Scientia Horticulturae 277 (2021) 109732

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Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

Characteristics of the pollen morphology and viability of

Bougainvillea (Nyctaginaceae)
Shengxin Chang a, b, Chunxia Li c, Yongzhou Jiang d, Yan Long d, Ya Li d, Junmei Yin a, b, *
a
Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences (CATAS), Key Laboratory of Crop Gene Resources and Germplasm
Enhancement in Southern China, Ministry of Agriculture, Danzhou 571737, Hainan Province, China
b
Engineering Technology Research Center of Tropical Ornamental Plant Germplasm Innovation and Utilization, Danzhou 571737, Hainan Province, China
c
Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, Hainan Province, China
d
College of Forestry, Hainan University, Haikou 570228, Hainan Province, China

A R T I C L E I N F O A B S T R A C T

Keywords: Pollen grains of 92 Bougainvillea genotypes were examined using a light microscope (LM) and scanning electron
Bougainvillea microscope (SEM) to identify taxonomic characteristics and the relationship between pollen morphology and
Pollen morphology germination capacity. The shapes of fresh pollen grains of Bougainvillea genotypes are spheroidal to prolate-
Pollen viability
spheroidal or suboblate after infolding. The sexine of Bougainvillea pollen grains consists of columellae and a
reticulate cristatum, with or without microechini. The distribution pattern of the tectum microechini is uniform
on the pollen grains within each genotype, yet it is variable among the different genotypes. Pollen with a smooth
reticulate cristatum was observed in the genotypes of B. peruviana, while pollen with muri covering the cristatum
were found in the genotypes of B. glabra and B. spectabilis. The pollen grains of each Bougainvillea germplasm
have a wide range of diameters. The mean diameter of the pollen grains of the diploid genotypes ranged from
22.2 to 32.1 μm, while that of the polyploid genotypes ranged from 26.7 to 40.4 μm. The germinated pollen
grains were observed in the diploid and tetraploid genotypes, the pollen germination rate of which was
commonly less than 20 %. The size of germinated pollen grains was significantly larger than that of the silent
ones, which indicated that the nonuniform distribution of grain size is a key reason for the low germination
capacity of Bougainvillea pollen.

1. Introduction To classify Bougainvillea genotypes, some studies have been carried

Bougainvillea (Nyctaginaceae, Caryophyllales) is a betaine-
containing plant originating from Brazil and Peru in South America.
Bougainvillea exhibits a rich bract colour, long flowering period, and
high tolerance to poor soil and high temperatures and is therefore
widely planted in the main tropical and subtropical regions of the world.
At least 31 regions of the world have selected Bougainvillea as their
representative flower, including Zambia, Grenada, Hainan Province in
China and Guam, a territory of the USA (Shengxin et al., 2018). The
horticulturally important Bougainvillea cultivars are mainly domesti­
cated from three basal species (B. glabra, B. spectabilis and B. peruviana)
as well as their hybrid varieties (Zadoo et al., 1975a). The Royal Hor­
ticultural Society (https://www.rhs.org.uk/) has catalogued approxi­
mately 300 Bougainvillea varieties.
out on the morphology of leaves, stems and flowers and reproductive
biology (Zadoo et al., 1975a; Zadoo et al., 1976a; Zhou et al., 2011;
Kumar et al., 2015; Yang et al., 2016); however, because of the weak
phenotypic convergence of different Bougainvillea genotypes, it is diffi­
cult to judge their taxonomic status according to these morphological
characteristics, especially for hybrid genotypes. A few DNA makers
(7–25) have also been used to classify Bougainvillea genotypes (Li et al.,
2011; Wu, 2012; Kumar et al., 2014), resulting in obvious differences
between genotypes and differences from the traditionally classified
groups, which may be because of the limit of classification accuracy
when using few markers.
Pollen is diverse in monad shape and exine sculpturing, which is
useful for plant classification and evolution studies (Shivanna, 2003).
Some four-decade-old pollen data, which address the pollen morphology

* Corresponding author.
E-mail addresses: Brownman87@foxmail.com (S. Chang), 568076816@qq.com (C. Li), 827583863@qq.com (Y. Jiang), 2315391296@qq.com (Y. Long),
1178657481@qq.com (Y. Li), yinjunmei2011@sina.com (J. Yin).

https://doi.org/10.1016/j.scienta.2020.109732
Received 30 May 2020; Received in revised form 25 August 2020; Accepted 8 September 2020
Available online 3 November 2020
0304-4238/© 2020 Published by Elsevier B.V.
S. Chang et al.
of Bougainvillea and its closely related Nyctaginaceae species, are
available (Nair, 1965). A recent study encompasses pollen morpho­
metric analyses of four Bougainvillea species, including B. glabra,
B. spectabilis, B. peruviana and B. buttiana (B. × glabra-peruviana), and 22
Indian varieties (Tripathi et al., 2017). However, until now there has
been a lack of powerful pollen markers to distinguish different
Bougainvillea species and cultivars, and the ultrastructure of Bougain­
villea pollen morphology is still insufficient compared with the size of
Bougainvillea germplasm resources. To evaluate the detailed taxonomic
characteristics of Bougainvillea pollen, this study selected as many as 92
Bougainvillea genotypes (Table S1) for scanning electron microscope
(SEM) observations, and the different features of pollen grains were
analysed using qualitative and quantitative methods.
Moreover, the germination capacity of pollen grains is quite low in
the three basal species of Bougainvillea and their hybrids (Zhou et al.,
2016), which results in difficulty in Bougainvillea crossbreeding. Pollen
morphology and ultrastructure have been shown to be correlated with
growth habitat and pollination biology (Osborn et al., 2001; Calic et al.,
2013). Germination capacity and vigour of pollen tube growth are
correlated with grain size in Dianthus (Caryophyllaceae, Caryophyllales)
(Tejaswini, 2002). To determine the relationship between pollen
viability and morphology in Bougainvillea, the pollen grains of 18
representative genotypes were selected to perform germination vigour
tests, and the morphological differences between the viable and
non-viable pollen grains were analysed.
2. Material and methods
2.1. Plant materials
The study was conducted on 92 Bougainvillea genotypes growing at
the germplasm nursery of the Tropical Crop Genetic Resource Institute
of the Chinese Academy of Tropical Agricultural Science (N 109.5◦ , E
19.5◦ ). The name and source of each genotype were based on the records
of the Royal Horticultural Society and Singh et al. (1999).
2.2. Scanning electron microscopy
Three tubiform florets of each genotype were collected randomly
from different inflorescences between 9:00 and 10:00 on a sunny
morning and stored in a 4 ℃ refrigerator for two days at most before the
SEM observations. Three anthers were peeled from different florets and
mounted on the surface of aluminium stubs with double-sided sticky
tape. Each anther was dragged on a small region of the stub to ensure
enough observable pollen grains. Sputter coating with gold/palladium
was performed with a sputter coater (SC7620, Quorum, England) and
the samples were subsequently examined with a scanning electron mi­
croscope (ΣIGMA, Carl Zeiss, Germany).
2.3. In vitro pollen viability
The pollen germination medium contained 10 g/100 mL sucrose, 10
mg/L boric acid and 0.5 g/100 mL agar. Three anthers selected from
different inflorescences as described in Section 2.2 were mounted on
solid medium in a 22 ℃ environment (the most appropriate germination
temperature was verified by a gradient temperature test, not shown).
The pollen germination rate and pollen tube growth process were
evaluated under a light microscope (DM2000, Leica, Germany) after a 3
h incubation period. An I2-KI staining method was used to determine the
starch content of the pollen grains according to Li (2000). Three repli­
cates (slides) were used for each genotype. The stained pollen grains
were photographed, and the mean luminance value of each pollen grain
was calculated using Photoshop version 13 (Adobe Inc., California, USA)
to estimate the relative content of starch.
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Scientia Horticulturae 277 (2021) 109732
2.4. Statistical analysis
The measurements of the pollen features were performed in ImageJ
version 1.52o (https://imagej.nih.gov/ij/). The one-sample Kolmo­
gorov-Smirnov test was used to test whether a data set comes from a
specific distribution. Comparisons between multiple data sets were
performed using the nonparametric Kruskal-Wallis test with post-hoc
Dunn’s test to confirm significant differences. Statistical analyses were
performed using XLSTAT (Addinsoft Inc., New York, USA). P values of <
0.05 were considered significant.
3. Results
3.1. Pollen shape and size
The pollen grains of Bougainvillea are developed in four pollen sacs of
the anthers (Fig. 1). After flower blooming, the pollen sacs are dehisced
to release the mature pollen grains (Fig. 1C). The majority of the pollen
grains were severely infolded and exhibited a concave “pie” shape or an
asymmetrical subspheroidal shape (Fig. 1C-D and 2) as a consequence of
harmomegathy, which is beneficial for protecting the male gametophyte
against desiccation (Grant-Downton, 2010). Only a small amount of
pollen grains were fully or partially turgescent with sub-oblate, sphe­
roidal or prolate-spheroidal shapes (Fig. 1C-D and 2). Based on a
large-scale observation, the shapes of the pollen grains did not show
obvious differences among the 92 Bougainvillea genotypes (File S1).
The sizes of the pollen grains of the different Bougainvillea genotypes
vary widely, ranging from 22.2 μm (B. glabra var. Mrs. Eva, 2n) to 40.4
μm (B. var. Odd leaves, 3n) in mean diameter. Except B. var. Odd leaves
(3n), the first three pollens were also from polyploid genotypes, ranking
as B. buttiana var. Chitra (4n), B. buttiana var. Begum Sikander (3n) and
B. buttiana var. Poultonii (3n). However, Kruskal-Wallis test showed that
these polyploidy genotypes didn’t have significant difference in pollen
size with other three diploid germplasms, including B. buttiana var.
Begum Sikander, B. var. Elizabeth Angus Fantasy and B. spectabilis var.
Splendens; and a polyploidy genotype, B. buttiana var. Louis Wathen
Intermediate (3n) was significantly grouped into the pollen-size group of
diploid genotypes (Table S1). These indicated that the pollen size of
polyploid genotypes wasn’t absolutely superior to that of diploid
germplasms. Moreover, based on extensive distribution fitting, an
interesting phenomenon was found: the pollen grains of Bougainvillea
had an obviously inhomogeneous distribution in monad size regardless
of having diploid, triploid or tetraploid genotypes (Fig. 2). For example,
the pollen grains of B. glabra var. Eva (2n) had a mean diameter of 22.2
μm; some of the grains were larger (> 27.5 μm), some were smaller (<
17.5 μm) and a large number had an intermediate size (20–25 μm). The
size distribution of the pollen grains of the diploid Bougainvillea geno­
types were significantly consistent with a Gaussian distribution model
with a large dispersion (σ > 0.03, Fig. 3A-C) based on a Kolmogorov-
Smirnov test, while pollen grains of the triploid and tetraploid geno­
types had a long left tail, which indicated a tendency for an asymmet­
rical distribution that favoured small sizes over large sizes (Fig. 3D-F).
From a polar view, the recognizable pollen grains usually have a 3-
zonicolpate aperture (Fig. 4A, Supplementary File 1), while a previous
study also found a kind of 4-zonicolpate aperture in B. buttiana var.
Chitra and B. var. Tetra Mrs. Mcclean (Tripathi et al., 2017). From an
equatorial view, the aperture was found with a layer of smooth mem­
brane, and the exine sculpturing was entirely disconnected (Fig. 4B).
During germination, one of the 3–4 apertures can act as the germination
site, from which the sprouting of a single pollen tube commonly occurs
(Fig. 5, Fig. S2).
3.2. Exine sculpturing
The sexine structure of Bougainvillea pollen grains, which consists of
the columellae, tectum and supratectual elements, contributes to the
S. Chang et al.
Fig. 1. Scanning electron micrographs of Bougainvillea anther morphology. Representative samples were collected from B. var. New River. The anthers in A, B and C
correspond to the three developmental stages (shown in order in the inset in A). D shows the dissected structure of the mature anther.
tectate type (Grant-Downton, 2010) (Figs. 4C and 8A). A portion of the
columellae distributed in the infratectal layer are covered by a coherent
reticulum, while more columellae are free-standing and exposed. The
lumina in the network-like tectum of Bougainvillea pollen grains are
independent and structurally closed, except for those in the ‘Ratana Red’
variety, which have open and malformed lumina (Fig. 4D). The pollen
lumina of 51 % of the Bougainvillea genotypes were greatly filled by
pollenkitt, which covered the surfaces of the columellae (Fig. 6A). A
total of 42 % of the Bougainvillea genotypes had pollen lumina that were
half-overlaid such that the tops of the columellae were exposed
(Fig. 6B), and the remaining Bougainvillea genotypes (no more than 10
%) lacked pollenkitt, thus exposing the outline of the foot layer (Fig. 6C).
For the pollen grains with visible columellae, the individual columellae
commonly maintain an obvious distance, and only in a special case
(B. peruviana var. Vishakha, were the columellae packed compactly in­
side the lumina (Tripathi et al., 2017).
The surface of the reticulum cristatum is the most easily observed
component of Bougainvillea pollen grains that are not affected by har­
momegathy or covered by pollenkitt. Through the large-scale compari­
sons of Bougainvillea genotypes, the reticulum surface of the pollen
grains was found to be smooth or covered with supratectal elements in
different distributions and with different morphologies (Fig. 7A), while
for the different pollen grains of the same genotype, the pattern of
supratectal elements was very stable. Based on this characteristic, the
Bougainvillea genotypes can be classified into two large groups: type I,
reticulum with smooth surface, and type II, reticulum covered with
microechinis. Type II can be further classified as follows: type II1, re­
ticulum covered with one or two short microechinis at each intersection
of the reticulum threads, type II2, reticulum covered with 1–4 short
microechinis at each intersection of the threads and free-standing
microechinis on the middle part of the threads, and type II3, reticulum
covered with 1–4 long spinous microechinis at each intersection of the
threads (Fig. 7A). For type II2, the locations of microechinis did not
correspond with those of the columellae below (Fig. 7A).
The pollen morphology of the majority (70.5 %) of Bougainvillea
genotypes, including the genotypes of B. peruviana and its hybrid vari­
eties with B. glabra and B. spectabilis, is consistent with type I (Fig. 7D).
The rest of the Bougainvillea genotypes, including the genotypes of
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Scientia Horticulturae 277 (2021) 109732
B. glabra and B. spectabilis and the interspecific hybrids of the three basic
Bougainvillea species, have morphologies consistent with type II. Type
II1 pollen is found in 9.5 % of the genotypes, including B. glabra var. Mrs.
Eva, B. spectabilis var. New River, B. buttiana var. Barbara Rose and so on
(Table S1). Type II2 pollen is found in 19.0 % of the genotypes, including
B. glabra var. Formosa, B. spectabilis var. Lateritia, B. buttiana var. Dr. P.
V. Sane and so on. Only a B. glabra var. Sanderiana possesses type II3
pollen grains.
3.3. Pollen viability
Eighteen Bougainvillea varieties were selected to perform the pollen
germination detection in vitro at 22 ◦ C. Germinated pollen grains were
found in the diploid and tetraploid genotypes (Fig. S1), whereas no
germinated pollen grains were found in the triploid genotypes, which
may be because of the chaotic chromosome constitution led by the
abnormal meiosis of the microspore mother cells (Qin et al., 2014; Yan
et al., 2020). The pollen germination rate of the fertile male Bougain­
villea genotypes was generally lower than 20 % in the 15 selected ge­
notypes (Fig. S1), which was in accord with the 18 previously studied
genotypes (Zhou et al., 2016). Furthermore, it was interesting to find
that the mean diameters of the germinated pollen grains were all
distributed in the right tail of the diameter distribution (Fig. 3), indi­
cating that the germinated pollen grains had grains were larger than
silent pollen grains (Fig. S2).
The I2-KI staining method was also used to determine the viability of
the fresh pollen grains based on the presupposition that the viability
strength of pollen grains is positively associated with their starch con­
tent, which can be indicated by the dyeing depth of iodine (Du et al.,
2019). In the present study, no relationship between staining degree and
grain size was found; deep staining was observed in both large and small
grains. Furthermore, the nonviable pollen grains of triploid genotypes
have an even deeper staining depth than that of the viable pollen grains
of the diploid and tetraploid genotypes (Fig. S3). According to the depth
of the dye, we found that the pollen grains of B. glabra var. Mrs. Eva
White, B. buttiana var. Chitra, B. glabra var. Zinna Barat and B. buttiana
var. Mrs. McClean had the lowest starch contents, while B. glabra var.
Formosa, B. buttiana var. Pixie Pink, B. var. Monan and B. peruviana var.
S. Chang et al. Scientia Horticulturae 277 (2021) 109732

Fig. 2. Scanning electron microscope (SEM) micrographs of pollen grains of B. var. Monan (A, 2n), B. var. Tropical Bouquet (B, 2n), B. × buttiana var. Rainbow Red
(C, 3n), B. × buttiana var. Poultonii (D, 3n) and B. × buttiana var. Chitra (E and F, 4n). The yellow and red circles indicate typical small and large pollen grains,
respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3. The mean diameter distribution of pollen grains of B. glabra var. Mrs. Eva (2n, A), B. spectabilis var. Splendens (2n, B), B. peruviana var. Partha (2n, C), B. var.
Odd leaves (3n, D), B. buttiana var. Poultonii (3n, E) and B. buttiana var. Chitra (4n, F) based on light microscopy (LM) observations. The green lines show the
diameters of the germinated pollen grains. Statistical estimation was performed based on a Kolmogorov-Smirnov test with the H0 hypothesis that the sample follows
a normal distribution. The significance level was set as 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)

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S. Chang et al. Scientia Horticulturae 277 (2021) 109732

Fig. 4. Scanning electron micrographs of Bougainvillea pollen grains. A and B show the polar and equatorial views, respectively. C and D show the normal and
abnormal exine sculpturings of the pollen grains, respectively. The genotypes from A to D are B. spectabilis var. New River (A-I), B. glabra var. Mrs Eva Ice-cream (A-
II), B. buttiana var. Chitra (B-I), B. spectabilis var. Red Triangle (B-II), B. glabra var. Formosa (C-I), B. buttiana var. Rainbow Red (C-II) and B. var. Ratana Red (D-I and
II), respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5. Germination and tube growth of Bougainvillea pollen (Mrs. Eva cultivar).

Mary Palmer had the highest starch contents. There was no relationship
between starch content and the Bougainvillea genotypes (Fig. S4).
4. Discussion
The configuration of Bougainvillea pollen is not unique in the Plantae,
and similar structures are also distributed widely in different eudicot
taxa, such as Dipteracanthus in the Acanthaceae, Impatiens in the Balsa­
minaceae, Erophila in the Brassicaceae, Viburnum in the Caprifoliaceae
and Thladiantha in the Cucurbitaceae (Grant-Downton, 2010). These
findings indicate the superiority of this pollen configuration in the
sexual propagation of plants. In general, a spheroidal shape, rather than
a branching shape, is beneficial for pollen grains, as it allows them to
resist physical injuries from random collisions during the pollination
process. The smallest superficial area is found on spherical objects; this
results in the smallest area of exposure to sunlight, which is beneficial
for pollen grains because it allows them to avoid serious ultraviolet
damage and water desorption. The complex reticulum structure and
multiple columellae can increase the area of contact between pollen
grains and the cuticle hairs or prickles of pollinating insects, which may
promote the fixation of pollen grains on the insect body. The close-set
lumina on the pollen exine (which act like reservoirs) have a strong
ability to absorb and store water during pollen germination.
For Bougainvillea pollen grains, these lumina may also be helpful for
pollenkitt storage, as indicated by the high content of pollenkitt in most
of the Bougainvillea pollen grains (Fig. 6). Ettore Pacini (2005) reviewed

5
S. Chang et al.
Fig. 6. Different contents of pollenkitt on the surface of Bougainvillea pollen grains. The histogram shows the percentage of different pollen types in the 92
Bougainvillea genotypes.
the important functions of pollenkitt to pollen grains between anther
opening and the hydration of the pollen on stigma; these functions
include protecting pollen from hydrolysis and exocellular enzymes,
making pollen attractive to animals and enabling pollen collection by
bees. These functions are especially important for the existence and
evolution of Bougainvillea, which is self-incompatible and has low
cross-compatibility (Zadoo et al., 1975b; Zadoo et al., 1976b). During
our microexamination, Bougainvillea pollen grains was found to be very
popular with thrips, which is an important kind of pollinating insect
(Fig. S5). The pollen grains were frequently found adhered to the cuti­
cles of the thrips, which is a benefit of the Bougainvillea pollen structure
and abundant pollenkitt.
Despite the benefits to sexual propagation provided by the pollen
configuration of Bougainvillea, the successful fertilization and setting
rates (commonly less than 10 %) of Bougainvillea genotypes are not
impressive, which has troubled Bougainvillea breeders over the years.
Based on the determination of the germination rate, the low viability of
Bougainvillea pollen grains, which act as a gamete in sexual propaga­
tion, may be one of the reasons for the difficulty producing hybrids of
Bougainvillea. Furthermore, the relationship between pollen grain
germination and pollen grain size indicated that monad size has a direct
influence on pollen viability. The low number of large pollen grains in a
single genotype might explain the low pollen germination rate of
Bougainvillea pollen grains. Grain size was also found to affect germi­
nation in Dianthus, which was considered a strategy to maximize
reproductive fitness (Tejaswini, 2002). Hoekstra (1986) has proved that
pollen grains can be dried to a certain water content without a loss in
viability. Based on this finding, we speculate that through specific dry­
ing treatment and screen filtration, the big pollen grains of Bougainvillea
genotypes may be separated and applied to improve the cross-breeding
efficiency of Bougainvillea. In addition, the starch content is commonly
considered an indicator of the viability of pollen germination; however,
the present study did not find an obvious correlation between these
variables, which suggests that true germination tests are more accurate
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Scientia Horticulturae 277 (2021) 109732
than the detection of the starch content in pollen grains.
There is a range of possible aspects influencing grain size, including
climate and environmental factors, nutrient availability and genome
size (Ejsmond et al., 2011; Lau and Stephenson, 1993; Dyer et al., 2013),
yet these aspects may be difficult to lead the large variation of pollen
grain size in a single Bougainvillea genotype. In order to illuminate a
similar phenomenon in size variation of Cedrus pollen, Bell et al. (2018)
have put forward a hypothesis that the irregular development of grains
may contribute to the size variation, if pollen release occurs while some
grains are still developing and others are fully developed.In principle,
Bougainvillea pollen grains can be analyzed according to numerous
qualitative and quantitive characteristics, including features related to
shape, size and exine sculpturing. A previous study used a large number
of parameters, such as aperture number, sexine and nexine thickness,
and distribution of columellae density, to describe Bougainvillea pollen
(Tripathi et al., 2017). However, when the number of Bougainvillea ge­
notypes under study is large, the different pollenkitt contents and de­
grees of dehydration render the measurement of many parameters
infeasible. Through general survey, this study completed the measure­
ment of five characteristics, including the number and distribution of
microechinis, pollenkitt content, starch content, mean diameter of the
pollen grains and mean lumina area of the pollen grains. An analysis of
the association between the pollen parameters and the traditional clas­
sification of Bougainvillea genotypes revealed that only the pattern of the
reticulum has a strong meaning for classifications, which suggests the
restricted applicability of pollen features to the classification of small
taxons. Moreover, the pattern of the reticulum was not considered
strongly enough in former studies on Bougainvillea pollen (Nair, 1965;
Tripathi et al., 2017), which may have resulted from the deterioration of
the microechinis due to a drying pretreatment.
The similarities in the pattern of the reticulum surface and micro­
echinis of the pollen grains indicate that B. glabra and B. spectabilis have
a close genetic relationship, while B. peruviana is obviously distantly
related to them. DNA evidence also supports this finding: B. glabra and
S. Chang et al.
Fig. 7. Exine sculpturing pattern of Bougainvillea pollen. A shows the four types of exine sculpturing. The yellow and red arrows denote the microechinis and covered
columellae, respectively. B and C show the distributions of the numbers of microechinis at the intersections and between the intersections of the reticulum,
respectively. D shows the Bougainvillea groups with the different pollen types. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
B. spectabilis have 17 pairs of euchromosomes in the nucleus and similar
genome sizes (8.25 and 8.91 pg, respectively), while B. peruviana has 15
pairs of euchromosomes and a chromosome ring consisting of four
euchromosomes and a genome size of 7.0 pg (Zadoo S N, 1976; Ohri and
Khoshoo, 1982). Although the subunits of pollen type II (II1 - II3) are not
specific to the species B. glabra and B. spectabilis (Fig. 7D), this classifi­
cation may be valuable for detecting the uniformity of different
Bougainvillea genotypes. Traditionally, Mrs. Eva is considered a wild
genotype, and the other Eva genotypes are considered mutations;
however, the SEM observations showed that Mrs. Eva and Mrs Eva Ice
cream have type II1 pollen, while Mrs. Eva White, Mrs. Eva Light Pink
and Mrs. Eva Variegata II have type II2 pollen (File S1). These results
may indicate that there are at least two genetic groups in the Mrs. Eva
series and the conjecture of single genetic origin for the Mrs. Eva series
should be denied.
In addition to B. glabra and B. spectabilis, microechinis were also
found on the reticulum surface in some genotypes of B. buttiana
(B. glabra-peruviana, Fig. 7D and File S1), which indicates that this
characteristic can be inherited by interspecies crosses. In a popular se­
ries of B. buttiana (Firecracker), the genotypes mainly differ in bract
colour, including red, pink, yellow, orange and white-pink colours, and
this difference has roused the argument of whether these varieties are
sourced from bud mutations of a common ancestor or hybridization
from the same parental pair. The SEM observation showed a separation
of Firecracker yellow, which had type II2 pollen, and the other geno­
types, which had type I pollen (File S1), which indicated that these
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Scientia Horticulturae 277 (2021) 109732
Firecracker genotypes are more likely to be selected from coeno-species.
For those genotypes for which the source is unknown, the pollen pattern
can be used to judge whether they have the genetic material of
B. peruviana, B. glabra or B. spectabilis. Based on the type I pollen found in
Elizabeth Angus and Imperial Delight, we can conclude that B. peruviana
served as the genetic background for these two genotypes. In particular,
Elizabeth Angus, which has smooth and leathery leaves, is commonly
mistaken for a genotype of B. glabra. Furthermore, the use of pollen
morphology may be limited to determining the sources of genotypes
with abnormal pollen. The Ratana genotype is known to have been
derived from a B. buttiana variety by γ-radiation, and its abnormal pollen
morphology is difficult to classify in the normal classification system
(Fig. 4D).
Credit author statement
Shengxin Chang: Experimental design, SEM observation, Data anal­
ysis and MS writing. Chunxia Li: Pollen collection, SEM observation and
MS writing. Yongzhou Jiang: Pollen germination test. Yan Long: Starch
content determination and Pollen features measurements. Ya Li: Pollen
diameter measurements and Data analysis. Junmei Yin: Study design.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
S. Chang et al.
the work reported in this paper.
Acknowledgements
We sincerely thank Mr. Xia Zhihui (Szhualao) for sharing the back­
ground knowledge about Bougainvillea. This work was supported by
grants from the National Natural Science Foundation of China (Grant
No. 31701947).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.scienta.2020.109732.
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