Grana
ISSN: 0017-3134 (Print) 1651-2049 (Online) Journal homepage: https://www.tandfonline.com/loi/sgra20
Pollen morphology of Brazilian species of Vriesea
(Bromeliaceae, Tillandsioideae)
Valéria Leobina dos SantosVALÉRIA LEOBINA DOS SANTOS, Maria das
Graças Lapa WanderleyMARIA DAS GRAÇAS LAPA WANDERLEY, Leonardo M.
VersieuxLEONARDO M. VERSIEUX & Cynthia Fernandes Pinto da LuzCYNTHIA
FERNANDES PINTO DA LUZ
To cite this article: Valéria Leobina dos SantosVALÉRIA LEOBINA DOS SANTOS, Maria
das Graças Lapa WanderleyMARIA DAS GRAÇAS LAPA WANDERLEY, Leonardo M.
VersieuxLEONARDO M. VERSIEUX & Cynthia Fernandes Pinto da LuzCYNTHIA FERNANDES
PINTO DA LUZ (2019): Pollen morphology of Brazilian species of Vriesea (Bromeliaceae,
Tillandsioideae), Grana, DOI: 10.1080/00173134.2019.1676305
To link to this article: https://doi.org/10.1080/00173134.2019.1676305
Published online: 07 Nov 2019.
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Grana, 2019
https://doi.org/10.1080/00173134.2019.1676305
Pollen morphology of Brazilian species of Vriesea (Bromeliaceae,
Tillandsioideae)
VALÉRIA LEOBINA DOS SANTOS1, MARIA DAS GRAÇAS LAPA WANDERLEY2,
LEONARDO M. VERSIEUX3 & CYNTHIA FERNANDES PINTO DA LUZ 1
1
Núcleo de Pesquisa em Palinologia, Instituto de Botânica, São Paulo, Brazil, 2Núcleo de Pesquisa Curadoria do Herbário,
Instituto de Botânica, São Paulo, Brazil, 3Departamento de Botânica e Zoologia, Universidade Federal do Rio Grande do
Norte, Natal, Brazil
Abstract
As a contribution to the infrageneric delimitation in the genus Vriesea pollen morphology of 17 species of the genus is described,
with original data for 14 of the species. The pollen grains are heteropolar, with ellipsoidal amb, monosulcate, sulci marginate,
reticulate with calota microreticulate or psilate-perforate. Variations in the dimensions of the structures, presence or absence of
microreticula surrounding the lumina of the reticulum, type of ornamentation on the equatorial calota, difference in the exine
thickness, and walls simplecolumellate or duplicolumellate, have helped in the separation of groups, and were important for the
Palynotaxonomy of the genus. Proximity between species previously grouped by vegetative, reproductive and/or anatomical
structures similarity, was confirmed for V. friburgensis and V. neoglutinosa, and for V. medusa and V. nanuzae. Vriesea bituminosa
and V. neoglutinosa have demonstrated to share palynological similarities, although belonging to different sections, Xiphion and
Vriesea, respectively. The pollen grains of V. flava and V. philippocoburgii, both from section Vriesea, were very similar, despite
neither presenting similarities in the reproductive characters nor a close molecular phylogenetic relationship, thus suggesting a
convergent evolution for this character. Such findings confirm the weakness of the delimitations of these two sections reported
by previous studies. Our results allowed an improved morphological characterisation of genus Vriesea and could contributed to
a biossytematic approach towards the resolution of species complexes.
Keywords: Brazil, monocotyledons, palynotaxonomy, Poales, Vrieseinae
Vriesea (Tillandsoideae) is the third largest genus in
the Bromeliaceae, with approximately 226 species distributed between two sections: Vriesea and Xiphion
(Gouda et al. 2018 continously updated). The genus
includes mostly epiphytes, but also a fewer lithophytes
and terrestrials. Flowers have free petals, bearing a pair
of basal petal appendages, and convolute blade stigma
delimiting Vriesea from the remaining genera of Tillandsioideae (Smith & Downs 1977; Smith & Till
1998; Wanderley & Martins 2007; Costa et al. 2014;
Leme et al. 2017).
As traditionally defined, Vriesea has two centres of
diversity: one in northern South America, Central
America and Caribe; and the other in eastern Brazil
(Smith & Downs 1977; Costa et al. 2015). After a deep
systematic revision of the entire subfamily (see Barfuss
et al. 2016 for detailed rearrangements affecting Vriesea sensu lato), Vriesea has become now restricted to
eastern Brazil, where its richness is concentrated in the
eastern region, in the Atlantic Forest Domain. However, species are also found in the Amazon, Caatinga,
Campos Rupestres, Cerrado and Pampas (Costa et al.
2007, 2015; Wanderley & Martins 2007; Versieux &
Wanderley 2008; Forzza et al. 2015).
The two sections Vriesea and Xiphion have been reevaluated based on more recent phylogenetic studies
(Barfuss et al. 2005, 2016). These sections are traditionally separated by floral characteristics (Smith & Till
1998), and by the time of floral anthesis, and flower
scent (Leme et al. 2010, 2017). The typical section, V.
sect. Vriesea, is recognised by the presence of odourless
diurnal flowers, with ligulate petals and tubular corol-
Correspondence: Valéria Leobina dos Santos, Núcleo de Pesquisa em Palinologia, Instituto de Botânica, São Paulo, Brazil. E-mail: valerialeobina@hotmail.com
(Received 25 March 2019; accepted 21 July 2019)
© 2019 Collegium Palynologicum Scandinavicum
2
V. L. Santos et al.
las, brightly coloured floral bracts and calyx, varying
from red to yellow colour, exserted stamens, and pollination mainly by hummingbirds (Leme et al. 2010,
2017). In contrast, section Xiphion comprises species
with nocturnal flowers, oboval sepals, floral bracts
faintly coloured (e.g. green to brown), included stamens, and pollination mostly by bats (Leme et al.
2010, 2017). Both sections present an overlap in the
colour of the floral bracts and position of stamens in
respect to the corolla (Costa et al. 2007, 2015; Wanderley & Martins 2007; Gomes-da-Silva et al. 2012),
detected as homoplastic characters in the cladistic analysis of Gomes-da-Silva and Souza-Chies (2018).
Other studies (morphological/molecular phylogenies),
have also reported inconsistencies in the circumscription of Vriesea or both of its sections (Barfuss et al.
2005, 2016; Givnish et al. 2011; Versieux et al. 2012;
Gomes-da-Silva & Souza-Chies 2018).
A recent monograph for subfamily Tillandsioideae highlighted the importance of the morphological data in the recognition of the new genera
segregated from Vriesea sensu lato (Barfuss et al.
2016), mainly based on the ovary position, stigma,
ovule and seed morphology, but also highlighting
the importance of pollen data. Barfuss et al. (2016)
have also noted that the use of the name section
Xiphion presents a nomenclatural problem and
needs to be revised.
The pollen literature documents that the pollen
grains of Vriesea are monosulcate, reticulate heterobrochate, of lumina filled with granula or pila, and the
presence of equatorial microreticulate calota, characterising Vriesea as a stenopalynous genus (Mez 1934–
1935; Erdtman 1952; Wanderley & Melhem 1991;
Halbritter 1992; Costa 2002; Melhem et al. 2003;
Vervaeke et al. 2003; Souza et al. 2004, 2017; Moreira
2007; Silva et al. 2016). Nonetheless, these authors
analysed the pollen of a small number of species, often
represented only by electromicrographs and lacking
measurement of the structures. Wanderley and Melhem (1991) did present measurements and 12 species
of a preserved forested area in the State of São Paulo,
Brazil and distinguished differentiated pollen patterns
in eight species, regarding the ornamentation of the
exine and pollen size, while the remaining four species
were stenopalynous. Moreira (2007) demonstrated
that the four species of Vriesea separated in the pollen
key, mainly for the used details of the exine sculpture,
which helped in the taxonomic distinction to separate
these species. For Alcantarea, a genus very closely
related to the species of Vriesea here studied, Santos
et al. (2018) confirmed the existence of groupings
between the species. Leme et al. (2017) segregated
the new genus Waltillia from the former Alcantarea,
and also used the presence of a sulcus covered with a
kind of operculum of almost smooth exine elements
with some perforation.
The purpose of this work was to describe the pollen
morphology of a wide sampling of Brazilian species of
Vriesea, from areas of Atlantic Forest and Campos de
Altitude, or Campos Rupestres, including representatives from its two traditional sections (i.e. V. sect.
Vriesea and V. sect. Xiphion), to aid in the taxonomic
characterisation and verify if there is any grouping
between the species belonging to the same sections,
which in theory would have different pollinators (predominantly bats in V. sect. Xiphion and hummingbirds
in V. sect. Vriesea) appear grouped.
Material and methods
Pollen morphology of 17 Brazilian endemic species of
Vriesea were surveyed, represented by 32 specimens, of
which six taxa belong to section Vriesea (V. cacuminis, V.
flava, V. friburgensis, V. neoglutinosa, V. philippocoburgii,
V. stricta) and 11 in section Xiphion (V. atropurpurea, V.
bituminosa, V. itatiaiae, V. jonghei, V. longicaulis, V.
medusa, V. minarum, V. aff. minor, V. nanuzae, V. oligantha, V. pseudoatra). Among the analysed taxa, 14
species had not been palynologically described before.
Wanderley (1984), Wanderley and Melhem (1991),
Halbritter (1992) and Melhem et al. (2003) previously
described the pollen grains of V. bituminosa, Wanderley
and Melhem (1991) and Souza et al. (2017) of V.
friburgensis and Souza et al. (2004) of V. neoglutinosa.
Vriesea oligantha will possibly be transfered in the future
to the new genus Stigmatodon (see Barfuss et al. 2016;
Leme et al. 2017) but no formal taxonomical change
has been made for this species.
Flower buds were removed from specimens deposited in the following herbaria: ‘Maria Eneyda P. Kauffmann Fidalgo’ (acronym SP), Instituto de Botânica,
São Paulo (Brazil); ‘Jardim Botânico do Rio de
Janeiro’ (RB), Rio de Janeiro (Brazil); ‘Departamento
de Botânica da Universidade de São Paulo’ (SPF),
São Paulo (Brazil) and ‘Departamento de Botânica e
Zoologia da Universidade Federal do Rio Grande do
Norte’ (UFRN), Rio Grande do Norte (Brazil).
The pollen grains were treated with classic Erdman’s acetolysis (1960) and measured within three
days after preparation, to avoid swelling and standardise the size variations (Salgado-Labouriau et al.
1965), using an Olympus OSM-4 (10 ×) micrometre
drum coupled to the eyepiece of an Olympus BX50
light binocular microscope.
The axis length averages are based on measurements of 25 pollen grains per sample. Dimensions
of other morphological structures were obtained from
measurements of ten pollen grains per sample. Pollen
was removed from at least three specimens per taxon
Pollen morphology of Vriesea
to account for any possible intraspecific variation and
standardise size variations.
Statistical analyses included arithmetic average (x),
average standard deviation (sx), sample standard deviation (s), coefficient of variability (VC%) and 95% confidence interval (CI) (Vieira 1981). Three classes of
lumima size were established based on Santos et al.
(2018), according to the mininum and maximum limits
of the observed averages of all analysed specimens
(‘medium’ [2.25–3.44 μm], ‘large’ [3.45–5.00 μm]
and ‘very large’ [≥ 5.01 μm]), and additionally two
classes of width of reticulum muri were recognised
(‘narrow’ [≤ 0.89 μm] or ‘large’ [≥ 0.90 μm]).
To test whether these pollen grain characteristics
allow for the grouping of species, a principal component
analysis (PCA) was performed on 13 metric variables:
larger equatorial axis in polar view (VPEM), shorter
equatorial axis in polar view (VPEm), equatorial axis
in equatorial view (VEEM), polar axis in equatorial view
(VEEm), sulcus length (SULC), tectum thickness on
the calota (TETA) and on the central area of pollen
grain (TETC), height of the columellae on the calota
(SEXA) and on the central area of pollen grain (SEXC),
nexine thickness on the calota (NEXA) and on the
central area of pollen grain (NEXC), width of the
lumina (LUMC) and width of the muri (MURC).
Initially, Fitopac software (Shepherd 1996) was used
for conversion of the metric measurements of the pollen
grains by natural logarithm (log [x + 1]), and afterwards,
PC-ORD version 7 (McCune & Melfford 1999) was
used for ordination from the covariance matrix.
Photomicrographs were obtained digitally with an
Olympus BX50 light binocular microscope with a
video camera (Olympus U-CMAD-2) and the Olympus CellSens 1.5 software. For detailed observation of
the exine features, non-acetolysed pollen from the
same samples was dehydrated in an ascendant hydroalcoholic series using ethanol, dripping the material at
the end directly on the microscope stubs and sputtered
with a 20 nm thick gold layer in a sputter coating
system (Melhem et al. 2003). The scanning electromicrographs were obtained using a Philips XL 20
scanning electron microscope at the Laboratory of
Electron Microscopy of the Instituto de Botânica,
São Paulo, a Quanta FEG 250 FEI scanning electron
microscope from the Universidade Federal de São
Paulo and a Jeol JSM5800LV scanning electron
microscope from the Universidade de Campinas.
Pollen terminology follows Barth and Melhem
(1988), Punt et al. (2007) and Hesse et al. (2009).
In this study, the term ‘calota’ refers to the exine
ornamentation difference in the apexes of the equatorial axis, according to the definition of Robyns (1963)
for Bombacaceae (now included within Malvaceae)
and adopted by Santos et al. (2018) for Alcantarea
3
pollen. Microscopic slides are deposited in the palynotheca at the Núcleo de Pesquisa em Palinologia of
the Instituto de Botânica, São Paulo, Brazil.
Results
General description
Pollen grains range from medium to very large (Tables
I, II), the longest axis reaching 112.46 µm, ellipsoidal in
polar view (Figures 1A, B, F, G, I, 2A, F, 3A, B, D, E,
G, H, J, K, 4A, B, D, E, G, H, J, K, 5A, B, D, E, H, I,
6A, D, J, 7A, 8D, G, 9D, G, J), flattened/convex
(Figures 1C, H, J, K, 2B, C, G, 3C, I, L, 4C, I, 5C,
G, 7J) or biconvex (Figures 4F, L, 6G) in equatorial
view. Pollen is monosulcate with a long sulcus (Table
III). The sulcus margin is differentiated by its psilateperforate margin or reticula finer than the remainder of
the sexine on the central area of the pollen grains, and
with the same type of ornamentation as the equatorial
calota.
The sulcus is covered by an insulate type membrane
(Figures 2A, 3D, 5A, D, 6D, J, 7A, 8E, J, 9D) (the
sulcus is covered by few, or several, isolated or scattered
exine elements). The exine is semitectate, reticulate,
heterobrochate (Figures 1D, E, H, J, 2B, D, E, K, 3F,
I, 4C, K, 5C, F, J, 6A, B, E, G, H, K, 7B, D, E, H, J, K,
8A, B, D, G, H, K, 9A, B, E, G, H, J, K), with rounded
lumina (Figures 1J, 6H) or angulated (polygonals)
lumina (Figures 1C–E, H, 2B, D, E, K, 3C, D, I, 4C,
K, 5C, F, J, 6B, E, K, 7B, E, H, K, 8B, H, K, 9B, E, H,
K), with lumina presenting bacula (Figure 7D) or granula scattered within the lumina (Figures 6B, 7H, K, 8H,
K, 9E). Bacula inside the lumina were observed in 96%
of the pollen grains of Vriesea itatiaiae RB474136 and in
78% of those of V. jonghei RB474136. The muri are
straight (Figures 1C–E, 2D, E, 5F, J, 6B, E, K, 7B, K,
8B, H, K, 9E, H) or curved (Figures 1J, 6H), narrow or
broad (Table III), smooth (Figures 6E, 7H, 9H, K) or
with sparse perforations (Figures 6B, 8H, K, 9E), continuous (Figures 6B, 7B, 8K, 9H, K) or discontinuous
in some areas (Figures 6E, 7H, K). The sexine on the
central area of the pollen grains are thicker than the
sexine on the equatorial calota (Figures 1K, 2C, H, I,
L, 3E, K, L, 4B, D, E, 5B, E, G, K, L, Table III). In the
specimen RB193801 of V. oligantha we also observed a
very small frequency of abnormal pollen grains
(Figure 9A).
Two main pollen types could be recognised based on
the exine ornamentation on the equatorial calota. Given
the variation observed among species and specimens,
seven pollen subtypes were distinguished based on the
presence of simplecolumellate or duplicolumellate
muri, the dimensions of the lumina, thickness of the
sexine in the central area of the pollen grain and pre-
4
Larger equatorial axis in equatorial view (VEEM)
Species
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
atropupurea Versieux 296
bituminosa SP195325
bituminosa SP195516
cacuminis SP392004
cacuminis RB584635
flava RB329116
flava RB612972
flava RB612973
friburgensis SP225698
friburgensis SP333339
friburgensis SP340277
itatiaiae RB474136
itatiaiae RB565884
jonghei SP167550
jonghei RB46532
longicaulis SP169371
medusa SP412686
minarum RB193450
minarum RB324783
aff minor SP412846
nanuzae SP465860
neoglutinosa SP387922
oligantha RB193801
oligantha RB484319
philippocoburgii SP196610
philippocoburgii SP224892
philippocoburgii SP351897
pseudoatra SP382076
pseudoatra RB96088
stricta SP169988
stricta SP169994
stricta SP169995
Variation
89.80–112.46
66.97–82.80
63.47–79.97
65.97–80.47
56.64–71.14
66.64–78.47
66.64–79.14
69.97–79.97
64.14–90.13
63.47–75.80
66.64–78.80
38.48–65.64
63.31–72.30
64.81–86.47
66.64–79.97
72.14–86.13
66.64–77.47
83.30–95.80
64.97–79.97
66.64–83.30
64.14–77.47
62.14–90.96
72.80–93.30
73.97–90.63
59.58–89.13
66.64–84.30
62.48–82.13
66.64–83.30
66.64–79.97
69.97–83.30
56.48-.80.63
66.64–79.97
x ± sx
99.59
74.51
72.20
71.87
63.91
71.87
71.62
75.28
74.82
69.71
70.93
60.44
67.33
75.32
72.21
76.97
72.41
87.35
74.29
71.31
68.57
68.88
82.48
82.70
77.96
74.90
74.10
74.73
74.28
76.18
69.74
72.36
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.69
0.73
0.87
0.67
0.73
0.78
0.67
0.60
1.05
0.77
0.64
1.06
0.45
1.15
0.68
0.78
0.69
0.69
0.72
0.76
0.60
1.01
1.07
0.88
1.38
0.95
1.08
0.96
0.57
0.60
1.11
0.84
Shorter equatorial axis in equatorial view (VEEm)
s
VC (%)
CI
Variation
8.43
3.67
4.36
3.36
3.65
3.89
3.35
3.01
5.23
3.83
3.22
5.29
2.25
5.76
3.39
3.88
3.43
3.47
3.62
4.14
3.01
7.12
7.29
4.49
6.88
4.75
5.41
4.78
3.48
4.63
5.55
4.20
8.47
4.92
6.04
4.68
5.71
5.41
4.68
4.00
6.98
5.49
4.54
8.75
3.34
7.65
4.69
5.04
4.74
3.97
4.87
5.80
4.39
10.33
8.84
5.43
8.82
6.35
7.30
6.40
4.69
6.07
7.95
5.80
97.82–101.36
73.00–76.02
70.40–74.00
70.49–73.26
62.41–65.42
70.27–73.47
70.24–73.01
74.04–76.52
72.67–76.98
68.13–71.28
69.61–72.26
58.26–62.61
66.40–68.25
72.94–77.69
70.82–73.61
75.37–78.57
71.00 − 73.82
85.92–88.78
72.80 − 75.78
69.74–72.88
67.33–69.81
66.71–71.06
80.18–84.78
80.89–84.51
75.13–80.80
72.94–76.86
71.88–76.33
72.76–76.70
73.08–75.48
74.87–77.50
67.45–72.02
70.63–74.09
54.98–61.64
42.48–51.65
39.98–56.64
43.32–60.98
33.32–44.98
39.98–46.48
38.32–46.98
37.32–43.98
36.49–51.48
39.98–48.81
37.49–45.98
33.32–41.65
39.98–48.31
37.15–49.98
41.65–51.65
42.15–52.48
37.49–49.15
41.98–54.15
37.49–47.48
37.49–48.48
34.99–45.15
33.32–46.65
39.82–60.31
39.98–49.98
42.98–49.98
40.82–53.48
33.32–61.64
42.65–53.65
42.48–53.31
39.48–49.98
36.32–48.31
33.32–49.98
x ± sx
58.08
47.14
47.54
49.52
40.43
43.13
42.48
39.82
42.98
44.12
41.58
36.74
44.18
43.40
46.60
48.19
41.73
46.57
40.96
42.42
39.92
38.31
48.64
44.99
47.35
46.95
41.74
48.27
48.06
44.80
42.94
42.44
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.27
0.42
0.88
0.80
0.51
0.40
0.47
0.41
0.74
0.48
0.48
0.42
0.44
0.72
0.47
0.51
0.62
0.70
0.51
0.60
0.52
0.57
0.77
0.60
0.47
0.68
1.16
0.53
0.47
0.47
0.73
0.77
s
VC (%)
CI
3.29
2.11
4.40
4.01
2.55
2.00
2.37
2.03
3.70
2.39
2.41
2.12
2.20
3.59
2.33
2.57
3.11
3.51
2.53
3.26
2.58
4.04
5.26
3.08
2.33
3.40
5.79
2.66
2.90
3.61
3.65
3.84
5.66
4.48
9.25
8.09
6.30
4.64
5.58
5.10
8.61
5.41
5.81
5.77
4.98
8.27
5.00
5.32
7.46
7.53
6.18
7.70
6.47
10.56
10.81
6.85
4.93
7.23
13.87
5.50
6.03
8.07
8.51
9.05
57.39–58.77
46.27–48.01
45.73–49.35
47.87–51.17
39.38–41.48
42.31–43.95
41.50–43.45
38.99–40.66
41.45–44.50
43.14–45.11
40.59–42.58
35.87–37.61
43.28–45.09
41.92–44.88
45.64–47.56
47.14–49.25
40.45–43.01
45.12–48.01
39.92–42.01
41.18–43.66
38.86–40.99
37.07–39.54
46.98–50.29
43.75–46.23
46.39–48.31
45.55–48.35
39.36–44.13
47.18–49.37
47.06–49.06
43.77–45.83
41.44–44.45
40.85–44.02
Note: Arithmetic average (x), average standard deviation (sx), sample standard deviation (s), coefficient of variability (VC%) and confidence interval 95% (CI).
V. L. Santos et al.
Table I. Measures (μm) of Vriesea pollen grains in equatorial view through light microscopy (n = 25).
Table II. Measures (μm) of Vriesea pollen grains in polar view through light microscopy (n = 25).
Larger equatorial axis in polar view (VPEM)
Species
atropupurea Versieux 296
bituminosa SP195325
bituminosa SP195516
cacuminis SP392004
cacuminis RB584635
flava RB329116
flava RB612972
flava RB612973
friburgensis SP225698
friburgensis SP333339
friburgensis SP340277
itatiaiae RB474136
itatiaiae RB565884
jonghei SP167550
jonghei RB46532
longicaulis SP169371
medusa SP412686
minarum RB193450
minarum RB324783
aff minor SP412846
nanuzae SP465860
neoglutinosa SP387922
oligantha RB193801
oligantha RB484319
philippocoburgii SP196610
philippocoburgii SP224892
philippocoburgii SP351897
pseudoatra SP382076
pseudoatra RB96088
stricta SP169988
stricta SP169994
stricta SP169995
81.63–96.63
69.97–79.30
62.81–78.14
63.31–82.63
56.64–73.47
66.64–79.97
66.64–78.30
68.47–81.63
64.64–79.97
62.14–75.64
66.64–76.64
54.15–64.14
60.31–70.64
64.47–83.30
66.64–78.97
70.81–88.13
63.47–81.80
83.30–95.80
66.64–81.13
68.14–79.14
61.64–78.30
54.98–69.97
69.14–99.96
71.64–87.30
63.31–86.47
66.81–83.30
66.31–83.30
66.31–83.30
69.14–80.63
66.64–85.80
63.81–78.64
66.64–79.14
x ± sx
89.32
74.68
71.78
70.88
65.10
72.76
70.04
75.01
73.67
69.77
69.99
58.67
66.03
74.20
71.69
77.68
73.02
87.35
73.32
73.20
69.27
65.04
81.86
80.79
76.82
75.12
72.35
73.30
73.49
75.00
70.04
71.16
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.62
0.43
0.68
1.00
0.60
0.72
0.70
0.75
0.75
0.69
0.65
0.62
0.54
1.00
0.65
0.89
0.77
0.70
0.70
0.60
0.70
0.56
1.20
0.66
1.12
0.91
1.02
1.04
0.59
1.04
0.89
0.68
s
VC (%)
CI
Variation
5.36
2.13
3.41
4.99
3.94
3.59
3.49
3.73
3.77
3.43
3.27
3.09
2.69
4.98
3.25
4.46
3.84
3.51
3.51
2.98
3.51
4.60
7.62
4.04
5.59
4.55
5.12
5.19
2.95
6.36
4.43
3.38
6.00
2.85
4.75
7.04
6.05
4.94
4.99
4.98
5.11
4.92
4.67
5.27
4.08
6.71
4.54
5.74
5.25
4.02
4.79
4.07
5.07
7.07
9.31
5.00
7.27
6.06
7.08
7.08
4.01
8.47
6.32
4.75
87.92–90.71
73.80–75.55
70.37–73.18
68.83–72.94
63.82–66.38
71.28–74.24
68.60–71.48
73.47–76.55
72.12–75.22
68.36–71.19
68.64–71.33
57.40–59.94
64.92–67.14
72.15–76.26
70.35–73.03
75.84–79.51
71.44 − 74.60
85.91–88.80
71.88 − 74.77
71.97–74.42
67.82–70.71
63.78–66.30
79.31–84.42
79.40–82.18
74.51–79.12
73.24–76.99
70.24–74.46
71.16–75.44
72.28–74.41
72.81–77.19
68.21–71.86
69.76–72.55
49.98–62.48
44.98–54.15
35.82–56.64
44.65–61.14
38.32–49.98
41.65–60.48
41.48–49.98
38.32–49.15
39.15–64.97
42.98–51.81
38.32–52.15
33.32–43.98
41.15–53.31
40.82–64.31
38.32–55.48
47.48–59.98
39.48–53.31
42.98–57.14
39.15–49.98
39.82–49.98
37.49–47.31
35.65–49.98
44.15–59.98
42.15–49.98
43.82–63.31
42.32–81.30
36.82–49.98
44.98–58.81
46.65–56.48
42.48–52.48
38.98–56.64
38.32–52.48
x ± sx
56.57
49.48
48.81
52.30
44.59
46.81
46.23
42.68
48.68
48.53
46.74
39.02
47.44
47.83
48.05
50.47
45.96
49.53
44.32
45.36
41.83
40.53
52.92
46.73
49.89
53.03
41.98
50.92
51.50
48.68
45.85
45.52
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.59
0.45
0.95
0.84
0.53
0.77
0.59
0.51
1.07
0.53
0.71
0.58
0.61
1.12
0.63
0.50
0.73
0.62
0.50
0.64
0.50
0.54
0.59
0.51
0.79
1.66
0.77
0.75
0.50
0.50
0.80
0.79
s
VC (%)
CI
5.10
2.25
4.74
4.18
3.50
3.84
2.95
2.56
5.34
2.65
3.54
2.88
3.03
5.61
3.17
2.50
3.65
3.09
2.51
3.22
2.52
4.40
3.73
3.11
3.97
8.32
3.84
3.74
2.52
3.07
3.99
3.97
9.01
4.54
9.71
7.98
7.85
8.20
6.39
5.99
10.97
5.47
7.57
7.38
6.39
11.73
6.60
4.96
7.95
6.25
5.67
7.10
6.01
10.85
7.05
6.66
7.95
15.69
9.15
7.35
4.90
6.30
8.70
8.72
55.24–57.90
48.56–50.41
46.86–50.76
50.58–54.02
43.45–45.73
45.23–48.39
45.02–47.45
41.63–43.74
46.48–50.88
47.44–49.63
45.28–48.20
37.83–40.20
46.19–48.69
45.52–50.14
46.75–49.36
49.44–51.50
44.46–47.47
48.26–50.81
43.28–45.35
44.04–46.69
40.79–42.87
39.33–41.73
51.67–54.17
45.65–47.80
48.25–51.52
49.60–56.45
40.39–43.56
49.38–52.46
50.46–52.54
47.62–49.73
44.21–47.49
43.89–47.16
Note: Arithmetic average (x), average standard deviation (sx), sample standard deviation (s), coefficient of variability (VC%) and confidence interval 95% (CI).
Pollen morphology of Vriesea
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
Variation
Shorter equatorial axis in polar view (VPEm)
5
6
V. L. Santos et al.
Figure 1. Light microscopy (LM) images of pollen grains of Vriesea. A–E. Vriesea atropurpurea Silveira Versieux 296 (SPF). A. Polar view,
aperture. B. Polar view, optical section. C. Equatorial view, surface. D. LO1 (high focus). E. LO2 (low focus). F–H. Vriesea bituminosa
Wawra. F. Polar view, aperture, SP195325. G. Polar view, optical section, SP195325. H. Equatorial view, surface, SP195516. I–L.
Vriesea cacuminis L.B. Sm. SP392004. I. Polar view, aperture. J. Equatorial view, surface. K. Equatorial view, optical section. L. Frontal
equatorial view, aperture. Scale bars – 10 µm (A–C, F–L), 2 µm (D, E).
sence or absence of a microreticulum surrounding the
lumina. The presence or absence of bacula and granules
within the lumina, the presence or absence of perforations on the muri and continuous versus discontinuous
muri were not used in the typification, because of the
great variation in these characters that was observed on
the same specimen.
Twenty-five specimens exhibited granules within the
lumina on some pollen grains: Vriesea atropurpurea
(100%), V. bituminosa SP195325 (64%), V. cacuminis
Pollen morphology of Vriesea
7
Figure 2. Light microscopy (LM) images of pollen grains of Vriesea. A–E. Vriesea flava A.F. Costa. A. Polar view, aperture,
RB612973. B. Equatorial view, surface, RB612973. C. Equatorial view, optical section, RB612973. D. LO1 (high focus),
RB612972. E. LO2 (low focus), RB612972. F–I. Vriesea friburgensis Mez. F. Polar view, aperture, SP333339. G. Equatorial view,
optical section, SP225698. H. Calota, optical section, SP333339. I. Central area of pollen grain, optical section, SP333339. J–L.
Vriesea itatiaiae Wawra. RB565884. J. Polar view, aperture. K. Equatorial view, surface. L. Equatorial view, optical section. Scale
bars – 10 µm (A–C, F, G, J–L), 2 µm (D, E, H, I).
SP392004 (10%), V. cacuminis RB584635 (69%), V.
flava RB612972 (62%), V. flava RB612973 (91%), V.
flava RB329116 (94%), V. friburgensis SP225698
(86%), V. friburgensis SP340277 (68%), V. friburgensis
SP333339 (80%), V. jonghei SP46532 (60%), V. longiscaulis SP169371 (94%), V. medusa SP412656 (16%),
V. minarum RB324783 (72%), V. minarum RB193450
(84%), V. aff minor SP412846 (100%), V. nanuzae
8
V. L. Santos et al.
Figure 3. Light microscopy (LM) images of pollen grains of Vriesea. A–C. Vriesea jonghei (K.Koch) E. Morren. A. Polar view, aperture,
SP46532. B. Polar view, optical section, SP46532. C. Equatorial view, optical section, SP167550. D–F. Vriesea longicaulis (Baker) Mez.
SP169371. D. Polar view, aperture. E. Polar view, optical section. F. Equatorial view, optical section. G–I. Vriesea medusa Versieux.
SP412656. G. Polar view, aperture. H. Polar view, optical section. I. Equatorial view, optical section. J–L. Vriesea minarum L.B. Sm.
RB324783. J. Polar view, aperture. K. Polar view, optical section. L. Equatorial view, optical section. Scale bars – 10 µm.
SP465860 (82%), V. neoglutinosa SP387922 (65%), V.
oligantha RB484319 (100%), V. philippocoburgii
SP224892 (76%), V. philippocoburgii SP196610
(80%), V. philippocoburgii SP351897 (100%), V. pseudoatra SP382076 (100%), V. stricta SP169988 (66%)
and V. stricta SP169995 (92%). Three specimens presented a combination of granules and bacula inside the
lumina of some pollen grains: V. jonghei SP167550
(78%), V. itatiaiae RB474136 (100%) and V. itatiaiae
RB565884 (100%).
Pollen morphology of Vriesea
9
Figure 4. Light microscopy (LM) images of pollen grains of Vriesea. A–C. Vriesea aff. minor (L.B. Sm.) Leme. SP412846. A. Polar view,
aperture. B. Polar view, optical section. C. Equatorial view, optical section. D–F. Vriesea nanuzae Leme. SP465860. D. Polar view,
aperture. E. Polar view, optical section. F. Equatorial view, optical section. G–I. Vriesea neoglutinosa Mez. SP387922. G. Polar view,
aperture. H. Polar view, optical section. I. Equatorial view, surface. J–L. Vriesea oligantha (Baker) Mez. RB193801. J. Polar view,
aperture. K. Polar view, optical section. L. Equatorial view, surface. Scale bars – 10 µm.
Description of the pollen types
Type I – Reticulate exine on the central area, psilate-perforate calota – Subtype Ia. — Muri simplecolumellate
(Figure 2D, E), without microreticulum surrounding
the lumina in some areas, sexine thickness on the
central area 1.03–2.42 μm (Table III). Lumina predominantly medium sized and angular; muri narrow and
straight: Vriesea minarum RB193450, V. aff. minor
10
V. L. Santos et al.
Figure 5. Light microscopy (LM) images of pollen grains of Vriesea. A–C. Vriesea philippocoburgii Wawra. SP224892. A. Polar view,
aperture. B. Polar view, optical section. C. Equatorial view, optical section. D–G. Vriesea pseudoatra Leme. D. Polar view, aperture,
RB96088. E. Polar view, optical section, RB96088. F. Equatorial view, surface, SP382076. G. Equatorial view, optical section,
SP382076. H–L. Vriesea stricta L.B. Sm. SP169995. H. Polar view, aperture. I. Polar view, optical section. J. Equatorial view, surface.
K. Calota, optical section. L. Central area of pollen grain, optical section. Scale bars – 10 µm (A–J), 2 µm (K, L).
SP412846, V. stricta SP169988. Lumina predominantly medium sized and angular; muri wide and
straight: V. itatiaiae RB474136, V. oligantha
RB193801. Lumina predominantly large and angular;
muri narrow and straight: V. jonghei SP46532. Lumina
predominantly large and angular; muri wide and
straight: V. pseudoatra SP382076, V. stricta SP169995.
Type I – Reticulate exine on the central area, psilate-perforate calota – Subtype Ib. — Muri duplicolumellate,
Pollen morphology of Vriesea
11
Figure 6. Scanning electron microscopy (SEM) images of pollen grains of Vriesea. A–C. Vriesea atropurpurea Silveira. Versieux 296 (SPF).
A. Proximal polar view. B. Ornamentation in the central area. C. Psilate-perforate calota. D–F. Vriesea bituminosa Wawra. SP195516. D.
Distal polar view, aperture. E. Ornamentation in the central area. F. Microreticulate calota. G–I. Vriesea cacuminis L.B. Sm. G. Equatorial
view, RB584635. H. Ornamentation in the central area, SP392004. I. Microreticulate calota, RB584635. J–L. Vriesea flava A.F. Costa.
RB612972. J. Distal polar view, aperture. K. Ornamentation in the central area. L. Microreticulate calota. Scale bars – 20 µm (A, D, G,
J), 10 µm (C, F, I, L), 5 µm (B, E, K), 1 µm (H).
without microreticula surrounding the lumina, sexine
thickness on the central area of 1.66 µm (Table III).
Lumina predominantly medium sized and angular;
muri wide and straight: Vriesea jonghei SP167550.
Comments. — In Vriesea jonghei SP167550, we
observed dimorphism in the ornamentation of the
calota; therefore, this specimen was also characterised as Type II.
12
V. L. Santos et al.
Figure 7. Scanning electron microscopy (SEM) images of pollen grains of Vriesea. A–C. Vriesea friburgensis Mez. A. Distal polar view,
aperture, SP225698. B. Ornamentation in the central area, SP340277. C. Microreticulate calota, SP340277. D–F. Vriesea itatiaiae
Wawra. RB474136. D. Proximal polar view. E. Ornamentation in the central area. F. Psilate-perforate calota. G–I. Vriesea jonghei (K.
Koch) E. Morren. G. Distal polar view, aperture, SP167550. H. Ornamentation in the central area, SP46532. I. Psilate-perforate calota,
SP167550. J–L. Vriesea longicaulis (Baker) Mez. SP169371. J. Equatorial view. K. Ornamentation in the central area. L. Microreticulate
calota. Scale bars – 30 µm (D), 20 µm (G, J), 10 µm (A, C, F, H, I, L), 5 µm (B, E, K).
Type I – Reticulate exine on the central area, psilateperforate calota – Subtype Ic. — Muri duplicolumellate (Figure 1D, E), with microreticulum surrounding the lumina in some areas (Figure 1C),
sexine thickness in the central area 1.32 µm
(Table III). Lumina predominantly large and
angular, muri narrow and straight: Vriesea atropurpurea Versieux 296.
Pollen morphology of Vriesea
13
Table III. Arithmetic average of measures (μm) of the lumina, muri, sulcus and thickness of the exine layer of Vriesea pollen grains through
light microscopy (n = 10).
Species
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
atropurpurea Versieux 296
bituminosa SP195325
bituminosa SP195516
cacuminis SP392004
cacuminis RB584635
flava RB329116
flava RB612972
flava RB612973
friburgensis SP225698
friburgensis SP333339
friburgensis SP340277
itatiaiae RB474136
itatiaiae RB565884
jonghei SP167550
jonghei SP46532
longicaulis SP169371
medusa SP412686
minarum RB193450
minarum RB324783
aff minor RB324783
nanuzae RB465860
neoglutinosa SP387922
oligantha RB193801
oligantha RB484319
philippocoburgii SP196610
philippocoburgii SP224892
philippocoburgii SP351897
pseudoatra SP382076
pseudoatra RB96088
stricta SP169988
stricta SP169994
stricta SP169995
Thickness of the exine layer
at the equatorial calota
Thickness of the exine layer at
the central area of pollen
grains
Lumina and muri at
the central area of
pollen grains
TETA
SEXA
NEXA
TETC
SEXC
NEXC
LUMC
MURC
SULC
0.27
0.26
0.23
0.40
0.21
0.22
0.21
0.23
0.22
0.16
0.19
0.28
0.16
0.32
0.36
0.35
0.30
0.30
0.33
0.32
0.25
0.27
0.32
0.26
0.24
0.25
0.26
0.25
0.27
0.29
0.29
0.32
0.71
1.15
0.97
1.70
0.82
0.90
0.85
0.92
0.92
0.64
0.92
0.81
0.90
0.87
1.01
0.99
0.91
0.81
0.86
0.89
1.22
0.68
1.33
1.16
1.18
1.08
1.01
1.27
1.43
0.80
0.79
0.64
0.48
0.73
0.56
0.78
0.55
0.61
0.67
0.63
0.58
0.42
0.60
0.57
0.55
0.66
0.79
0.79
0.63
0.60
0.65
0.66
0.85
0.49
0.60
0.61
0.55
0.56
0.57
0.59
0.69
0.65
0.65
0.63
0.56
1.16
0.69
0.73
0.37
0.58
0.42
0.51
0.45
0.36
0.64
0.47
0.71
0.67
0.91
0.71
0.61
0.62
0.74
0.69
0.83
0.68
0.61
0.42
0.76
0.89
0.61
0.85
0.78
0.65
0.62
0.37
0.76
1.44
1.00
1.80
0.94
0.93
0.97
0.94
1.02
0.91
1.00
0.91
0.92
0.99
1.20
1.10
0.94
0.94
0.95
1.12
1.28
0.78
1.81
1.20
1.32
1.23
1.14
1.38
1.50
1.00
0.97
0.66
0.54
0.77
0.59
0.81
0.64
0.68
0.71
0.75
0.64
0.67
0.73
0.72
0.88
0.70
0.91
0.88
0.66
0.68
0.69
0.79
0.91
0.62
0.77
0.72
0.75
0.60
0.68
0.68
0.74
0.77
0.72
0.37
4.92
2.74
4.70
5.33
2.25
3.42
3.61
3.35
3.09
2.77
3.85
2.64
3.80
3.28
4.95
3.13
3.35
3.43
3.29
2.81
3.58
3.35
2.98
3.50
4.56
4.76
2.49
4.15
4.43
3.01
3.02
3.76
0.87
0.77
0.93
0.98
0.77
0.67
0.85
0.76
0.75
0.57
0.81
1.02
0.82
0.90
0.87
0.83
0.89
0.83
1.09
0.78
0.95
0.57
0.93
1.04
0.63
0.68
0.59
1.02
0.85
0.67
0.85
0.94
78.02
70.67
66.01
63.99
60.58
68.79
65.99
71.34
67.49
69.14
68.89
56.91
61.08
67.56
67.99
73.45
69.36
83.75
68.71
71.14
64.92
65.14
80.63
74.15
71.45
69.17
69.39
67.87
70.92
69.39
64.14
67.72
Sulcus length
Note: TETA and TETC, thickness of the tectum; SEXA and SEXC, high of the columellae; NEXA and NEXC, thickness of the nexine;
LUMC, width of the lumina; MURC, width of the muri; SULC, length of the sulcus.
Type II – Reticulate exine on the central area, microreticulate calota – Subtype IIa. — Muri simplecolumellate, without microreticulum surrounding the
lumina, sexine thickness on the central area 1.27–
2.60 µm (Table III). Lumina predominantly medium sized and angular, muri narrow and straight:
Vriesea bituminosa SP195325, V. friburgensis
SP333339, V. medusa SP412656, V. neoglutinosa
SP387922. Lumina predominantly large and
angular, muri narrow and straight: V. friburgensis
SP340277, V. itatiaiae RB565884, V. pseudoatra
RB96088. Lumina predominantly large and angular, muri wide and straight: V. bituminosa
SP195516. Lumina predominantly large and
angular, muri wide and straight to curved: V.
nanuzae SP465860. Lumina predominantly very
large and rounded, muri wide and curved: V.
cacuminis SP392004.
Type II – Reticulate exine on the central area, microreticulate calota – Subtype IIb. — Muri simplecolumellate,
with microreticulum surrounding the lumina in some
areas (Figures 2B, D, E, 9E), or lumina predominantly
surrounded by microreticulum, sexine thickness in the
central area 1.39–2.43 µm (Table III). Lumina predominantly medium sized and angular, muri narrow
and straight: Vriesea flava RB612973, V. flava
RB329116, V. friburgensis SP225698, V. philippocoburgii SP351897. Lumina predominantly large and angular, muri narrow and straight: V. flava RB612972, V.
philippocoburgii SP224892, V. philippocoburgii
SP196610.
Type II – Reticulate exine on the central area, microreticulate calota – Subtype IIc. — Muri duplicolumellate, without microreticulum surrounding the
lumina, sexine thickness on the central area 1.31–
14
V. L. Santos et al.
Figure 8. Scanning electron microscopy (SEM) images of pollen grains of Vriesea. A–C. Vriesea minarum L.B. Sm. A. Equatorial
view, RB193450. B. Ornamentation in the central area, RB324783. C. Microreticulate calota, RB324783. D–F. Vriesea aff. minor
(L.B. Sm.) Leme. SP412846. D. Proximal polar view. E. Insulae type sulcus membrane. F. Psilate-perforate calota. G–I. Vriesea
nanuzae Leme. SP465860. G. Proximal polar view. H. Ornamentation in the central area. I. Microreticulate calota. J–L. Vriesea
neoglutinosa Mez. SP387922. J. Sub-equatorial view. K. Ornamentation in the central area. L. Microreticulate calota. Scale bars –
30 µm (A), 20 µm (D, G, J), 10 µm (C, E, F, H, I, L), 5 µm (B, K).
1.81 µm (Table III). Lumina predominantly medium
sized
and
angular,
muri
narrow
and straight: Vriesea cacuminis RB584635, V. longiscaulis SP169371, V. stricta SP169994. Lumina
predominantly medium and angular, muri wide
and straight: V. jonghei SP167550. Lumen predominantly large and angular, muri wide and straight: V.
oligantha RB484319.
Pollen morphology of Vriesea
15
Figure 9. Scanning electron microscopy (SEM) images of pollen grains of Vriesea. A–C. Vriesea oligantha (Baker) Mez. A. Distal polar
view, aperture, very small abnormal pollen grain, RB193801. B. Ornamentation in the central area, RB484319. C. Microreticulate calota,
RB484319. D–F. Vriesea philippocoburgii Wawra. D. Distal polar view, aperture, SP224892. E. Ornamentation in the central area,
SP351897. F. Microreticulate calota, SP196610. G–I. Vriesea pseudoatra Leme. SP382076. G. Proximal polar view. H. Ornamentation
in the central area. I. Psilate-perforate calota. J–L. Vriesea stricta L.B. Sm. J. Proximal polar view, SP169988. K. Ornamentation in the
central area, SP169988. L. Psilate-perforate calota, SP169995. Scale bars – 20 µm (A, J), 10 µm (C, D, F, G, L), 5 µm (B, K), 2 µm (E),
1 µm (H, I).
Type II – Reticulate exine on the central area, microreticulate calota – Subtype IId. — Muri duplicolumellate, with microreticulum surrounding the
lumina in some areas (Figure 8B), sexine thick-
ness on the central area 1.69 µm (Table III).
Lumina predominantly medium sized and angular, muri wide and straight: Vriesea minarum
RB324783.
16
V. L. Santos et al.
Figure 10. Principal component analysis (PCA) performed with the pollen metrical variables from Vriesea. 1. Vatr, V. atropurpurea; 2. Vbit,
V. bituminosa; 3. Vcac, V. cacuminis; 4. Vfla, V. flava; 5. Vfri, V. friburgensis; 6. Vita, V. itatiaiae; 7. Vjon, V. jonghei; 8. Vlon, V. longiscaulis;
9. Vmed, V. medusa; 10. Vmin, V. minarum; 11. Vmir, V. aff. minor; 12. Vnan, V. nanuzae; 13. Vneo, V. neoglutinosa; 14. Voli, V.
oligantha; 15. Vphi, V. philippocoburgi; 16. Vpse, V.pseudoatra; 17. Vstr, V. stricta.
Principal component analysis (PCA)
Four groups of Vriesea pollen grains were formed by the
PCA based on the 13 metric variables (Figure 10). The
pollen variability among the species and specimens
comprised 64.05% on the two first axis in the PCA.
The first axis summed 42.99% of variance according to
the highly correlated values (> 0.01) especially polar
axis in equatorial view (VEEm), shorter equatorial axis
in polar view (VPEm), width of the lumina (LUMC),
thickness of the tectum on the central area (TETC)
and width of the muri (MURC) (Table IV).
The second component explained 21.06% of the
variability of the data, in which the most important
variables (> 0.02) for ordination of specimens (Table
IV) were: equatorial axis in equatorial view (VEEM),
the larger equatorial axis in polar view (VPEM),
length of sulci (SULC), high of the columellae on
the central area of the pollen grains (SEXC), the
equatorial calota (SEXA), nexine thickness on the
central area (NEXC) and on the calota (NEXA)
and on the calota (TETA) of the pollen grains.
On the bottom right side of the graph, the two
specimens with higher values of sexine (height of the
columellae on the centre of the pollen grain and on
the apex of the calota, and thickness of the tectum
on the centre of the pollen grain) were ordered.
Pollen morphology of Vriesea
Table IV. Pearson and Kendall correlation coefficients for pollen
grains metric variables of the first and the second axis of principal
component analysis (PCA) ordination in Vriesea Lindl.
Principal
components
Variables
VPEM
VPEm
VEEM
VEEm
SEXA
TETA
NEXA
SEXC
TETC
NEXC
LUMC
MURC
SULC
Variables (meaning)
Axis 1
Axis 2
Major equatorial axis in polar
view
Minor equatorial axis in polar
view
Equatorial axis in equatorial
view
Polar axis in equatorial view
Height of the columellae at
the equatorial calota
Tectum thickness at the
equatorial calota
Nexine thickness at the
equatorial calota
Height of the columellae at
the centre area of the pollen
grain
Tectum thickness at the
centre area of the pollen
grain
Nexine thickness at the centre
area of the pollen grain
Lumen width at the centre
area of the pollen grain
Murus width at the centre
area of the pollen grain
Length of sulcus
0.1903
0.2618
0.2684
0.1815
0.2023
0.3410
0.3069
0.3842
0.2173
−0.4091
0.0164
−0.0257
0.0931
−0.1794
0.3817
−0.4797
0.2850
−0.2551
0.0828
−0.2857
0.5815
0.3331
0.0915
−0.0507
0.1405
0.2107
In contrast, on the top left side, the specimens
with the lowest values of these characteristics were
ordered. Vriesea stricta (SP169995) presented the
lowest dimensions of the exine, among the analysed
pollen grains. The specimens that were ordered on
the positive side of the second axis presented the
largest pollen grains. V. atropurpurea Versieux 296
was the specimen with the largest dimensions, while
V. itatiaiae (RB474136) was ordered on the botton
left side of the graph for presenting the smallest
pollen grains (Figure 10).
Discussion
Vriesea is considered a stenopalynous genus, based
on the reticulate ornamentaion and monosulcate
aperture, as was described for Tillandsioideae by
Mez (1934–1935), Erdtman (1952), Ehler and
Schill (1973), Halbritter (1992) and Moreira
(2007). However, the data obtained in the present
investigation indicate variations in the ornamentation of the equatorial calota, which, together with
other characteristics, can be used for grouping some
species by similarity.
17
Most species could not be separated by the size of
the pollen grains, due to an overlap of the measurements, in which the variation of the minimum and
maximum values of the equatorial axis in equatorial
view and longest equatorial axis in polar view ranged
between 38.48–112.46 μm and 54.15–99.96 μm,
respectively.
In most species, it was possible to analyse several
specimens, which allowed documenting intraspecific
variation. Only in Vriesea atropurpurea, V. longicaulis,
V. medusa, V. aff. minor, V. nanuzae and V. neoglutinosa a single specimen was analysed. Of the 32 analysed specimens of Vriesea, ten (31%) exhibited
psilate-perforate ornamentation on the equatorial
calota, while 22 (69%) presented microreticulate
calota. The simplecolumellate muri prevailed in a
total of 25 specimens (78%), while seven (22%)
were duplicolumellate. In 20 specimens (63%), no
microreticulum were found surrounding the lumina,
while in 12 others (37%), they were present. In nine
specimens of Vriesea (28%), the measurements
obtained for the sexine thickness on the central area
of the pollen grain were above 2 μm, while in 23
specimens (72%), they were lower than this.
Some pollen characters varied on the same plant,
and among specimens of the same species, such as
the presence or absence of bacula or granules inside
the lumina, and muri continuous or discontinuous
in some areas of the pollen grains.
In comparison with the data obtained in the literature, Wanderley (1984) and Wanderley and Melhem (1991) described the pollen grains of Vriesea
bituminosa as having duplicolumellate muri, and an
average exine thickness of 2.5 μm, which diverged
from the present study, since the two specimens
investigated here exhibited simplecolumellate muri,
and thinner exine. Halbritter (1992) used electromicrographs for analysing the pollen grains of V. bituminosa and described the exine ornamentation as
reticulate and the sulcus as presenting an insulate
type membrane, which corroborated our observations in both specimens here analysed. This species
was also analysed by Melhem et al. (2003); however,
the specimen analysed was the same which had been
described in Wanderley (1984) and Wanderley and
Melhem (1991).
Wanderley and Melhem (1991) characterised the
pollen grains of Vriesea friburgensis as presenting
reticulum with small lumina, with pila inside and
muri large, perforate and simplecolumellate. In the
three specimens here analysed, the lumina were
medium sized, and the muri narrow and simplecolumellate, with pila inside the lumina, but presenting
granules. Souza et al. (2017) performed lactic acetolysis to treat the pollen material of V. friburgensis
18
V. L. Santos et al.
(ESA121282) and measured it using an image digitisation software, describing them as large size
(87.77 μm). They reported also the presence of
granules on the inside of lumina, with reticula network decreased towards the equatorial apex of the
pollen grains. These data corroborate in part our
results, since the pollen grains were also characterised as large, although with lower average values
(69.71 μm, 70.93 μm and 74.82 μm, respectively, in
the three specimens here analysed). This fact can be
explained for the pollen measurements having been
made based on photographs, which can generate
inconsistencies in the limits of the exine layers,
unlike in this study, where the micrometric drum
coupled to the binocular microscope was used,
which offers higher visual precision to the observer.
The presence of microreticulum surrounding the
lumina in some areas was also not mentioned by
the earlier-mentioned authors.
Both species cited earlier can be considered of
wide distribution in Brazil, and part of taxonomic
complexes, presenting phenotypic polymorphisms
or distinct varieties in the case of Vriesea friburgensis
(Smith 1952; Moura & Costa 2014), what could
explain in part such variation reported for their pollen grains in different works.
Souza et al. (2004) described the pollen grains of
Vriesea neoglutinosa as medium sized, in contrast with
the large size pollen grains found in the present analysis. These differences in the pollen dimensions can be
explained by the different chemical treatments
adopted: lactic acetolysis (ACLAC) by Raynal and
Raynal (1971) cited in Souza et al. (2004) and Erdtman’s (1960) acetolyis in the present study. It is
reported that acetolysis increases the pollen grain size
in relation to ACLAC. The cited authors neither
documented the presence of exine elements inside
the lumina nor microreticulum surrounding them, as
here described. Our results and those of Souza et al.
(2004) characterise the sexine as thicker than the nexine, however, Souza et al. (2004) show almost identical thickness values for the sexine and nexine, while in
the present study, the sexine value was much higher to
that of the nexine.
Vriesea friburgensis and V. neoglutinosa are related by
the similarities in the reproductive structures (Wanderley & Martins 2007). The pollen grains of the three
specimens of V. friburgensis presented great morphological similarity, with microreticulate calota and simplecolumellate muri, with only one specimen
(SP225698) differing for the present of microreticulum surrounding the lumina. The pollen grains of V.
neoglutinosa were classified in the same pollen type as
V. friburgensis, and, this way, the palynology supports
the morphological affinity between the two taxa, both
representing section Vriesea, and are bird pollinated.
Vriesea atropurpurea, V. bituminosa, V. jonghei, V.
minarum and V. minor (section Xiphion) are phylogenetically closely related species by the inflorescence characters (Costa et al. 2015). Also,
according to Barfuss et al. (2005), V. bituminosa
and V. jonghei are closely related species based on
DNA sequences. The present study demonstrated
the similarity in pollen morphology between V. jonghei SP46532, V. minarum RB193450 and V. aff.
minor, which exhbited the same type of ornamentation in the equatorial calota and in the centre of the
pollen grains. Vriesea jonghei SP167550 differed
from the remaining specimens for presenting wide
duplicolumellate muri. Besides, this specimen presented heteromorphism in its pollen grains, presenting also microreticulate ornamentation on the
equatorial calota. The two specimens of Vriesea jonghei were collected in Paranapiacaba, São Paulo and
they are highlighted here for further taxonomic
investigation, given the morphological variations
found in their pollen grains.
Vriesea atropurpurea, although fitting in the same
pollen type of V. jonghei SP167550 as both of them
present duplicolumellate muri and psilate-perforate
calota, the first one stood out with larger pollen grains,
wider lumina and the presence of microreticulum surrounding the lumina. The two specimens of V. bituminosa exhibited great pollen similarity, differing only
in the size of the lumina and width of the muri. However, V. minarum showed heterogeneity in pollen morphology, since their two specimens, which were
collected in two different municipalities in the state
of Minas Gerais, were classified, by the calota ornamentation and muri simplecolumellate or duplicolumellate, in two distinct pollen types.
It was possible to recognise morphological variations corresponding to four pollen subtypes distributed across two palynotaxonomical groups, in the
earlier-mentioned species of section Xiphion, which
should be taken into consideration by taxonomists,
demonstrating even variation in the pollen patterns
among specimens (in Vriesea minarum) and even
within the same specimen (V. jonghei). The species
V. atropurpurea, V. itatiaiae and V. pseudoatra
demonstrated a preference for the grassland habitat
called ‘Campo de Altitude’. The specimens of V.
itatiaiae and V. pseudoatra exhibited variation in the
sculpture of the calota of their pollen grains, hence
each was classified in two main pollen types simultaneously, demonstrating the morphological heterogeneity in the two species. In addition, each
specimen of these two species shared similarities
Pollen morphology of Vriesea
with one of the specimens of the other species, not
helping in distinguishing these taxa, and reinforcing
the existence of taxonomic problems between them
or a strong convergence in pollen characters in this
case. Santos et al. (2018) also reported differences
in the ornamentation of the equatorial calota betwen
the specimens of a same species in Alcantarea, genus
segregated from Vriesea, such as Alcantarea extensa
(L.B. Sm.) J.R. Grant, A. geniculata (Wawra) J.R.
Grant, A. nahoumii (Leme) J.R. Grant, A. turgida
Versieux et Wand, revealing that it is common to
find intraspecific variation in their pollen grains.
Concerning Vriesea atropurpurea, the pollen morphology separated it from both V. itatiaiae and V.
pseudoatra by their distinct pollen pattern of duplicolumellate muri, and differences in the characteristics of
the reticulum in the central area of the pollen grain.
Vriesea bituminosa and V. neoglutinosa belong to different sections, Xiphion and Vriesea, respectively, with
different reproductive characters (Wanderley & Martins 2007). However, the pollen characters showed to
be homogeneous between the specimens of the two
species, grouping them in a single palynotaxonomic
group. These evidences indicate the existence of a
morphopolynic overlap between both sections,
which, therefore, does not help in the distinction of
such taxa and also suggests that pollen traits in Vriesea
are not directly driven or selected by interactions with
bird or bat pollinators.
Vriesea cacuminis, V. minarum and V. stricta show
similarities in reproductive and vegetative structures,
making it difficult to separate these two species
which constitute the ‘Vriesea minarum complex’
(Versieux 2011). As mentioned earlier, the palynological results demonstrated morphological differences between the pollen grains of the studied
specimens of V. minarum, which were classified in
the two main pollen types for the differences in the
calota ornamentation. The same has happened with
three specimens of V. stricta, which were collected in
the same municipality of Jaboticatubas, in the state
of Minas Gerais, but found to have either microreticulate or psilate-perforate calota.
The two specimens of Vriesea cacuminis, both collected in the municipality of Lima Duarte, in the state
of Minas Gerais, were set apart for the simplecolumellate or duplicolumellate muri, and details of the reticulum pattern, while sharing the same ornamentation
of the calota. The pollen morphology of species of the
‘Vriesea minarum complex’ showed to be heterogeneous, placing them in four pollen subtypes: V. atropurpurea was the most distinctive of all species, what
was already expected, since it has very different flowers
from the remaining species. The two specimens of V.
cacuminis presented differences in the ornamentation
19
of the equatorial calota and were placed in different
pollen types. The specimen V. cacuminis SP392004
did not group with any of its more closely related
species, while V. cacuminis SP584635, V. minarum
RB324783 and V. stricta SP169994 formed a group,
for sharing microreticulate calota and duplicolumellate muri. In addition, the specimen V. minarum
SP19350 grouped with two specimens of V. stricta
(SP169988 and SP169995), for the psilate-perforate
calota and simplecolumellate muri. This variability of
pollen patterns between species and specimens reflects
that the pollen grains are not informative for the species distinction in the ‘Vriesea minarum complex’.
Vriesea flava is related to V. eltoniana E. Pereira et
Ivo, V. interrogatoria L.B.Sm. and V. paraibica Wawra,
integrating the ‘Vriesea paraibica complex’, which is
characterised by a dense inflorescence that is shared
by all the species (Costa et al. 2009). In this study, only
pollen grains of the first species were analysed, and no
literature data was found for the remaining species of
the group. We verified that the three specimens of V.
flava were classified in the same pollen type and
showed to be very similar for the several characters
which were shared (microreticulate calota, simplecolumellate muri, with microreticulum surrounding the
lumina). Souza et al. (2017) studied the pollen of V.
paraibica, which exhibited a similar pattern to that of
V. flava. We suggest that further investigations should
be conducted with the remaining species, to elucidate
possible palynological affinities.
Vriesea philippocoburgii is morphologically related to
V. reitzii Leme et Costa due to the reddish yellow
bracts, both occuring in allopatry or sympatry, in
Santa Catarina (Machado & Semir 2006). The three
specimens here studied of V. philippocoburgii demonstrated to be very similar, grouping in a single pollen
type; only the specimen SP351897 differed a little
from the remainder for the smallest values in certain
metric variables. No data was found in the literature
about the pollen morphology of V. reitzii, thus we
suggest additional analysis for this species.
Surprisingly, the following pollen grains presented
a difficult taxonomic circumscription for their great
overall morphological similarity: the three specimens
of Vriesea flava, which were collected in Paraná and
Santa Catarina; the three specimens of V. philippocoburgii, which were collected in three municipalities
in São Paulo, and the specimen V. friburgensis
SP225698 collected in Piracicaba, São Paulo.
These three species are inclued in the section Vriesea
but are well separated by their flower morphology
and/or inflorescence type, and there is no molecular
phylogenetic evidence of their close relationship
(Costa et al. 2015), suggesting a convergent evolution of similar pollen patterns in these species.
20
V. L. Santos et al.
Vriesea longicaulis is related to the five other taxa
which were not analysed in the present study (V.
hydrophora Ule, V. longiscapa Ule, V. pabstii McWill.
et L.B.Sm., V. platzmannii E.Morren and V. unilateralis (Baker) Mez) (Costa et al. 2015). Only one specimen of V. longicaulis was analysed here while all the
possibly related species mentioned earlier were not.
Vriesea unilateralis was studied in Souza et al. (2017),
and their results partially corroborated our data, since
the pollen grains of the specimen analysed by these
authors presented microreticulum surrounding the
muri, while ours did not. Here, V. longicaulis was
placed next to V. cacuminis RB584635 and V. stricta
SP169994, with duplicolumellate muri, microreticulate calota, and without microreticulum surrounding
the muri. According to Costa et al. (2015), these
species present differences in the reproductive structures, and there are no records of the proximity
between these taxa.
Vriesea medusa is related to V. nanuzae by the
morphology of the reproductive structures (Versieux
2008). In the present study, the similar pollen pattern put into evidence the morphological similarity
of these two species considered taxonomically close,
both in section Xiphion.
Vriesea oligantha is vegetatively related to V. pseudoligantha Philcox and V. vellozicola Leme et J.A.
Siqueira (Ribeiro et al. 2014), but the two latter species
were not investigated in the present work. The pollen
grains of two specimens of V. oligantha here analysed,
which were collected in different municipalities of the
state of Minas Gerais, showed morphological variations, especially in the ornamentation of the equatorial
calota, placing them in different pollen types. The
specimens V. oligantha RB193801 and V. itatiaiae
RB474136 were grouped in the same pollen type for
the similarities in their characters. According to Coffani-Nunes et al. (2010) and Wanderley and Martins
(2007), these two species present a distinction in the
reproductive and vegetative characters, not demonstrating affinities between them. Nevertheless, it is
important to mention that V. oligantha is possibly
going to be transfered to the new genus Stigmatodon,
where other more related and comparable species are
expected to be found.
Conclusions
The pollen morphology of Vriesea presented little
variation and, therefore, it was considered as a stenopalynous genus, as already pointed out by the
literature. However, characteristic differences in the
ornamentation of the equatorial calota, and details
of the reticulum in the central area of the pollen
grains demonstrated to be useful in the infrageneric
palynotaxonomy.
Some pollen groups which were formed confirmed the relationship between certain species
which had already been delimited by reproductive
and vegetative structures, or by molecular evidence,
i.e. Vriesea friburgensis and V. neoglutinosa; V. medusa
and V. nanuzae. Other comparisons may be more
clearly understood in the future, when well resolved
phylogenies for the genus become available.
The pollen grains of Vriesea flava were very similar to those of V. philippocoburgii, which although
endemic to the Serra do Mar corridor, in southern
and southeast part of Brazil, neither presented similarities in the reproductive characters, nor in DNA
sequences, suggesting a homoplastic origin.
It is notorious that most species analysed in section
Vriesea (V. cacuminis, V. flava, V. friburgensis, V. neoglutinosa and V. philippocoburgii) presented similar
ornamentation in the equatorial calota (microreticulate), except in V. stricta, in which the specimens presented the calota either psilate-perforate, or
microreticulate. These two types of calota were also
present in most taxa analysed from section Xiphion (V.
atropurpurea, V. itatiaie, V. jonghei, V. minarum, V. aff.
minor, V. oligantha and V. pseudoatra), while V. bituminosa, V. longicaulis, V. medusa and V. nanuzae
exhibited only microreticulate calota.
Although both sections Vriesea and Xiphion are
delimited based on floral characters, anthesis time
and flower scent, we observed overlap in the pollen
grains morphology, suggesting that the palynotaxonomy based on measurements and qualitative morphological characteristics do not help in their distinction
and also suggesting that pollinators, either bats or
hummingbirds, appear to have a weak selective pressure over the pollen morphology in Vriesea.
Acknowledgements
The authors are grateful to the curators and staff members of the herbaria who provided the specimens (RB,
SP, SPF and UFRN). The authors are also grateful to
Luciana Benjamim Benatti of the Laboratory of Scanning Electron Microscopy of the Instituto de Botânica
and André Haraguti Aguillena of the Scanning Electron Microscopy Centre of the Universidade Federal
de São Paulo, São Paulo, Brazil, for the technical support and access to the scanning electron microscopes.
Disclosure statement
No potential conflict of interest was reported by the
authors.
Pollen morphology of Vriesea
Funding
This work was supported by the Coordenadoria de
Aperfeiçoamento em Pesquisa e Ensino Superior
(CAPES) (grant number PNADB 17/2009); Fundação de Amparo à Pesquisa do Estado de São Paulo
(FAPESP) (grant numbers 2015/15359-0, 2016/
24015-5, 2017/50341-0). The authors are thankful
to the Fundação de Amparo à Pesquisa do Estado de
São Paulo (FAPESP) for providing the Master’s
degree scholarship (grant number 2013/22881-9) to
the first author. The authors also extend their thanks
to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the fellowship of
‘Productivity in research’ to the third and fourth
authors (grant numbers 304778/2013-3, 302766/
2016-2).
21
Vriesea oligantha (Baker) Mez. Brazil: Minas Gerais, Santana de
Pirapama, E.M.C. Leme 7224, 23 August 2007 (RB484319);
Brazil: Minas Gerais, Diamantina, G. Martinelli 6281, 15
December 1979 (RB193801).
Vriesea philippocoburgii Wawra. Brazil: São Paulo, L.S.R. Duarte
35, 29 April 1982 (SP196610); Brazil: São Paulo, Cananéia,
M.G.L. Wanderley et al. 997, 6 May 1988 (SP224892); Brazil:
São Paulo, São Miguel Arcanjo, G. Martinelli & M.G.L. Wanderley 15791, 1 October 1999 (SP351897).
Vriesea pseudoatra Leme. Brazil: Rio de Janeiro, Nova Friburgo,
L.M. Versieux & A.M. Calvente 234, 23 November 2005
(SP382076); Brazil: Espírito Santo, E. Pereira 2230, 6 December 1956 (RB96088).
Vriesea stricta L.B. Sm. Brazil: Minas Gerais, Jaboticatubas, M.G.L.
Wanderley (Flora da Serra do Cipó collection 5549), 14 August
1979 (SP169988); Brazil: Minas Gerais, M.G.L. Wanderley et al.
216, 5 August 1980 (SP169994); Brazil: Minas Gerais, Jaboticatubas, M.G.L. Wanderley et al. 217, 5 August 1980 (SP169995).
ORCID
Specimens investigated
Vriesea atropurpurea Silveira. Brazil: Minas Gerais, Santana do
Riacho. L.M. Versieux 296, 8 December 2006 (SPF).
Vriesea bituminosa Wawra. Brazil: São Paulo Biritiba Mirim, A.C.
Filho 1780, 11 November 1983 (SP195325); Brazil: São
Paulo, Biritiba Mirim, A.C. Filho 1892, 24 November 1983
(SP195516).
Vriesea cacuminis L.B. Sm. Brazil: Minas Gerais, Lima Duarte, L.
M. Versieux et al. 345, 13 January 2007 (SP392004); Brazil:
Minas Gerais, Lima Duarte, A. Cancio 3, 7 December 2010
(RB584635).
Vriesea flava A.F. Costa. Brazil: Paraná, Campina Grande do Sul,
M.L. Brotto 1718, 9 July 2014 (RB612972); Brazil: Paraná,
Piraquara, M.L. Brotto 1621, 4 August 2014 (RB612973);
Brazil: Santa Catarina, Campo Alegre, G. Martenelli 14973,
20 May 1998 (RB329116).
Vriesea friburgensis Mez. Brazil: São Paulo, Itanhaém, R.J.F. Garcia et al. 1688, 2 December 1988 (SP333339); Brazil: São
Paulo, Garulhos, M.A Campacci s.n, 28 January 2000
(SP340277); Brazil: São Paulo, Piracicaba, E.L Catharino s.
n., 28 December 1987 (SP225698).
Vriesea itatiaiae Wawra. Brazil: Minas Gerais, Passa Quatro, L.D.
Meireles 3105, 24 April 2007 (RB474136); Brazil: Rio de
Janeiro, Nova Friburgo, A. Bonnet & E. Caglioni 69, 11 May
2012 (RB565884).
Vriesea jonghei (K.Koch) E. Morren. Brazil. São Paulo, Paranapiacaba, M.G.L. Wanderley 214, 27 August 1980 (SP167550); Brazil: São Paulo, Paranapiacaba, O. Handro 599, 3 July 1956
(SP46532).
Vriesea longicaulis (Baker) Mez. Brazil: São Paulo, Cunha, M.G.
L. Wanderley 281, 13 November 1981 (SP169371).
Vriesea medusa Versieux. Brazil: Minas Gerais, Rio Vermelho, M.
G.L. Wanderley et al. 2719, 23 September 2008 (SP412656).
Vriesea minarum L.B. Sm. Brazil: Minas Gerais, Itabirito, G.
Martinelli 3608, 20 January 1978 (RB193450); Brazil:Minas
Gerais, Caeté, P.I.S. Braga 2210, 27 February 1986
(RB324783).
Vriesea aff. minor (L.B. Sm.) Leme. Brazil: Minas Gerais, Igarapé,
F.F Carmo 3485, 29 September 2008 (SP412846).
Vriesea nanuzae Leme. Brazil: Minas Gerais, São Gonçalo do Rio
Preto, N.F.O. Mota et al. 1265, 29 February 2008 (SP465860).
Vriesea neoglutinosa Mez. Brazil: Paraná, Matinhos, N. Kuhlmann
s.n., 1 September 1939 (SP387922).
Cynthia Fernandes Pinto da Luz
0001-7908-155X
http://orcid.org/0000-
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