and Biodiversity 4: Stehmann JR, Mentz LA, Agra MF, Vignoli-Silva M, Giacomin L, Rodrigues IMC Solanaceae. In: Lista de Especies da Flora do Brasil [Internet]. Rio de Janeiro: Jardim Bot^anico do Rio de Janeiro. Available from: Vignoli-Silva M, Batista-Franklim CP, Correa DSM, Mentz LA, Mendonça CBF, Gonçalves-Esteves V Pollen diversity in Cestrum L. Solanaceae from extra- Amazonian Brazil. Palynology Available from: org/ / Wanderley MGL, Melhem TS Flora Polınica da Reserva do Parque Estadual das Fontes do Ipiranga (S~ao Paulo Brasil). Famılia: Bromeliaceae. Hoehnea 18:5 42. Zamberlan PM, Rodrigues IMC, Mader G, Castro L, Stehmann JR, Bonatto SL, Freitas LB Re-evaluation of the generic status of Athenaea and Aureliana (Withaniinae, Solanaceae) based on molecular phylogeny and morphology of the calyx. Bot J Linn Soc. 177(3):