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PollenmorphologyinAthenaeaSendtn.andAurelianaSendtn.(Solanaceae)

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Pollen morphology in Athenaea Sendtn. and AurelianaSendtn. (Solanaceae)Izabella Martins da Costa Rodriguesa, Bruno Fernandes Falcãoa, João Renato Stehmanna &Soraia Girardi Bauermannb

a Laboratório de Sistemática Vegetal, Universidade Federal de Minas Gerais, Belo Horizonte,Brazilb Laboratório de Palinologia, Universidade Luterana do Brasil, Canoas, BrazilPublished online: 30 Jul 2015.

To cite this article: Izabella Martins da Costa Rodrigues, Bruno Fernandes Falcão, João Renato Stehmann & SoraiaGirardi Bauermann (2015): Pollen morphology in Athenaea Sendtn. and Aureliana Sendtn. (Solanaceae), Palynology, DOI:10.1080/01916122.2015.1022908

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Pollen morphology in Athenaea Sendtn. and Aureliana Sendtn. (Solanaceae)

Izabella Martins da Costa Rodriguesa*, Bruno Fernandes Falc~aoa, Jo~ao Renato Stehmanna and Soraia Girardi

Bauermannb

aLaborat�orio de Sistem�atica Vegetal, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil; bLaborat�orio 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 inthe Brazilian Atlantic Rain Forest. They are difficult to distinguish using gross morphology, and are traditionallysegregated based on differences in fruiting calyx size. Pollen grains of all taxa were acetolysed, treated and examinedwith light and scanning electron microscopy, with the aim of identifying diagnostic characters. Microphotographsand illustrations of pollen grains are presented. The species analysed present small- to medium-sized monad pollengrains of varying morphology with long to extremely long colpi, and lalongate endoapertures. The analysis showedthat the pollen grains of both genera are very similar, differing in size-related characters. These data contributed tothe 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 com-

prises five families of flowering plants: Convolvulaceae,Hydroleaceae, Montiniaceae, Sphenocleaceae and Sola-

naceae (APG III 2009), of which Solanaceae is the larg-

est 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€arkinenet al. 2013). It contains food crops of global economicimportance such as the cultivated potato (Solanum

tuberosum L.), tomato (S. lycopersicum L.), eggplant

(S. melongena L.) and pepper (Capsicum L.), and impor-

tant 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 estimated470 species in 34 genera, many of them endemic (Hun-

ziker 2001; Stehmann et al. 2014). Among the Brazilian

genera that are endemic or near endemic are Aureliana

Sendtn. (eight species) and Athenaea Sendtn. (seven spe-

cies), both of which have their centres of diversity in the

Brazilian Atlantic rainforest (Mata Atlantica s.l.). Spe-

cies of these two genera are concentrated in southeast-

ern and southern Brazil, with only Aureliana fasciculata

(Vell.) Sendtn. reaching southeastern Paraguay and

Argentina (eastern Misiones), with a few from Bolivia

and Peru, as shown in the map (Figure 1).

Aureliana and Athenaea have traditionally been dis-

tinguished 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 angustifo-

lia (Carvalho & Bovini 1995; Almeida-Lafet�a 2000)

and one change in taxonomic rank: Aureliana sellowi-

ana (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 hasbeen rigorously examined, and a new circumscription

is about to be published (Zamberlam et al. 2015). Olm-

stead 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 com-

prises, in addition to Aureliana and Athenaea, the gen-

era 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. Email: izabellamcr@yahoo.com.br

� 2015 AASP � The Palynological Society

Palynology, 2015

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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 dis-

tinctness of Aureliana and Athenaea. Palynological

studies have been a rich source of taxonomic informa-

tion in the Solanaceae in a variety of genera, such asSolanum (Murray & Eshbaugh 1971; Sharma 1974;

Anderson & Gensel 1976; Anderson 1977; Srivatstava

1977; Edmonds 1984; Knapp et al. 1998), Brunfelsia

L. (Plowman 1998; Batista-Franklim & Goncalves-

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 sub-

tribe, the traditional Aureliana and Athenaea, describ-

ing pollen morphology, and evaluating its taxonomicsignificance, 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 Man-oel Teixeira da Costa, (CPMTC)�Instituto de Geo-

ciencias, UFMG. Slides (three for each specimen) were

mounted in glycerin jelly and examined by light

Figure 1. Occurrence map of Aureliana fasciculata.

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microscopy (LM) analysis under a Zeiss Primo Star

microscope (Zeiss, Germany). Pollen grains were mea-

sured and microphotographed up to 7 days after ace-tolysis (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 diam-

eter in equatorial view of pollen grains. Ten measure-

ments were noted for the comparison specimens, aswell 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. Sta-tistical analysis was conducted to obtain means and

standard deviations; coefficients of variation were cal-

culated 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 micro-scope at Centro de Microscopia Eletronica of UFMG.

Here, pollen grains were directly transferred to a metal-

lic stub using double-sided sellotape and coated with

gold (thickness 10 nm) in a sputtering chamber (Mel-

hem 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. longifoliaand Athenaea (with the exception of Athenaea micran-

tha) 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, 33.28 mm and 30.66 mm, while Aureliana

tomentosa presented the lowest averages, 23.48 mm and

23.96 mm, respectively, for the polar axis and equato-rial diameters (Tables 1�2; supplemental online mate-

rial). Evaluating the shape, all species showed

subspheroidal pollen grains, with the majority prolate

Figure 2. A. Aureliana tomentosa fruit. B. Athenaea cuspidata fruit.

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spheroidal; Aureliana tomentosa was the exception and

showed only oblate spheroidal pollen grains

(Table 2�3; supplemental online material).

Pollen grains of all species were typically tricolpo-

rate, 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 endoaperturewith acute ends, geniculum (non-acetolysed grains; Plate

1, figure 8; Plate 3; Plate 4; Plate 5, figures 3, 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 ano-

nacea, Aureliana angustifolia, Aureliana fasciculata var.

longifolia and Aureliana tomentosa. One of the three

Aureliana wettsteiniana specimens presented only ina-

perturate pollen grains; otherwise, 25% of these grains

were found in one specimen of Athenaea martiana andAthenaea anonacea (Plate 1, figure 6; Plate 5, figure 6).

A long colpus was found in pollen grains of Athe-

naea anonacea, Aureliana fasciculata var. tomentella

and Aureliana fasciculata var. longifolia. In Athenaea

Plate 1. Detail of endoaperture and colpus in equatorial and polar view of mature pollen grains of Athenaea and Aureliana (lightmicroscopy). 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.

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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 angustifo-lia: tetracolporate pollen grain in polar view. 6. Aureliana brasiliana: polar view. Scale bars: 10 mm.

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Plate 3. Mature tricolporate pollen grains of Aureliana (scanning electron microscopy): 1, 2. Aureliana fasciculata var. fascicu-lata: 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.

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

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Plate 5. 1�4. 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. Athenaeamartiana. 5, 6. Anthers: 5. Aureliana tomentosa. 6. Aureliana wettsteiniana: anther with inaperturate pollen grains. Scale bars:1 mm (1�4); 1 mm (5�6).

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anonacea, we observed some parasyncolporate pollen

grains in which the endoapertures were so wide that

the ends of two adjacent endoapertures crossed with-out 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. fascicu-

lata, 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 dis-

rupted by acetolysis, creating a cavity, often called a

fastigium. Athenaea martiana and Aureliana fasciculata

var. longifolia had the most prominently developed fas-

tigium 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 andAureliana (standard specimens).

Taxa Range X § SD CV (%) IC 95 (%) Shape

Athenaea anonacea 25.2�32.8 29.14 § 1.75 6.00 28.45�29.83 prolate spheroidal

Athenaea cuspidata 25.6�32.1 29.2 § 1.32 4.52 28.68�29.72 prolate spheroidal

Athenaea martiana 29.1�33.2 31.3 § 1.09 3.48 30.87�31.73 prolate spheroidal

Athenaea micrantha 24.5�29.4 26.88 § 1.59 5.91 26.25�27.5 prolate spheroidal

Athenaea anonacea 25.2�32.8 29.14 § 1.75 6.00 28.45�29.83 prolate spheroidal

Athenaea picta 25�33.8 30.61 § 1.95 6.37 29.84�31.37 prolate spheroidal

Athenaea pogogena 28.3�34.6 30.66 § 1.44 4.7 30.1�31.22 prolate spheroidal

Aureliana angustifolia 24.3�30 27.51 § 1.48 5.38 26.93 �28.09 prolate spheroidal

Aureliana brasiliana 21.2�27.9 24.58 § 1.61 6.55 23.95�25.21 prolate spheroidal

Aureliana darcyi 21.5�28.1 25.04 § 1.79 7.15 24.34�25.74 prolate spheroidal

Aureliana fasciculata var. fasciculata 24�27.4 25.45 § 0.9 3.54 25.1�25.80 prolate spheroidal

Aureliana fasciculata var. longifolia 25.5�31.3 28.08 § 1.59 5.66 27.45�28.70 prolate spheroidal

Aureliana fasciculata var. tomentella 26�33.8 31.10 § 1.94 6.24 30.34�31.86 prolate spheroidal

Aureliana tomentosa 21.2�25.8 23.96 § 1.34 5.59 23.44�24.48 oblate spheroidal

Aureliana velutina 24.2�29.9 26.46 § 1.64 6.2 25.82�27.10 prolate spheroidal

Aureliana wettsteiniana 25�30 27.77 § 1.56 5.62 27.16�28.38 oblate spheroidal

Note: Arithmetic average (X ), standard deviation (SD), variability coefficient (CV), confidence interval (IC) (n D 25).

Table 1. Polar diameter (P) and exine thickness (EX) measurements (in mm) of pollen grains of the Brazilian species of Athenaeaand Aureliana (standard specimens).

Taxa Range X § SD CV (%) IC 95 (%) P/E EX (mm)

Athenaea anonacea 27�33.4 29.7 § 1.61 5.42 29.07�30.33 1.01 1.9

Athenaea cuspidata 27.5�34.2 32.12 § 1.63 5.07 31.48�32.76 1.1 1.6

Athenaea martiana 30.3�35 32.4 § 1.38 4.25 31.86�32.94 1.03 1.8

Athenaea micrantha 24.7�30.2 27.84 § 1.84 6.61 27.12�28.56 1.03 1.7

Athenaea picta 27�36 32.36 § 1.98 6.12 31.58�33.13 1.05 1.9

Athenaea pogogena 29.6�36.6 33.28 § 1.59 4.78 32.66�33.90 1.08 1.7

Aureliana angustifolia 25.1�30.8 28.13 § 1.47 5.22 27.55�28.71 1.02 1.7

Aureliana brasiliana 22.5�30.1 25.51 § 1.62 6.35 24.87�26.14 1.04 1.5

Aureliana darcyi 22.6�30.9 26.3 § 2.11 8.02 25.47�27.13 1.05 1.8

Aureliana fasciculata var. fasciculata 24�28.6 26.38 § 1.22 4.62 25.90�26.86 1.04 1.5

Aureliana fasciculata var. longifolia 27.8�35.7 31.11 § 1.7 5.46 30.44�31.78 1.11 1.6

Aureliana fasciculata var. tomentella 27.7�35.4 31.81 § 1.78 5.6 31.11�32.51 1.02 1.5

Aureliana tomentosa 20.5�25.5 23.48 § 1.34 5.71 22.95�24.00 0.98 1.4

Aureliana velutina 24.5�34.2 28.54 § 2.12 7.43 27.71�29.37 1.08 1.9

Aureliana wettsteiniana 24.3�30.4 27.07 § 1.69 6.24 26.41�27.73 0.97 1.4

Note: Arithmetic average (X ), standard deviation (SD), variability coefficient (CV), confidence interval (IC) and exine thickness (EX), (nD 25).

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Table 3. Pollen grains of Athenaea and Aureliana: polar (P) and equatorial diameter (E) measurements (in mm) and shape (com-parison specimens, n D 10).

Taxa Registry P E P/E Shape

Athenaea anonacea BHCB 125569 BHCB 145085 31.08 29.97 30.3 28.87 1.02 1.03 prolate spheroidal prolatespheroidal

Athenaea cuspidata BHCB 38700 BHCB 38701 32.12 28.74 31.54 29.19 1.02 0.98 prolate spheroidal oblatespheroidal

Athenaea martiana BHCB 26284 BHCB 124213 31.37 36.43 32.58 33.06 0.96 1.1 oblate spheroidal prolatespheroidal

Athenaea micrantha BHCB 126146 BHCB 45271 29.96 29.44 28.33 28.61 1.06 1.03 prolate spheroidal prolatespheroidal

Athenaea picta BHCB 38697 BHCB 133520 34.21 36.02 31.38 32.63 1.09 1.1 prolate spheroidal prolatespheroidal

Athenaea pogogena BHCB 106743 BHCB 93326 26.33 30.94 26.61 31.94 0.98 0.96 oblate spheroidal oblatespheroidal

Aureliana angustifolia BHCB 134381 27.03 25.63 1.05 prolate spheroidal

Aureliana brasiliana BHCB 145053 BHCB 133647 28.79 28.69 29.34 26.44 1.04 1.08 prolate spheroidal prolatespheroidal

Aureliana darcyi BHCB 100186 BHCB 48387 29.76 28.03 29.52 27.47 1.01 1.02 prolate spheroidal prolatespheroidal

Aureliana fasciculatavar. fasciculata

BHCB 126175 BHCB 126176 26.41 23.85 24.54 23.56 1.08 1.01 prolate spheroidal prolatespheroidal

Aureliana fasciculatavar. longifolia

BHCB 25266 BHCB 145020 30.9 29.03 28.21 26.23 1.09 1.11 prolate spheroidal prolatespheroidal

Aureliana fasciculatavar. tomentella

BHCB 48367 BHCB 89804 30.34 31.70 29.03 31.00 0.98 1.02 oblate spheroidal prolatespheroidal

Aureliana tomentosa BHCB 124211 BHCB 87656 24.5 28.83 24.99 29.02 0.98 0.99 oblate spheroidal oblatespheroidal

Aureliana velutina BHCB 61184 BHCB 127290 28.95 29.69 25.54 28.08 1.13 1.06 prolate spheroidal prolatespheroidal

Aurelianawettsteiniana

BHCB 10519 28.32 28.27 1.01 prolate spheroidal

Table 4. Arithmetic average (in mm) of aperture measurements of pollen grains of the Brazilian species of Athenaea and Aureli-ana (n D 10).

Colpus Endoaperture

Taxa Length Width Width

Athenaea anonacea 24.50 2.30 3.10

Athenaea cuspidata 27.91 2.55 4.30

Athenaea martiana 27.98 3.20 3.67

Athenaea micrantha 24.37 2.47 2.93

Athenaea picta 28.47 2.26 6.00

Athenaea pogogena 28.23 2.91 4.82

Aureliana angustifolia 24.44 2.73 2.35

Aureliana brasiliana 25.50 2.40 10.81

Aureliana darcyi 22.06 2.50 2.14

Aureliana fasciculata var. fasciculata 22.26 2.30 1.80

Aureliana fasciculata var. longifolia 24.92 2.60 4.10

Aureliana fasciculata var. tomentella 22.51 2.53 2.31

Aureliana tomentosa 20.61 2.11 1.64

Aureliana velutina 24.83 2.61 3.16

Aureliana wettsteiniana 22.98 2.50 2.52

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PAI varied from 0.22 to 0.36. 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; supplemen-

tal online material).

Exine sculpturing varied from scabrate (Plate 3, fig-

ures 1, 2, 4; Plate 4, figures 3, 6) in Aureliana fasciculata

var. fasciculata and Aureliana sellowiana to scabra-te�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 deter-

mine if such elements are artifacts of the technique of

scanning microscopy or particular exine structures.

Exine thickness ranged from 1.5 mm in Aureliana fasci-

culata var. tomentella, Aureliana fasciculata var. fasci-

culata 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 sharedbetween 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, Tubo-

capsicum 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 complexupon palynological study, providing only a slight sepa-

ration through pollen grain measurements. However,

these data confirm the position of the Athenaea/Aureli-

ana clade in the subtribe Withaniinae. Overall, the pol-

len 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 con-

sistently different from other species, eventually recog-

nized 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-Bar-

ros and colleagues (2011) investigated Athenaea picta.

The description of the pollen grains of the studied spe-

cies 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 undu-lated in A. fasciculata var. longifolia, probably because

of the lack of SEM analysis. Small- to medium-sized,

prolate to oblate spheroidal, tri- or tetracolporate

pollen grains with scabrate, granulate, reticulate and

striate ornamentation were observed in Withania som-

nifera (L.) Dunal, W. coagulans (Stocks) Dunal,

W. aristata Pauq., Nothocestrum longifolium A. Gray

and N. subcordatum H. Mann (Murray & Eshbaugh1971; Alwadie 2002; Al-Quran 2004; Perveen & Qaiser

2007). Tubocapsicum anomalum pollen grains have

been described as medium-sized, subspheroidal and tri-

colporate, 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’ H�er., C. sclhechten-dahlii G.Don., Lycium makranicum Schoenbeck-Tem-

esy, L. ruthenicum Murray, Sessea brasiliensis Toledo,

Solanum americanum Mill., S. anguivi Lam, S. argen-

teum 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 &

Table 5. Arithmetic average (in mm) of distance between theapices of two ectocolpi (d), equatorial diameter in polar view(D) and polar area index (PAI), of pollen grains of the Brazil-ian species of Athenaea and Aureliana (n D 10).

Taxa D d PAI

Athenaea anonacea 28.82 8.34 0.29

Athenaea cuspidata 29.60 9.82 0.33

Athenaea martiana 31.42 8.42 0.27

Athenaea micrantha 29.86 6.04 0.22

Athenaea picta 31.14 8.13 0.26

Athenaea pogogena 31.03 9.25 0.30

Aureliana angustifolia 26.55 5.94 0.22

Aureliana brasiliana 30.38 10.57 0.35

Aureliana darcyi 26.24 7.69 0.29

Aureliana fasciculata var. fasciculata 25.21 6.53 0.26

Aureliana fasciculata var. longifolia 28.52 9.41 0.33

Aureliana fasciculata var. tomentella 25.42 6.20 0.24

Aureliana tomentosa 23.15 5.28 0.23

Aureliana velutina 27.93 6.87 0.24

Aureliana wettsteiniana 27.75 10.04 0.36

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Duarte 2008; Batista-Franklin & Goncalves-Esteves

2008), and a punctate exine with at least seven species

of Salpiglossideae (Stafford & Knapp 2006). Exine

ornamentation is the most variable pollen character inSolanaceae palynology; however,Stafford and Knapp

(2006) suggest caution in using it to determine relation-

ships 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 gen-

iculum in genera of about 13 families of angiosperms

has been described by Erdtman (1952). The term refersto the similarity of a knee (Potoni�e 1934) and is

described by Punt et al. (2007) as ‘a bulge in the equa-

torial exine of the colpus, often associated with separa-

tion of the sexine from the nexine and the rupturing of

the later. If the separation forms a cavity, the term fas-

tigium should be applied’. A geniculate aperture has

been described only rarely in Solanaceae. Stafford and

Knapp (2006) found a central transversely orientedridge 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 endoaper-

ture 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 thegenera 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 & Goncalves-

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 adetailed 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 ana-

lysed is the presence of an endocingulum, which some-

times, 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 widethat 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 &

Goncalves-Esteves 2008), although, in fact, it is cor-

rectly applied in Brugmansia and Datura (Persson et al.

1999).

Some species in this study had inaperturate pollen

grains. These are usually associated with derived breed-ing 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 inaperatu-

rate 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 pol-

len 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 individ-

ual � relatively common in Solanum, Deprea, Witha-

nia, etc. (Sawyer & Anderson, 2000; Hunziker 2001;

Anderson et al. 2006). So, the biology behind these ina-

peraturate grains needs detailed study. Male sterility in

other Solanaceae genera appears to involve viable pol-

len 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 weremarkedly 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 mem-

bers 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 macromorphologicaland 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 systemat-

ics 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 Cas-sino for the support and helpful guidance on the acetolysisprocess; Andr�eia Cardoso Pacheco Evaldt and Andr�e Ramosfor their valuable assistance, and for receiving the first author

12 I. M. da Costa Rodrigues et al.

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in the Palynology Laboratory of Universidade Luterana doBrasil; and Sandy Knapp for reviewing the English.

Funding

This work was supported by the Fundac~ao de Amparo �a Pes-quisa do Estado de Minas Gerais (FAPEMIG) under Grant[APQ-02015-11] and Conselho Nacional de DesenvolvimentoCient�ıfico e Tecnol�ogico (CNPq) under doctoral scholarshipto the first author [143004/2009-3].

Author biographies

IZABELLA MARTINS DA COSTA RODRIGUES gradu-ated in biology (emphasis on plant biology) from the Univer-sidade Federal de Vicosa, and has a Master’s degree in plantscience (agronomy) from the same institution (2008). She hasa PhD in plant biology from the Universidade Federal deMinas Gerais (2013), and part of this study was performed inLondon at the Natural History Museum. Izabella has experi-ence in plant biology (systematics and taxonomy), Solana-ceae taxonomy, palynology, plant morphology, reproductivebiology, allelopathy and weed management.

BRUNO FERRNANDES FALCeAO graduated in biologyfrom the Universidade Federal de Minas Gerais. He is cur-rently a Master’s student at the same institution. Bruno hasexperience in plant systematics and taxonomy, Solanaceaereproductive biology and pollination biology.

JOeAO RENATO STEHMANN has a PhD in plant biologyfrom the Universidade Estadual de Campinas. He is a spe-cialist on the Solanaceae family and is advisor for the post-graduate Program in Plant Biology at the UniversidadeFederal de Minas Gerais. Jo~ao researches flowering planttaxonomy, systematics in Solanaceae and floristic studies.Currently, he is curator of the collection of dicotyledons andthe BHCB herbarium database, contributes to the manage-ment committee of the Institutos Nacionais de Ciencia e Tec-nologia (INCT) Virtual Herbarium of Flora and Fungi ofBrazil, and operates in coordination of the Species List of theflora of Brazil.

SORAIA GIRARDI BAUERMANN graduated in biologyfrom the Pontif�ıcia Universidade Cat�olica do Rio Grande doSul, and has a Master’s degree in botany from the Universi-dade Federal do Rio Grande do Sul (1989) and a PhD in geo-sciences from the same institution (2003). Currently, she is aprofessor at the Universidade Luterana do Brasil, UlbraCanoas, coordinator of the Palynology Laboratory andresearch coordinator. Soaraia has experience in botany andpalaeontology, acting on the following topics: pollen mor-phology, surface samples and palaeoenvironmental reconsti-tution of Quaternary palynology.

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