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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: [email protected]
� 2015 AASP � The Palynological Society
Palynology, 2015
http://dx.doi.org/10.1080/01916122.2015.1022908
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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
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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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