A taxonomic aid arising from pollen grains of Cestrum L. and Solanum L. (Solanaceae): differences in ornamentation and aperture

ABSTRACT

Solanaceae is represented by herbs, shrubs, trees and climbing plants and has cosmopolitan distribution, with a large number of native species in the Neotropical region. This study aimed to characterize the pollen morphology of ten species of Solanaceae (especially species of Cestrum and Solanum) from Cerrado forest fragments in Brazil, in order to contribute to the palynology, taxonomy and conservation of degraded areas. Pollen grains were acetolysed, measured and photographed using light and scanning electron microscopy. Qualitative data were described, and quantitative data were analyzed statistically according to sample size. Morphologically, the studied pollen grains vary in size (small to medium), amb (subcircular to subtriangular), shape (oblate spheroidal to subprolate), aperture details (very long, long or narrow colpi, rounded or tapered at the polar ends, colpi with margo, sometimes with a fastigium, endoapertures can present costa and median constriction) and ornamentation (psilate, rugulate, striate or microreticulate). We observed rugulate or striate pollen grains without fastigium for Cestrum species, whereas psilate or microreticulate pollen grains with fastigium were observed for Solanum species. Qualitative data on diameters and aperture measurements were also found to be important in characterizing the two genera. The results obtained here confirm Solanaceae as an eurypalynous family.

Keywords:
eurypalynous; multivariate analysis; pollen grains; pollen morphology; Solanales

Introduction

In tropical regions, there is an increasing process of fragmentation of forest habitats, and understanding the transformation of these areas is fundamental for the conservation of regional biodiversity, in addition to assisting in the management of the sustainable use of the remaining biological resources (Necchi 2012). In order to identify and reinforce the importance and need to preserve and conserve local biodiversity, Ranga et al. (2012) conducted a project in fragmented areas of Cerrado in the Northwestern region of the state of São Paulo, Brazil, listing more than 460 plant species, revealing the occurrence of exclusive species and unique characteristics of the analyzed regions. Based on projects similar to these, studies aiming to morphologically characterize native species in these areas of Cerrado contribute with important and useful data in the identification for the preservation of these fragments.

Solanaceae is one of five families of Solanales within the Lamiids group (APG IV 2016) and includes 2,480 species distributed into 102 genera (Stevens 2001 onwards). Solanaceae species are cosmopolitan, but their greatest biodiversity occurs in the western hemisphere, concentrated in the Neotropical region (Olmstead et al. 2008; Souza & Lorenzi 2008), with the main dispersion centers in Australia and Latin America (Barroso et al. 1991). In Brazil, the family is represented by 504 species, 237 of which are endemic, distributed in its 36 genera, and Solanaceae species can be found in all Brazilian biomes, being widely distributed in the main ecosystems: Atlantic Forest, Amazon and Cerrado (BFG 2015; Flora do Brasil 2020 2020).

The family includes species of great economic importance used in food, such as potatoes (Solanum tuberosum) and tomatoes (Solanum lycopersicum), or ornamental (Petunia hybrida). Many species accumulate alkaloids, being characterized as extremely toxic plants, like Atropa belladona Among those native to Brazil, we can highlight Solanum americanum - “maria-pretinha”, an invader of cultures; Solanum lycocarpum, part of the diet of the maned wolf (Chrysocyon brachyurus); and Solanum paniculatum (“jurubeba”), widely used for medicinal purposes; and the ornamental Cestrum spp. “ladies of the night” (Judd et al. 2009; Souza & Lorenzi 2008).

Traditionally, only two subfamilies were recognized in Solanaceae, based on macro-morphological characteristics: Cestroideae, with its straight or slightly bent embryos in small, prismatic to subglobose seeds and typically capsular fruits, and Solanoideae, with its curved embryos contained in flattened discoid seeds and typically berry-like fruits (D'Arcy 1979; 1991; Hunziker 1979; 2001; Olmstead & Palmer 1992). With the advancement of molecular studies to clarify phylogenetic relationships, new subfamilies have been proposed within Solanaceae. Currently, the family is mainly divided into four subfamilies, viz. Cestroideae, Goetzeoideae, Nicotianoideae and Solanoideae (Martins & Barkman 2005; Olmstead et al. 1999, 2008; Särkinen et al. 2013), in addition to confirming Cestroideae as a well-supported clade and the ambiguity of characters found in Solanoideae (Jamil et al. 2014).

The pollen morphology of various genera and species of Solanaceae has been described by different types of studies: Pollen morphology of the family in general (Guang-Fang et al. 1985; Bernadello & Luján 1997; Khatamsaz & Zangirian 1998; Batista-Franklim & Gonçalves-Esteves 2002; Rojas & Laportte 2004; Rodrigues et al. 2016; Dhanya & Devipriya 2016); Pollen morphology of Cestrum or Solanum (Murry & Eshbaugh 1971; Silva et al. 2003; Al-Quran 2004; Perveen & Qaiser 2007; Batista-Franklim & Gonçalves-Esteves 2008; Lashin 2011; Kumar et al. 2015; Vignoli-Silva et al. 2015; Song et al. 2018) and Pollen flora of some species (Erdtman 1952; Chung & Huang 1972; Salgado-Labouriau 1973; Rao & Ling 1974; Punt & Monna-Brands 1977; Roubik & Moreno 1991; Velásquez & Rangel 1995; Melhem et al. 2003; Barth & Duarte 2008; Cruz-Barros et al. 2011; Mercado-Gómez et al. 2013; Silva et al. 2014, 2016; Lorente et al. 2017). These authors have observed the differences regarding pollen grain aperture and ornamentation of the exine, concluding that the family is eurypalynous.

Given the wide distribution and abundance of Solanaceae species, and even with several studies that have reported the pollen morphology of the family, a better characterization of the pollen grains is necessary in order to extend the existing data in the literature, and seek a better delimitation of species or groups of species. Studies like those of Edmonds (1984), Persson et al. (1994), Knapp et al. (1998), Stafford & Knapp (2006) and Du et al. (2017) use palynological characters to corroborate the phylogeny of Solanaceae, thus considering pollen morphology of extreme importance in the study of the relationship between species and genera of the family (Song et al. 2018).

Some studies on the pollen of native species from remaining forest fragments of Cerrado indicate variations in shape, aperture, amb, ornamentation and size of the pollen grains (Souza & Gasparino 2014; Belonsi & Gasparino 2015; Dutra & Gasparino 2018; Landi & Gasparino 2018; Souza et al. 2019; Bellonzi et al. 2020; Dutra et al. 2020 and Soares et al. 2021); therefore, the aim of this study was to identify morphological characteristics in pollen grains of Solanaceae species, in order to contribute with the palynology, taxonomy and conservation of these species in degraded areas. This analysis also intended to identify which morphological characters of the pollen grains are important in the segregation of native Solanaceae species in Cerrado forest fragments.

Materials and methods

We studied the pollen grains of 10 native species of Solanaceae (Tab. 1) from the remnant forest fragments of Cerrado in the northwest of the State of São Paulo, described by Ranga et al. (2012). This area was chosen to represent the remaining Cerrado forest fragments in the State of São Paulo, and the studied species occur throughout the Brazilian territory, mainly concentrated in the Southeast and South of Brazil (Specieslink 2020). We analyzed two genera (Cestrum L. and Solanum L.). All Solanaceae species listed in Ranga et al. (2012) are new records in the study area. Five of the species analyzed (Cestrum bracteatum Link & Otto, C. nocturnum L., Solanum argenteum Dunal., S. lycocarpum A. St.-Hil. and S. paniculatum L.) are on the IUCN list of threatened species in the category of Least Concern (IUCN 2020). The pollen material was obtained from samples collected mainly in the remnant forest fragments of the northwest of the State of São Paulo, deposited as dried herbarium specimens in JABU and SJRP herbaria (acronym by Thiers 2019, continuous adapted).

Table 1
Voucher specimens of Solanaceae from Brazilian forest fragments. SJRP = Institution: UNESP, Campus São José Rio Preto; Location: Brazil. São Paulo. São José do Rio Preto and JABU = Institution: UNESP, Campus Jaboticabal; Location: Brazil. São Paulo. São Paulo. * standard specimen.

Genus/Species	Locality	Collector/ Date	Herbarium
Cestrum L.
C. bracteatum Link & Otto	BRAZIL. GOIÁS: Vicentinopolis *	Ranga N. T. et al. n/n - Aug. 25, 2007	SJRP
	BRAZIL. SÃO PAULO: Potirendaba	Almella D. and Gimenez L. R. 034 - May 27, 1999	SJRP
C. nocturnum L.	BRAZIL. SÃO PAULO: São José do Rio Preto*	Stranguetti V. 756 - Nov. 28, 1996	SJRP
C. strigillatum Ruiz & Pav.	BRAZIL. PARANÁ: Jataizinho*	Silva J. M. and Ziller S. 1886 - Mar. 22, 1997	SJRP
	BRAZIL. SÃO PAULO: Pindorama	Ranga N. T. et al. n/n - Sept. 04, 2008	SJRP
Solanum L.
S. argenteum Dunal.	BRAZIL. PARANÁ: Londrina*	Rodas L. A. C. n/n - May 20, 1985	SJRP
S. grandiflorum Ruiz & Pav.	BRAZIL. GOIÁS: Vicentopolis*	Ranga N. T. et al. 10 - Oct. 25, 2007	SJRP
S. lycocarpum A. St.-Hil.	BRAZIL. SÃO PAULO: Barretos*	Ranga N. T. et al. 19 - Mar. 05, 2009	SJRP
	BRAZIL. MINAS GERAIS: São Roque	Mazzarelli C. C. M. and Almeida R. V. 01 - Nov. 14, 2009	JABU
S. mauritianum Scop	BRAZIL. SÃO PAULO: Barretos*	Ranga N. T. et al. 32 - Oct. 16, 2008	SJRP
S. paniculatum L.	BRAZIL. SÃO PAULO: Taiúva*	Rodrigues E. H. A. n/n - Nov. 18, 2010	JABU
	BRAZIL. SÃO PAULO: Jaboticabal	Rodrigues E. H. A. 109 - Oct. 27, 1990	JABU
S. pseudoquina A. St.-Hil.	BRAZIL. PARANÁ: Paranaguá*	Cordeiro J. and Cruz J. M. 1191 - Nov. 11, 1994	SJRP
S. ramulosum Sendtn.	BRAZIL. SÃO PAULO: Matão*	Ranga N. T. et al. 60 - May 27, 2007	SJRP
	BRAZIL. SÃO PAULO: Bebedouro	Ranga N. T. et al. 29 - Feb. 19, 2009	SJRP
	BRAZIL. SÃO PAULO: União Paulista	Ranga N. T. et al. 14 - Oct. 19, 2007	SJRP

Pollen grains of all 16 specimens (for each species, when possible, about three specimens were analyzed to confirm the data, considering only one specimen as the standard - Tab. 1) were studied using light microscopy (LM) and scanning electron microscopy (SEM). For the analysis, permanent slides were prepared with pollen material acetolyzed according to Erdtman (1960) modified by Melhem et al. (2003). The pollen grains were measured (pollen diameter, n = 25; aperture and exine thickness, n = 10) seven days after preparation, as established by Salgado-Labouriau et al. (1965). The slides were deposited in the pollen reference collection of the Departamento de Biologia Aplicada à Agropecuária, Unesp, Jaboticabal, SP, Brazil.

A statistical analysis was conducted with the measures taken to obtain the means (x), standard deviation (sx), standard error (s), 95 % confidence intervals (CI), coefficient of variability (V) and range (R) according to Vieira (2011) and Zar (2010). For measures of exine thickness, and length and width of the apertures, only the arithmetic mean was calculated. To compare the values of pollen grain diameters we used the graphs of the MINITAB program which represent the mean and the confidence interval values. A principal component analysis (PCA) and a cluster analysis (hierarchical cluster analysis - HCA) were performed using the programs FITOPAC (Shepherd 1996) and PC-ORD (McCune & Mefford 2011). PCA analysis aims to group the analyzed species according to the metric values of the pollen grains, while in the cluster analysis we want to identify the relationship among the species (similarity data).

We used 14 metric variables for the PCA and HCA: equatorial diameter in polar view (EDPV), polar diameter in equatorial view (PDEV), equatorial diameter in equatorial view (EDEV), length of colpus (CLEG), width of colpus (CWID), margo of colpus (CMAR), length of endoaperture (ELEG), width of endoaperture (EWID), shape (SHAP), exine (EXIN), nexine (NEXI), sexine (SEXI), polar area index (PAI) and width colpus index (WCI). In PCA, the Pearson and Kendall correlation coefficients were considered, and the dendrogram of HCA was produced using the Jaccard distance measure and the Group Average linkage method analyzed the percentage of information (variables) necessary to reach the final number of groups formed.

Pollen terminology follow Punt et al. (2007); we adopted the polar area index proposed by Faegri & Iversen (1966), colpus width index in accordance with Gasparino et al. (2013), and pollen description is based on Bellonzi et al. (2020). Photomicrographs were taken with a Bel Photonics light microscope, for LM photos, and electromicrographs were taken with a JEOL JSM5410.

Results

General description

The pollen grains of the studied Solanaceae species (Tabs. 2, 3, 4; Figs. 1, 2, 3) are monad, isopolar, small to medium, subcircular to subtriangular amb, very small or small polar area, oblate spheroidal to subprolate, 3-colporate with long or very long and narrow colpi, rounded or tapered at the polar ends, colpi with margo, with or without fastigium or ornamented colpus membrane; lalongate endoapertures, sometimes with costa and median constriction. Exine tectate (psilate, rugulate or striate) or semitectate (microreticulate). The sexine is thicker than the nexine.

Figure 1
Photomicrographs of Cestrum L. pollen grains. A-F. Cestrum bracteatum. A. Polar view, optical section; B. Equatorial view; C. Equatorial view, colpus and endoaperture; D’-D”. Ornamentation in high and low focus, rugulate exine; E. Polar view, ornamentation in MEV, verrucate exine; F. Ornamentation in SEM details. G-I. Cestrum nocturnum. G. Polar view, optical section; H. Equatorial view, colpus and endoaperture; I. Equatorial view, ornamentation in SEM, striate exine. J-L. Cestrum strigillatum. J. Optical section; K. Equatorial view, colpus and endoaperture; L’-L”. Ornamentation in high and low focus, striate exine. Scale bars: E-F, I = 2 μm; D, L = 5 μm; A-C, G-H, J-K = 10 μm.

Figure 2
Photomicrographs of Solanum L. pollen grains. A-E. Solanum argenteum. A. Polar view, optical section; B. Equatorial view, optical section; C. Equatorial view, colpus and endoaperture; D. Equatorial view, ornamentation in SEM, granulate exine; E. Ornamentation in SEM, details of the densely verrucate colpus membrane. F-H. Solanum grandiflorum. F. Polar view, optical section; G. Equatorial view, optical section; H. Equatorial view, colpus and endoaperture. I-L. Solanum lycocarpum. I. Polar view, optical section; J. Equatorial view, optical section; K. Equatorial view, colpus and endoaperture; L’-L”. Ornamentation in high and low focus, microreticulate exine. Scale bars: E = 1 μm; D = 2 μm; L = 5 μm; A-C, F-K = 10 μm.

Figure 3
Photomicrographs of Solanum L. pollen grains. A-C. Solanum mauritianum. A. Polar view, optical section; B. Equatorial view, optical section; C. Equatorial view, colpus and endoaperture. D-F. Solanum paniculatum. D. Polar view, optical section; E. Equatorial view, optical section; F. Equatorial view, colpus and endoaperture. G-I. Solanum pseudoquina. G. Polar view, optical section; H. Equatorial view, optical section; I. Equatorial view, colpus and endoaperture. J-L. Solanum ramulosum. J. Polar view, optical section; K. Equatorial view, optical section; L. Equatorial view, colpus and endoaperture. Scale bars: 10 μm.

Genera description

Cestrum (Fig. 1)

Studied species: Cestrum bracteatum (Fig. 1A-F); Cestrum nocturnum (Fig. 1G-I); Cestrum strigillatum (Fig. 1J-L).

Size: medium (Tabs. 2, 3).

Table 2
Morphological characterization of Solanaceae pollen grains.

Species	Size	P/E	Shape	PAI	AS	EO
Cestrum bracteatum	M	0.92	OS	0.30	11.11	RU
Cestrum nocturnum	M	1.05	PS	0.39	14.01	ST
Cestrum strigillatum	M	1.10	PS	0.21	8.49	ST
Solanum argenteum	S	1.16	SP	0.22	5.75	PS
Solanum grandiflorum	S-M	1.02	PS	0.25	5.85	PS
Solanum lycocarpum	S-M	1.10	PS	0.26	6.41	MR
Solanum mauritianum	M	1.05	PS	0.27	8.23	MR
Solanum paniculatum	M	1.07	PS	0.28	8.79	MR
Solanum pseudoquina	S	1.22	SP	0.30	5.75	PS
Solanum ramulosum	S-M	1.03	PS	0.30	7.14	PS

• P/E = ratio between polar and equatorial diameter (min=minimum-max=maximum); PAI= polar area index, AS = apocolpus side; EO = exine ornamentation; S = small; M = medium; OS = oblate spheroidal; PS = prolate spheroidal; SP = subprolate; MR = microreticulate; PS= psilate; RU= rugulate; ST = striate.

Table 3
Quantitative data of Solanaceae pollen grain, n = 25.

Species	(R) x̄ ± sx̄	s	CI	V
Equatorial diameter in polar view
Cestrum bracteatum	(32.50 - 42.50) 37.30 ± 0.50	2.49	(36.27 - 38.33)	6.68
Cestrum nocturnun	(32.50 - 37.50) 35.70 ± 0.37	1.84	(34.94 - 36.46)	5.16
Cestrum strigillatum	(32.50 - 47.50) 40.10 ± 0.67	3.34	(38.72 - 41.48)	8.84
Solanum argenteum	(15.00 - 20.00) 18.10 ± 0.30	1.49	(17.48 - 18.72)	8.25
Solanum grandiflorum	(20.00 - 27.50) 23.10 ± 0.46	2.31	(22.14 - 24.06)	10.02
Solanum lycocarpum	(17.50 - 30.00) 25.00 ± 0.52	2.60	(23.93 - 26.70)	10.41
Solanum mauritianum	(25.00 - 35.00) 30.20 ± 0.45	2.27	(29.26 - 31.14)	7.53
Solanum paniculatum	(27.50 - 35.00) 31.79 ± 0.37	1.87	(30.93 - 32.47)	5.90
Solanum pseudoquina	(15.00 - 22.50) 18.80 ± 0.36	1.79	(18.06 - 19.54)	9.50
Solanum ramulosum	(17.50 - 27.50) 24.10 ± 0.43	2.15	(23.21 - 24.99)	8.92
Polar diameter in equatorial view
Cestrum bracteatum	(32.50 - 42.50) 36.60 ± 0.54	2.69	(35.49 - 37.71)	7.35
Cestrum nocturnun	(32.50 - 42.50) 36.90 ± 0.55	2.73	(35.77 - 38.03)	7.39
Cestrum strigillatum	(37.50 - 55.00) 44.50 ± 0.80	4.02	(42.84 - 46.16)	9.03
Solanum argenteum	(17.50 - 25.00) 21.20 ± 0.33	1.63	(20.53 - 21.87)	7.70
Solanum grandiflorum	(22.50 - 30.00) 25.00 ± 0.38	1.91	(24.21 - 25.79)	7.64
Solanum lycocarpum	(25.00 - 30.00) 28.80 ± 0.29	1.46	(28.20 - 29.40)	5.09
Solanum mauritianum	(20.00 - 35.00) 29.60 ± 0.76	3.80	(28.03 - 31.17)	12.83
Solanum paniculatum	(30.00 - 37.50) 33.80 ± 0.41	2.06	(32.95 - 34.65)	6.08
Solanum pseudoquina	(20.00 - 27.50) 22.70 ± 0.35	1.76	(21.98 - 23.42)	7.74
Solanum ramulosum	(20.00 - 27.50) 24.63 ± 0.45	2.03	(23.31 - 25.94)	8.25
Equatorial diameter in equatorial view
Cestrum bracteatum	(27.50 - 47.50) 40.00 ± 0.88	4.39	(38.19 - 41.81)	10.97
Cestrum nocturnun	(30.00 - 40.00) 35.30 ± 0.49	2.43	(34.30 - 36.30)	6.88
Cestrum strigillatum	(32.50 - 47.50) 40.30 ± 0.86	4.29	(38.53 - 42.07)	10.64
Solanum argenteum	(15.00 - 22.50) 18.20 ± 0.45	2.23	(17.28 - 19.12)	12.23
Solanum grandiflorum	(20.00 - 30.00) 24.40 ± 0.44	2.20	(23.49 - 25.31)	9.01
Solanum lycocarpum	(20.00 - 30.00) 26.20 ± 0.52	2.61	(25.12 - 27.28)	9.98
Solanum mauritianum	(22.50 - 35.00) 28.20 ± 0.64	3.19	(26.88 - 29.52)	11.31
Solanum paniculatum	(27.50 - 35.00) 31.50 ± 0.35	1.77	(30.77 - 32.23)	5.61
Solanum pseudoquina	(15.00 - 22.50) 18.60 ± 0.36	1.78	(17.87 - 19.33)	9.57
Solanum ramulosum	(20.00 - 27.50) 23.88 ±.053	2.36	(22.34 - 25.41)	9.89

• R = range; x̄ = mean (µm); sx̄ = standard deviation (µm); s = standard error (µm); CI = confidential interval in 95 % (µm); V = coefficient of variability (%).

Amb and shape: subcircular to subtriangular amb or only subtriangular (Cestrum strigillatum) amb, very small (C. strigillatum) or small polar area, oblate spheroidal (C. bracteatum) to prolate spheroidal (Tab. 2).

Aperture: 3-colporate, long or very long (Cestrum strigillatum) and narrow colpi, rounded (C. strigillatum) or tapered at the polar ends (C. bracteatum and C. nocturnumFig. 1D-E, G), with margo and with ornamented colpus membrane only in C. bracteatum (Fig. 1E), without fastigium (all species). Endoaperture - lalongate (Fig. 1C, H, K -Tabs. 3, 4), without costa and with median constriction (Fig. 1C, H).

Exine: Tectate and rugulate in Cestrum bracteatum (presenting verrucate ornamentation in SEM, Figs. 1E-F); tectate and striate in C. nocturnum and C. strigillatum (Fig. 1H-I, K-L, Tab. 2). The sexine is thicker than the nexine (Tab. 4).

Solanum (Fig. 2-3)

Studied species: Solanum argenteum (Fig. 2A-E), Solanum grandiflorum (Fig. 2F-H), Solanum lycocarpum (Fig. 2I-L), Solanum mauritianum (Fig. 3A-C), Solanum paniculatum (Fig. 3D-F), Solanum pseudoquina (Fig. 3G-I) and Solanum ramulosum (Fig. 3JL).

Size: small (Solanum argenteum and S. pseudoquina), small to medium (S. grandiflorum, S lycocarpum and S. ramulosum) and medium (S. mauritianum and S. paniculatum - Tabs. 2, 3).

Amb and shape: subcircular and subtriangular (S. argenteum and S. ramulosum) amb, very small (S. argenteum) or small polar area, prolate spheroidal to subprolate (Tab. 2).

Aperture: 3-colporate, long or very long (Solanum argenteum) and narrow colpi, rounded (S. mauritianum and S. paniculatum) or tapered at the polar ends, with margo; with or without ornamented colpus membrane (in S. argenteum, the colpus membrane is verrucate under SEM, Figs. 2D-E); with fastigium (all species). Endoaperture - lalongate (Figs. 2C, H, K, 3C, F, I, J - Tab. 4) endoaperture with costa (in S. lycocarpum, S. mauritianum and S. paniculatum - Figs. 2J, 3B, E) or not and with median constriction (only in S. paniculatum - Fig. 3F).

Table 4
Measurements (in µm) of Solanaceae pollen grain, apertures and exine (n = 10).

Species	Colpus			Endoaperture		WCI	Ex	Sex	Nex
	Length	Width	Margo	Length	Width
Cestrum bracteatum	26.45	2.63	0.54	3.83	19.41	15.20	1.14	0.74	0.39
Cestrum nocturnun	28.72	0.83	0.54	1.59	15.47	42.32	1.50	0.89	0.62
Cestrum strigillatum	34.69	2.07	0.58	4.62*	17.84*	19.44	1.58	1.03	0.52
Solanum argenteum	14.48	1.05	0.43	1.10	6.95	17.28	1.25	0.84	0.45
Solanum grandiflorum	17.25	1.06	0.47	1.59	8.53	22.95	1.32	0.88	0.45
Solanum lycocarpum	20.27	1.25	0.53	1.41	11.88	20.94	1.28	0.88	0.45
Solanum mauritianum	23.06	1.44	0.47	2.04	13.74	19.61	1.34	0.84	0.49
Solanum paniculatum	25.03	1.58	0.48	3.31	14.99	19.94	1.27	0.84	0.47
Solanum pseudoquina	16.40	1.42	0.47	2.61	9.65	13.07	1.03	0.68	0.39
Solanum ramulosum	17.41*	1.32*	0.46*	2.23*	10.10*	18.15*	1.07	0.72	0.42

• Ex =exine thickness; Sex = sexine thickness; Nex = nexine thickness; WCI = width colpus index, *n <10.

Exine: Semitectate and microreticulate (Solanum lycocarpum, S. mauritianum and S. paniculatum - Figs. 2L, 3B-C, E-F); tectate and psilate (other species), sometimes psilate and slightly granulate in S. argenteum (Figs. 2D-E). The sexine is thicker than the nexine (Tab. 4).

Artificial Key to the Solanaceae taxa studied

1. Pollen grains without fastigium and rugulate or striate ornamentation ............................ 2

1’. Pollen grains with fastigium and psilate or microreticulate ornamentation .................... 4

2. Rugulate ornamentation..................................................................... Cestrum bracteatum

2’. Striate ornamentation ...................................................................................................... 3

3. Very long colpus, rounded at the polar ends ..................................... Cestrum strigillatum

3’. Long colpus, tapered at the polar ends .............................................. Cestrum nocturnum

4. Microreticulate ornamentation, endoaperture with costa ................................................. 5

4’. Psilate ornamentation, endoaperture without costa ........................................................ 7

5. Endoaperture with median constriction ……………………..……. Solanum paniculatum

5’. Endoaperture without median constriction ………………….………………………… 6

6. Colpus tapered at the polar ends …………………………………...Solanum lycocarpum

6’. Colpus rounded at the polar ends …………………….………… Solanum mauritianum

7. Pollen grains with subcircular amb …………………………………………………….. 8

7’. Pollen grains with subtriangular amb …………………………………………………. 9

8. Prolate spheroidal shape …………………………………………Solanum grandiflorum

8’. Subprolate shape ………………………….……………………. Solanum pseudoquina

9. Very long colpus, very small polar area and subprolate shape …..…Solanum argenteum

9’. Long colpus, small polar area and prolate spheroidal shape ……… Solanum ramulosum

Analysis of quantitative data

For the quantitative analysis of pollen grain size, we used the polar and equatorial diameters in equatorial view (Fig. 4). When we observe the mean and confidence interval of the pollen grain diameters, most species present the same order of increasing values for both diameters, with the exception of species Cestrum bracteatum and C. nocturnum, which interchange the values of equatorial diameter (Fig. 4B).

Figure 4
Representation of the confidence interval of the mean in 95 % of the Solanaceae pollen grains. A. Polar diameter in equatorial view. B. Equatorial diameter in equatorial view. The highest and lowest boundaries show the confidence interval; the average circle shows the arithmetic mean. C. brac= Cestrum bracteatum; C. noc= Cestrum nocturnum; C. str= Cestrum strigillatum; S. arg= Solanum argenteum; S. gra= Solanum grandiflorum; S. lyc= Solanum lycocarpum; S. mau= Solanum mauritianum; S. pan= Solanum paniculatum; S. pse= Solanum pseudoquina; S. ram= Solanum ramulosum. The values are in µm.

We found that Solanum argenteum and S. pseudoquina are separated from the other species by lower polar and equatorial diameters. Regarding polar diameter, Cestrum strigillatum has a larger diameter than all other species. When we consider the equatorial diameter, C. bracteatum and C. strigillatum are separated from the other species because they have similar and larger diameters than the others. The other species are in a continuous group of intermediate diameter values (Fig. 4A-B).

When we compare the diameter values, both equatorial and polar, it is evident that Cestrum species differ from Solanum species because they present the highest mean values of the diameters.

PCA is an exploratory analysis of quantitative data valued for Solanaceae pollen grains. This analysis summarizes in its two axes (Fig. 5) 94.28 % of total data variability. The first axis of the PCA explained 70.83 % of the total variability according to the metric variables. This component explained the variance based mainly on endoaperture width (EWID), diameters (EDEV and EDPV) and colpus length (CLEG) (Tab. 5). The species of the genus Cestrum are grouped on the negative side of axis 1, with the highest diameter values (especially equatorial diameter in equatorial view and equatorial diameter in polar view) and the species of the genus Solanum, mainly on the positive side, presenting the lowest values. The second component explains 23.45 % of the variability of the species studied, the values of colpus index (WCI), endoaperture length (ELEG) and colpus width (CWID). Cestrum nocturnum was separated from all other species because it has a high colpus index (WCI).

Figure 5
Principal component analysis performed with the pollen metrical variables from Solanaceae species. EDPV = equatorial diameter in polar view; PDEV = polar diameter in equatorial view; EDEV = equatorial diameter in equatorial view; CLEG = length of colpus; CWID = width of colpus; CMAR = margo of colpus; ELEG = length of endoaperture; EWID = width of endoaperture; SHAP = shape; EXIN = exine; NEXI = nexine; SEXI = sexine; PAI = polar area index; WCI = width colpus index. Species abbreviations in figure 4.

Table 5
Pearson and Kendall correlation coefficients for pollen grain metric variables of the first and the second axis of principal component analysis (PCA) in Solanaceae species.

Variables		Principal components
		Axis 1	Axis 2
EDPV	equatorial diameter in polar view	-0.3986	0.0959
PDEV	polar diameter in equatorial view	-0.3440	0.0697
EDEV	equatorial diameter in equatorial view	-0.4014	0.0814
SHAP	shape	0.0304	-0.0078
CLEG	length of colpus	-0.3940	0.1561
CWID	width of colpus	-0.1834	-0.3966
CMAR	margo of colpus	-0.0395	0.0085
ELEG	length of endoaperture	-0.3673	-0.4771
EWID	width of endoaperture	-0.4630	0.0216
EXIN	exine	-0.0637	0.1410
NEXI	nexine	-0.0330	0.1076
SEXI	sexine	-0.0340	0.0676
PAI	polar area index	-0.0105	0.0424
WCI	width colpus index	-0.1209	0.7394

The HCA based on the Jaccard linkage method produces a dendrogram with a linkage value of 10.00 %. When considering the percentage of similarity (based in pollen grain metric variables - Tab. 5), two groups were recognized, representing the two genera analyzed (with approximately 35 % of similarity - Fig. 6). With about 35 % of similarity, the Cestrum species were grouped for presenting larger diameter values. Cestrum bracteatum and C. strigillatum (approximately 80 % of similarity) form a group for presenting the highest values of endoaperture length (ELEG). The other species comprise the Solanum group, with about 53 % of similarity. The species Solanum argenteum and S. pseudoquina are closely related with approximately 68 % of similarity for presenting the smallest diameter values among all species. In addition, species S. grandiflorum, S. lycocarpum and S. ramulosum were 94 % similar, for presenting intermediate diameter values within Solanum. The group formed by S. grandiflorum and S. lycocarpum presents similar values for exine (EXIN) and lower values for endoaperture length (ELEG). The species S. mauritianum and S. paniculatum were grouped for presenting the highest diameter values within the genera and the highest values for colpus length (CLEG). Qualitative data were also optimized in Figure 6.

Figure 6
Dendrogram of the hierarchical cluster analysis (HCA) with the pollen metrical variables from Solanaceae species and optimization of qualitative pollen data. The triangle corresponds to the species of Cestrum and the squares correspond to Solanum. Species abbreviations in Figure 4.

Discussion

Several studies, including Erdtman (1952), Salgado-Labouriau (1973), Roubik & Moreno (1991), Velásquez & Rangel (1995), Melhem et al. (2003), Silva et al. (2003), Barth & Duarte (2008), Cruz-Barros et al. (2011), Mercado-Gómez et al. (2013), Vignoli-Silva et al. (2015), Silva et al. (2016) and Song et al. (2018), describe Cestrum pollen grains as: monad; isopolar or occasionally heteropolar; mostly medium sized; suboblate to prolate shape, circular to triangular amb; 3(4)-colporate or occasionaly syncolporate with margo, displaying equatorial constriction or not, very long or long colpus, narrow, sunken; with or without colpus membrane; distinctly lalongate endoaperture, fastigiated or not, or occasionally zonorate forming ring, with or without constriction; scabrate, striate, striate-rugulate, perfurate-scabrate, rugulate, rugulate-areolate rugulate-striate, or verrucate ornamentation and sexine slightly thicker than the nexine.

Cestrum bracteatum pollen grains have been previously studied by Velásquez & Rangel (1995), Silva et al. (2003), Cruz-Barros et al. (2011), Mercado-Gómez et al. (2013) and Vignoli-Silva et al. (2015). These studies disagree in terms of colpus structures (presence or absence of fastigium, operculum and constriction) and exine ornamentation (varying among psilate, striate, rugulate-striate and rugulate in the mesocolpus and psilate in the apocolpus, respectively). In our studies, the pollen grains of this species do not present fastigium or operculum, as previously noted, with the presence of the median constriction in the endoaperture; under LM the exine is regulate; however under SEM it is possible to observe verruca in the ornamentation, which is not seen under LM.

Roubik & Moreno (1991) analyzed the pollen grains of Cestrum nocturnum and described them as 3-colporate and syncolporate, long colpi and lalongate endoaperture, like a ring. We observed 3-colporate pollen grains and lalongate endoaperture without a ring. Roubik & Moreno (1991) also described rugulate pollen grains, while in our analysis exine ornamentation is striate, confirmed by scanning electron microscopy (SEM), in some cases it is difficult to define the ornamentation of Cestrum pollen grains only under LM.

Cestrum strigillatum pollen grains have been analyzed by Barth & Duarte (2008) and Vignoli-Silva et al. (2015). Vignoli-Silva et al. (2015) described the presence of fastigium and constriction in the endoaperture, and rugulate-striate exine (conspicuous in the mesocolpus and psilate in the apocolpium); for Barth & Duarte (2008), pollen ornamentation is striate. For this species we did not observe fastigium and we can see endoaperture with constriction and striate ornamentation in the pollen grains.

Solanum species had their pollen grains previously studied by Erdtman (1952), Murry & Eshbaugh (1971), Chung & Huang (1972), Salgado-Labouriau (1973), Rao & Ling (1974), Punt & Monna-Brands (1977), Edmonds (1984), Roubik & Moreno (1991), Velásquez & Rangel (1995), Knapp et al. (1998), Melhem et al. (2003), Al-Quran (2004), Perveen & Qaiser (2007), Batista-Franklim & Gonçalves-Esteves (2008), Barth & Duarte (2008), Cruz-Barros et al. (2011), Lashin (2011), Silva et al. (2014), Kumar et al. (2015), Silva et al. (2016), Du et al. (2017) and Lorente et al. (2017). The authors, in general, describe pollen grains as: monad; isopolar; small to medium size; oblate-spheroidal to subprolate shape; circular to triangular and lobate amb; mostly 3-colporate, occasionally 4-colporate, sometimes 2-6 colporate or pantocolporate, syncolporate or parasyncolporate, colpus very long, long or short, with or without margo, fastigium, operculum, granulate or tuberculate colpus membrane and medium constriction; lalongate endoaperture, sometimes elliptic, with or without medium constriction, occasionally forming constinuous ring, with or without margo, costa and fastigium; granulate, granulate-perforate, granulate-punctate, granulate-punctate-fossulate; granulate-verrucate-punctate, echinate, microechinate, microreticulate, psilate, spinulose, spinulose-perforate, spinulose-punctate, reticulate, rugulate-ganulate, scabrate, verrucate, verrucate-punctate, with or without ornamented operculum and aspis; sexine thicker or thinner than nexine or as thick as nexine.

Roubik & Moreno (1991) and Melhem et al. (2003) studied Solanum argenteum and described 3-colporate pollen grains without fastigium, lalongate endoaperture and psilate sexine; Batista-Franklim & Gonçalves-Esteves (2008) also observed 3-colporate pollen grains, but they described fastigium in the lalongate endoaperture (with constriction), scabrate and granulate sexine in SEM. Our study portrayed the pollen grains with margo in the colpus, endoaperture with median constriction and psilate exine ornamentation; in addition, we observed a higher diameter value than those mentioned by the authors. In SEM, it is possible to observe granules in the ornamentation of the pollen grains of this species, as well as an ornamented membrane densely verrucate in the colpus, different from the characteristics of the genus Solanum presented by Du et al. (2017), since the authors cite granulate or tuberculate ornamentation distributed on the surface of the colpus.

The pollen grains of Solanum grandiflorum were described by Salgado-Labouriau (1973) as 3-colporate with long colpi and prominent margo, lalongate endoaperture with margo and occasionally constricted, psilate or granulate ornamentation. For this species, the characteristics found by Salgado-Labouriau (1973) are very similar to those observed by us. We describe the endoaperture without costa for the pollen grains, following the definition proposed by Punt et al. (2007), since the term margo refers to a differentiated area in the ectocolpus (Iversen & Troels-Smith 1950).

The species Solanum lycocarpum has been analyzed by several authors (Salgado-Labouriau 1973; Cruz-Barros et al. 2011; Du et al. 2017; Lorente et al. 2017), which describe the pollen grains as 3-colporate, long colpi with margo, lalongate endoaperture with margo and constriction (Cruz-Barros et al. 2011; Salgado-Labouriau 1973) and with conspicuous fastigium (Cruz-Barros et al. 2011); nevertheless, the authors report differences in exine ornamentation: psilate (Salgado-Labouriau 1973), rugulate (Cruz-Barros et al. 2011), granulate (Du et al. 2017) and microreticulate (Lorente et al. 2017). We observed margo in the colpi and endoaperture with costa, ornamented membrane, granulate in the colpus, endoaperture without constriction, and psilate ornamentation for S. lycocarpum.

Batista-Franklim & Gonçalves-Esteves (2008), Cruz-Barros et al. (2011), Silva et al. (2014), Kumar et al. (2015) and Lorente et al. (2017) have studied the species Solanum mauritianum and described 3-colporate pollen grains with margo (ornamented by Lorente et al. 2017), ornamented membrane in the colpus and median constriction in the endoaperture (Cruz-Barros et al. 2011), lalongate endoaperture with fastigium, without constriction (Batista-Franklim & Gonçalves-Esteves 2008), or with constriction and operculum in the endoaperture (Kumar et al. 2015) or forming a continuous ring (Silva et al. 2014) and granulate, microreticulate and spinulose ornamentation. Our results for S. mauritianum described margo present in the colpus and endoaperture with costa (without constriction) and microreticulate ornamentation, data similar to those of Cruz-Barros et al. (2011).

Some studies have depicted the pollen grains of Solanum paniculatum: Batista-Franklim & Gonçalves-Esteves (2008) described 3-colporate pollen grains with fastigium, colpus with thin margo, lalongate endoaperture, without constriction, and granulate exine. Cruz-Barros et al. (2011) observed ornamented membranes in the colpus and microreticulate exine, while Silva et al. (2016) reported costa in the endoaperture and psilate ornamentation with granulum when observed in SEM. Other morphological characteristics, such as the presence of a vestibulum and endocingulum (Silva et al. 2014), and scabrate ornamentation (Lorente et al. 2017) have also been described for the pollen grains of S. paniculatum. In our study we observed 3-colporate pollen grains with margo and ornamented membrane in the colpus, lalongate endoaperture and microreticulate ornamentation, corroborating the data of Cruz-Barros et al. (2011). Analyzing the definitions of the palynological glossaries (especially Punt et al. 2007) we did not observe structures like vestibulum and endocingulum for the pollen grains of this species.

Solanum pseudoquina pollen grains have been previously studied by Melhem et al. (2003), Batista-Franklim & Gonçalves-Esteves (2008) and Cruz-Barros et al. (2011), who have described 3-colporate pollen grains, with fastigium, lalongate endoaperture and slightly granulate ornamentation (Melhem et al. 2003), scabrate in LM and rugulate in SEM (Batista-Franklim & Gonçalves-Esteves 2008) and scabrate (Cruz-Barros et al. 2011). We observed fastigium, margo in the colpus, lalongate endoaperture without costa and psilate ornamentation for S. pseudoquina.

Du et al. (2017) presented the only study for Solanum ramulosum pollen grains, and the authors described small pollen grains and granulate ornamentation. We observed for this species variation between small and medium pollen grains and slightly granulate or psilate ornamentation, corroborating the data found by Du et al. (2017).

The qualitative data on pollen grains are similar among the species of the same genera; however, they allow the differentiation between Cestrum and Solanum, mainly due to the presence or absence of fastigium (which was confirmed by the quantitative data of the pollen grains). Exine ornamentation patterns can vary among the species of the studied genera: rugulate or striate exine was observed for Cestrum species and microreticulate or psilate exine (sometimes finely granulate) for Solanum pollen grains (see the artificial pollen key). Nonetheless, previous studies have shown that ornamentation patterns in Solanaceae pollen grains can be very variable.

As observed by the quantitative data (PCA and cluster analysis), the measurements of the pollen grain diameters, as well as the length and width of the apertures, helped to distinguish among the studied genera. Although there are other studies on Solanaceae palynology, it is the first time that the importance of these detailed quantitative data in the separation of Cestrum and Solanum is reported. Among the Solanum species, it is interesting to highlight the qualitative similarity of S. grandiflorum and S. ramulosum (artificial pollen key); nonetheless, we can differentiate the pollen grains of these species by higher endoaperture values (in S. ramulosum) and higher exine values (in S. grandiflorum).

When we compare the quantitative data of Solanaceae pollen grains with recent studies conducted in the same area (forest fragments from Cerrado), we can verify that the values of diameters combined with aperture measurements are also important for species segregation in Fridericia (Souza & Gasparino 2014), Malpighiaceae (Belonsi & Gasparino 2015) and Sapindaceae (Bellonzi et al. 2020). For Bignoniaceae (Souza et al. 2019) and Rubiaceae (Dutra et al. 2020) species, the pollen grain diameters associated with other metric data help the recognition of some taxa. The same is true when considering the measurements of apertures for the studies with Rutaceae (Dutra & Gasparino 2018) and Caesalpinioideae (Soares et al. 2021).

Therefore, we can conclude that: 1) The main characteristics that differentiate the genera in this study is the absence of fastigium and rugulate or striate exine ornamentation for pollen grains of Cestrum, and pollen grains with fastigium with microreticulate or psilate exine ornamentation for Solanum species; 2) Among the quantitative data, the values of pollen grain diameter allow the grouping of the species, with the Cestrum species having the highest values and the Solanum species, the lowest values. In addition, the width of the endoaperture and the length of the colpus are always greater in the Cestrum species when compared to the pollen grains of Solanum; 3) The morphological variation found in this study, besides being important in the characterization of the genera, confirm Solanaceae as an eurypalynous family.

Acknowledgements

The authors are grateful to the JABU and SJRP herbaria for the support in the collection of plant materials and the Laboratório de Microscopia Eletrônica, FCAV for the analysis in SEM.

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Publication Dates

• Publication in this collection
  04 Feb 2022
• Date of issue
  Oct-Dec 2021

History

• Received
  09 June 2020
• Accepted
  16 Mar 2021