This research article examines pollen morphological variation in 10 Berberis species from Pakistan using light and scanning electron microscopy. A variety of qualitative and quantitative pollen characteristics were measured, including shape, size, presence/absence of colpi, colpi length and width, exine thickness, ornamentation, aperture type, and polar-equatorial ratio. Results showed variation in all characteristics studied. Five species had pantocolpate pollen while five had spiroaperturate pollen. Pollen size ranged from 29-35 μm to 60-65 μm. Ornamentation included psilate-regulate and foveolate-fossulate. This pollen morphological data provides useful information for taxonomic identification of

1. R E S E A R C H A R T I C L E
Pollen morphological variation of Berberis L. from Pakistan
and its systematic importance
Saeed ur Rahman1,2
| Shujaul Mulk Khan1
| Muhammad Zafar1
| Mushtaq Ahmad1
|
Raees Khan1
| Sajad Hussain1
| Muhammad Khalid3
| Sadaf-Ilyas Kayani2
1
Department of Plant Sciences, Quaid-i-Azam
University, Islamabad, Pakistan
2
Joint International Research Laboratory of
Metabolic & Developmental Sciences, Key
Laboratory of Urban Agriculture (South)
Ministry of Agriculture, Plant Biotechnology
Research Center, Fudan-SJTU-Nottingham
Plant Biotechnology R&D Center, School of
Agriculture and Biology, Shanghai Jiao Tong
University, Shanghai, China
3
School of Agriculture and Biology, Shanghai
Jiao Tong University, Shanghai, China
Correspondence
Shujaul Mulk Khan, Department of Plant
Sciences, Quaid-i-Azam University, Islamabad
44000, Pakistan.
Email: smkhan@qau.edu.pk
Review Editor: Alberto Diaspro
Abstract
Due to overlapping and diverse morphological characters, Berberis is among the most
taxonomically complex genera. Palynology is one of the taxonomic tools for delimitation
and identification of complex species. In this study, pollens of 10 Berberis species were
analyzed through light and scanning electron microscopy. Qualitative as well as quantita-
tive features (pollen shape, size, presence or absence of colpi, colpi length and width,
exine thickness, ornamentation, pollen class, aperture, and polar–equatorial ratio) were
measured. Five species were observed to have colpate (pantocolpate) with elongated
ends, radially symmetrical, isopolar, monads, and psilate-regulate pollens. In polar view,
six pollen were spheroidal, two were ovoid, one spherical, and one oblate. Similarly, varia-
tion in pollen length was prominent and the largest pollen on polar view was recorded
for B. psodoumbellata 60–65 μm (62.4 ± 0.9), while the smallest one was observed for B.
lycium 29–35 μm (32.2 ± 1). The observed variation in both quantitative and qualitative
features were important in taxonomic identification. This shows that palynological char-
acters are helpful in identification of Berberis genus at the species level.
K E Y W O R D S
Berberis genus, identification problems, Pakistan, pollen types
1 | INTRODUCTION
With 500 species, Berberis is an important genus of the family
Berberidaceae (Ahrendt, 1961; Frodin, 2004; Ghavipanje, Nasri,
Farhangfar, & Modaresi, 2016; Mokhber-Dezfuli, Saeidnia, Gohari, &
Kurepaz-Mahmoodabadi, 2014; Rao, Husain, Dutt, & Garg, 1998;
Rounsaville & Ranney, 2010; Schneider, 1905). The genus consists of
perennial shrubs, found in different countries of the world (Harber,
2012). From Pakistan, the genus is represented by 29 species (20 species,
6 subspecies, 2 varieties, and 1 forma) distributed mainly in the northern
mountainous ranges (Khan, Ahmad, Ahmad, & Azam, 2014; Khan, Shah,
et al., 2018). Different overlapping characters not only make the genus
complex but also problematic taxonomically which makes species identifi-
cation challenging (Sodagar et al., 2012). Environmental conditions also
play an important role in species variation (Ahrendt, 1961; Chapman,
1936; Khan et al., 2014; Rao et al., 1998). Leaf texture and serrations
show variation as the plant age increases (Rao et al., 1998; Tiwari,
Rawat, & Adhikari, 2012). Various morphological characters have been
extensively revised by taxonomists and concluded that the overlapping
characters not only vary from population to population but within the
population and even within the same plant (Rao et al., 1998). Various
studies have been conducted on the importance of morphology
(Ahrendt, 1961), palynology (Khan, Abidin, et al., 2017; Khan et al., 2018),
floral anatomy, (Rao et al., 1998; Khan, Ahmad, et al., 2017; Khan, Abidin,
et al., 2019), and DNA barcoding but still taxonomic problems exist in the
genus (Roy et al., 2010). Pollen characters have long been used to resolve
taxonomic problems in a number of plant families (Castro, Silveira,
Navarro, Paiva, & Coutinho, 2009). Pollen characters have not only
importance in taxonomic and phylogenetic study but also in paleobotany,
aeropalynology, pollination biology, pollen-pistil interaction, potential
Received: 26 December 2018 Revised: 30 April 2019 Accepted: 8 June 2019
DOI: 10.1002/jemt.23325
Microsc Res Tech. 2019;82:1593–1600. wileyonlinelibrary.com/journal/jemt © 2019 Wiley Periodicals, Inc. 1593

2. source of nectar, pollen allergy, and in the recognition of bee plants (Paul,
Essien, Idachaba, Edegbo, & Tamenku, 2014; Saensouk, Chantaranothai, &
Theerakulpisut, 2009; Yuanhui, 1988). At different taxonomic levels, pol-
len morphology has not only a crucial role in identification but also in the
interpretation of relationship among different species (Song, Park, &
Song, 2012). Blackmore and Heath (1984) have been studied the pollen
morphology of three species of the family Berberidaceae and observed
3-colpate, micro-reticulate pollen in Epimedium alpinum and syncolpate,
psilate or punctate pollen in Mahonia aquifolium and Berberis vulgaris.
Similarly, Perveen and Qaiser (2010) studied the pollen morphology of
12 species representing two genera of the family Berberidaceae from
Pakistan using light microscope (LM) and scanning electron microscope
(SEM). Their results show radially symmetrical, spheroidal, or subprolate
pollen, which was isopolar or apolar, foveolate-fossulate or sub-psilate
often regulate-reticulate. The taxonomy of Berberis is still contradictory
and perhaps the morphological variation of barberries is responsible (par-
tially) for the existing taxonomic confusion (Karlovic, Kremer, Liber,
Šatovic, Vršek, 2009). SEM and LM studies are critically important for
the taxonomic identification of complex species (Khan et al., 2018, Khan
et al., 2019). Considering these facts, the main aim of the current work
was to contribute to the knowledge of Berberis species identification,
especially on the palynological level.
TABLE 1 List of examined plants name its location and accession numbers
S# Taxa Collection site Collected by Flowering period Accession No.
1 B. aitchisonii Ahrendt Chitral (Boni) Saeed ur Rahman May–June 129521
2 B. baluchistanica Ahrendt Balochistan Zain ul Abidin and Raees Khan June 129522
3 B. calliobotrys Bien Chitral (Ziarat) Saeed ur Rahman April–June 129523
4 B. chitria Buch. -Ham Murree Saeed ur Rahman, Benazir Abbasi June–July 129524
5 B. glaucocarpa Stapf Kashmir (Rawlakot) Saeed ur Rahman, Sajad Hussain April–May 129525
6 B. kunawurensis Royle Kashmir (Ponch) Saeed ur Rahman May–July 129526
7 B. lycium Royle Dir Upper, Dir Lower, Murree Saeed ur Rahman, Fazal Ullah April–June 129527
8 B. orthobotrys Bien Kashmir (Rawlakot) Saeed ur Rahman May–June 129528
9 B. parkeriana C.K. Schneid Dir Upper Saeed ur Rahman June 129529
10 B. psodoumbellata R.Parker Gilgit Saeed ur Rahman, Siraj Khan June–July 129530
FIGURE 1 Light microscope (LM) photos of the studied pollens. (a) B. aitchisonii, (b) B. baluchistanica, (c) B. chitria, (d) B. calliobotrys,
(e) B. glaucocarpa, (f) B. kunawurensis, (g) B. lycium, (h) B. orthobotrys, (i) B. parkeriana, (j) B. psodoumbellata
1594 UR RAHMAN ET AL.

3. 2 | MATERIALS AND METHODS
2.1 | Taxon sampling, identification, preservation,
and herbarium deposition
Fresh plant specimens were collected between March and August 2016
from different areas of Pakistan. These areas were visited several times
during the flowering season to ensure the collection of taxa with mature
flowers. The collected plant species were identified with the help of taxon-
omists from Quaid-i-Azam University through personal communication.
These species were also confirmed using available literature. Flora of
Pakistan (http://www.efloras.org) was also reviewed to ensure confirma-
tion of exact species. Voucher specimens were deposited in the Herbar-
ium of Pakistan, Quaid-i-Azam University, Islamabad, Pakistan (Table 1).
2.2 | Light microscopy
For LM previously described method by Harley (1992) was used with
minor modifications. Glacial acetic acid was used to dehydrate the sam-
ple prior to acetolysis (nine parts acetic anhydride and one part sulphuric
acid). The samples were treated again with glacial acetic acid in the test
tube before washing. Anthers from mature flowers were separated care-
fully with the help of forceps and a piercing needle. The anthers were
then placed on microscope glass slides. After crushing with a small glass
rod, impurities from the glass slides were removed by using a soft camel
hair brush. A drop of glycerin jelly (safranine) was pipetted onto the glass
slides for visibility of the pollen grains. The prepared slides (four to six for
each species) were examined under Nikon microscope (E-40×/0.65).
Photographs were taken through a LM (MT 4300H) connected with
CCD digital camera (Model: HDCE-50B; Figure 1).
2.3 | Scanning electron microscopy
For SEM, pollens were dehydrated with ethanol mounted on metallic
stubs and sputter coated with gold at high vacuum (Ion-sputter Jeol
JFC-1100). Pollens were clearly observed and photographs were taken
at different magnifications with a JEOL-(Model-JSM5910) SEM at the
Physics Department, University of Peshawar, Pakistan. The observed
qualitative and quantitative characters were recorded as polar–equatorial
diameter, the shape of pollen, number of colpi, length and width of colpi,
P/E ratio, exine thickness, and so on (Table 2; Figures 3–7). Samples
(five to seven) were analyzed for each species. The terminologies used
were followed that of Erdtman (1986).
3 | RESULTS
Pollen of 10 Berberis species were studied for different quantitative and
qualitative palyno-morphological characteristics in which five species, that
is, B. aitchisonii, B. chitria, B. glaucocarpa, B. parkeriana, and B. orthobotrys
have been reported for the first time from Pakistan. For each pollen grain
(four to eight samples), qualitative and quantitative features such as pol-
len shape, size, presence or absence of colpi, colpi length and width, exine
thickness, ornamentation, aperture, pollen class, and P/E ratio were inves-
tigated (Table 2; Figures 3–7). Variation was seen in all noted characters
and the studied pollen grains were of large size. The quantitative
FIGURE 2 Scanning electron microscope (SEM) images of (a) B. aitchisonii, (b) B. baluchistanica, (c) B. chitria, (d) B. calliobotrys,
(e) B. glaucocarpa, (f) B. kunawurensis, (g) B. lycium, (h) B. orthobotrys, (i) B. parkeriana, (j) B. psodoumbellata
UR RAHMAN ET AL. 1595

4. characters are represented as a minimum–maximum (length and width)
μm (mean ± SE). Both polar and equatorial measurements were recorded
and largest pollen was observed in B. psodoumbellata (zonocolpate)
60–65 μm (62.4 ± 0.9) while the smallest one was observed in B. lycium
29–35 μm (32.2 ± 1) as shown in Table 2. Pollens were usually monad, radi-
ally symmetrical, isopolar, and predominantly a psilate-regulate sculpture
(Figure 2). Among the studied pollen grains five were colpate (pantocolpate).
Colpus length ranges from 2 μm in B. calliobotrys to 3 μm in
B. psodoumbellata. Colpus width was found to be variable and ranged from
1.2 μm in B. glaucocarpa to 2 μm in B. chitria. The shape index (P/E ratio) var-
ies from 1 μm in B. chitria to 2.6 μm in B. lycium. Shapes of pollen grains vary
from ovoid as observed in B. aitshisonii and B. chitria, spheroidal pollen
shapes were common as observed in B. orthobotrys, B. baluchistanica,
B. calliobotrys, B. kunawurensis, B. parkeriana, and B. psodoumbellata. Other
pollen types were oblate in B. lycium and spherical in B. glaucocarpa. Psilate-
regulate and foveolate-fossulate were the ornamentation observed in the
studied species. B. aitshisonii, B. chitria, B. glaucocarpa, and B. lycium have
psilate-regulate ornamentation and B. baluchistanica, B. calliobotrys,
B. orthobotrys, B. parkeriana, and B. psodoumbellata with foveolate-fossulate
ornamentation while B. kunawurensis was observed having sub-psilate orna-
mentation. Five pollen were pantocolpate while five were observed as
spiroaperturate with long spirals. Exine thickness ranges from 1 μm in
B. chitria to 2.5 μm in B. glaucocarpa and B. kunawurensis.
3.1 | Taxonomic key to the Berberis species
1 + Pollen grain pantocolpate, psilate-regulate Berberis aitchisonii.
− Pollen spiroaperturate 2
2 + Foveolate-fossulate, spheroidal Berberis
baluchistanica
− Psilate-regulate, pantocolpate 3
3 + Small colpi, ovoid, 40–45 μm in diameter Berberis chitria
− Pollen grain pantocolpate, small colpi 4
4 + Foveolate-fossulate, spheroidal, 50–53 μm in
diameter
Berberis calliobotrys
− Small copli, pantocolpate, spherical 5
5 + Psilate-regulate, exine thickness 2.5 μm Berberis
glaucocarpa
− Spiral long, sub-psilate 6
6 + Spiroaperturate, spheroidal, 41–48 μm in
diameter
Berberis
kunawurensis
− Oblate, psilate-regulate, colpi small 7
7 + Pantocolpate, exine thickness 1.9 μm Berberis lycium
− Spiroaperturate, spheroidal, 35–40 μm in
diameter
8
8 + Foveolate-fossulate, spheroidal, spiral long Berberis
orthobotrys
− Spheroidal, exine thickness 1.3 μm 9
9 + Foveolate-fossulate, spiroaperturate, 49–54 μm
in diameter
Berberis parkeriana
− Pollen 60–65 μm in diameter, spheroidal 10
10 + spiroaperturate, foveolate-fossulate,
P/E ratio 1.9
Berberis
psodoumbellata
TABLE2Qualitativeandquantitativecharactersofstudiedpollens
S#SpeciesPolardiameter(μm)Equatorialdiameter(μm)PollenclassApertureOrnamentationShapeofpollenNo.ofcolpiL.C(μm)W.C(μm)P/EratioE.T(μm)
1B.aitshisonii44–50(46.9±1.1)32–35(33.3±0.5)PantocolpateSmallcolpiPsilate-regulateOvoid52.51.41.41.5
2B.baluchistanica37–41(39±1)31–34(32.5±0.7)SpiroaperturateSpiral,longFoveolate-fossulateSpheroidal0001.22
3B.chitria40–45(42.6±0.9)28–31(29.5±0.5)PantocolpateSmallcolpiPsilate-regulateOvoid52211
4B.calliobotrys50–53(51.6±0.5)30–35(32.3±0.8)PantocolpateSmallcolpiFoveolate-fossulateSpheroidal521.31.41.2
5B.glaucocarpa55–60(57.8±0.8)33–36(34.7±0.5)PantocolpateSmallcolpiPsilate-regulateSpherica52.51.21.72.5
6B.kunawurensis41–48(44.2±1.1)21–25(23±0.7)SpiroaperturateSpiral,longSub-psilateSpheroidal0001.92.5
7B.lycium29–35(32.2±1)10.5–13.5(12±0.5)PantocolpateSmallcolpiPsilate-regulateOblate521.22.61.9
8B.orthobotrys35–40(37±0.8)25–30(27±0.8)SpiroaperturateSpiral,longFoveolate-fossulateSpheroidal0002.31.3
9B.parkeriana49–54(51.4±0.9)19–23(21±0.7)SpiroaperturateSpiral,longFoveolate-fossulateSpheroidal0002.41.3
10B.psodoumbellata60–65(62.4±0.9)29–34(31.2±0.8)SpiroaperturateSpiral,longFoveolate-fossulateSpheroidal0001.92
Abbreviations:E.T,exinethickness;L.C,lengthofcolpus;P/Eratio,polar/equatorialratio;W.C,widthofcolpus.
1596 UR RAHMAN ET AL.

5. FIGURE 3 Comparative number of
colpi in different pollen species [Color
figure can be viewed at
wileyonlinelibrary.com]
FIGURE 4 Comparative
length and width of colpi in
different pollen species [Color
figure can be viewed at
wileyonlinelibrary.com]
FIGURE 5 Comparative
polar–equatorial length in
different pollen species [Color
figure can be viewed at
wileyonlinelibrary.com]
UR RAHMAN ET AL. 1597

6. 4 | DISCUSSION
In this study, it was intended to assess Berberis pollen flora from
Pakistan using LM and SEM for its taxonomic importance in Berberis
species identification. Using different observed palyno-morphological
features, a taxonomic key was proposed for fast and correct identifi-
cation of collected Pakistani Berberis species. Because of species
identification problems in the field, the pollen morphology of family
Berberidaceae is still poorly understood. Our study sought to provide
important and useful palynological information and is the first report
of the characters studied in some species. In this part, we provide a
discussion on various variable characters observed during our study
which has implications in palynology. Price and Ayers (2008) stated
that pollen shape can be influenced by various factors such as drying
FIGURE 6 P/E ratio of different
pollen species. P/E ratio, polar/
equatorial ratio [Color figure can be
viewed at wileyonlinelibrary.com]
FIGURE 7 Variation in exine
thickness in different pollen species [Color
figure can be viewed at
wileyonlinelibrary.com]
1598 UR RAHMAN ET AL.

7. time, process, and preparation. Pollen of 10 species from the genus
Berberis in the family Berberidaceae shows a reasonable variation in
various aspects such as size, shape, pollen class, ornamentation, aper-
ture, and presence or absence of colpi. For a systematic study of a
genus, pollen characters such as shape, size, number of apertures, and
exine ornamentation are not only important but also deciding factor for
species identification (Myoung Yuon, 2012). Pollen varies in shape,
size (both polar and equatorial diameter), ornamentation, and other
aspects studied in the current study. These are the main findings
and identification characteristics on the basis of which species may
separate. The results show that LM and SEM palyno-morphological
characters are important in the identification of Berberis species. In
addition, LM and SEM studies can play an important role to identify
complex pollen flora without knowing its species and particularly in
the fields of geopalynology and aeropalynology. Perveen and
Qaiser (2010) studied pollen of 12 Berberis species and stated that
pollens were tri and pantocolpate. Our results are in accordance
with them based on palyno-morphological investigations for some
species. As pantocolpate pollen were observed in B. aitchisonii,
B. chitria, B. calliobotrys, B. glaucocarpa, and B. lycium. Our results
corroborate with information reported by Perveen and Qaiser
(2010) regarding pollen shapes as they reported spheroidal. The
current study shows that different palynological characters, studied
through LM and SEM are helpful tools in the identification of
Berberis species and will encourage palynologists to use LM and
SEM for identification of different plant species.
5 | CONCLUSION AND FUTURE
RECOMMENDATIONS
The current study demonstrates the importance of LM and SEM
studies for accurate and fast identification of Berberis flora using dif-
ferent palyno-morphological characters. The very specific objective
of the current study was simply to separate and/or identify Berberis
species based on palyno-morphological features. Of course, there
are some crucial limitations to this work, mainly, that we could not
collect the maximum number of species because of seasonal varia-
tion, that is, as flowering period varies from species to species.
In-depth studies of palynological characters are suggested to the
researchers to explore the remaining species which may lead to the
identification of some new species.
ACKNOWLEDGMENTS
The authors are thankful to Prof. Dr. Mir Ajab Khan for guidance,
encouragement, data analysis, and skilled technical support. Quaid-i-
Azam University Islamabad Pakistan is also acknowledged for provid-
ing facilities to complete this work.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interest to claim.
ORCID
Raees Khan https://orcid.org/0000-0003-1388-7141
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