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Citation: Xiao, F.; Wang, X.; Jiang, Y.;
Chen, C.; Chen, J.; Zhang, J.; Wen, Y.
Combined Morphological and
Palynological Classification for
Hibiscus syriacus L. (Malvaceae):
Construction of the Diagnostic
Classification Framework and
Implications of Pollen Morphological
Variation on Fruiting. Agronomy 2023,
13, 828. https://doi.org/10.3390/
agronomy13030828
Academic Editors: Guanglong Wang,
Lijun Ou and Aisheng Xiong
Received: 17 February 2023
Revised: 8 March 2023
Accepted: 10 March 2023
Published: 11 March 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
agronomy
Article
Combined Morphological and Palynological Classification for
Hibiscus syriacus L. (Malvaceae): Construction of the Diagnostic
Classification Framework and Implications of Pollen
Morphological Variation on Fruiting
Fen Xiao 1,2, Xiaohong Wang 1,2,*, Yun Jiang 3, Chulin Chen 1, Jiajia Chen 1, Jingwen Zhang 1and Yafeng Wen 1,2
1College of Landscape Architecture, Central South University of Forestry & Technology,
Changsha 410004, China
2Hunan Big Data Engineering Technology Research Center of Natural Protected Areas Landscape Resources,
Changsha 410004, China
3Department of Horticulture and Landscape, Shanghai Chenshan Botanical Garden, Shanghai 201602, China
*Correspondence: t19960222@csuft.edu.cn; Tel.: +86-156-1633-1155
Abstract:
Identifying useful taxonomic indicators for classifying Hibiscus syriacus L. (Malvaceae)
cultivars can help address challenges in their homonymy and synonymy. Moreover, analyzing
which pollen traits possibly lead to their successful fruiting can serve to guide the hybridization
and breeding of
H. syriacus
. For the first time, this study classified 24 cultivars of
H. syriacus
based
on 24 morphological and palynological indicators assessed for flowers, leaves, and pollen grains.
These indicators were a mixture of quantitative and qualitative traits, measured to contribute to the
identification and classification of
H. syriacus
cultivars. The results showed that the 24
H. syriacus
cultivars could be classified into 2–6 clusters according to different taxonomic criteria. The leading
diagnostic indicators were eight quantitative and eight qualitative traits, of which two new quantita-
tive traits—the width of the spine base (SW) and average of the pollen grain radius and spine length
(D-spine)—and five new qualitative traits—the amount of pollen surface spines (O-SA), whether
the petals have the red center (B-RC), whether the pollen surface ruffles strongly (B-RS), the degree
of pollen surface ruffling (O-DR), and relationship between calyx and bract (O-CB)—could be used
as defining traits for
H. syriacus
cultivars owing to their robust contribution to the classification.
The correlations between indicators for flowers, leaves, and pollen grains were explored, which
revealed that the O-SA in
H. syriacus
was strongly tied to quantitative pollen traits. Furthermore,
three qualitative morphological traits—whether the stamens are heterogeneous in terms of inner
petals (B-IP), O-CB, and whether the leaf lobing is strong (B-LL)—were correlated with partial quan-
titative pollen traits. We also found that those
H. syriacus
cultivars with micro-spines or granulate
on the pollen grain surface have higher fruiting rates; additionally, pollen diameter, spine length,
and spine spacing might also be potential factors influencing successful breeding. The insights
gained from this study could fill a key knowledge gap concerning the taxonomic criteria suitable
for distinguishing
H. syriacus
cultivars. Our findings also provide timely information on how to
understand the pollination process, especially those aspects leading to pollinator selection via pollen
grain features, which could influence breeding programs and outcomes.
Keywords:
classification; Hibiscus L.; morphology and palynology; pollen morphology variation;
quantitative and qualitative traits
1. Introduction
Hibiscus syriacus L., a species of the genus Hibiscus of the Malvaceae family, is one of
the traditional Chinese flowers and the national flower of South Korea [
1
,
2
]. It is noted
for its diverse floral morphological characteristics, wide distribution, and adaptability [
3
].
Agronomy 2023,13, 828. https://doi.org/10.3390/agronomy13030828 https://www.mdpi.com/journal/agronomy
Agronomy 2023,13, 828 2 of 18
Approximately 250 cultivars of
H. syriacus
are known worldwide [
2
]; however, suitable
cultivars for horticultural landscapes are few and limited [
4
], and still beset with challenges
of homonymy and synonymy. Therefore, a unified nomenclature and classification of
H. syriacus
cultivars is of particular importance to maximize the use of
H. syriacus
resources.
Only few studies have attempted to classify
H. syriacus
cultivars in terms of differing
color systems or pollen grains [
2
,
5
]. The spiny pollen grains of the Malvaceae are ben-
eficial for the classification of this group, mainly for Hibiscus species, as demonstrated
for
H. pernambucensis
and H. tiliaceus by the study of Oliveira et al. [
6
]. Accordingly, for
H. syriacus
cultivars that are homochromatic and have similar phenotypic characteristics,
palynological classification must be very useful. Sung et al. classified 22
H. syriacus
culti-
vars into six clusters based on six pollen feature indices [
5
]. We hypothesized that relying
solely on pollen traits for plant taxonomy is insufficient and not intuitive enough for a
species whose pollen morphology is relatively uniform. Instead, it seems more sensible
and acceptable to arrive at a quick classification directly gleaned from an examination of
external morphology, such as flower and leaf traits; however, that might not always be
compatible with palynological investigations. Taxonomic studies of this species in other
aspects such as palynological or molecular classification remain surprisingly limited. In
particular, a classification that integrates all aspects of morphology, such as the combination
of flower, leaf, and pollen features, is not yet available. Such studies are necessary as they
will allow a more intuitive and rapid classification of most
H. syriacus
cultivars and will be
essential for marketability and industrial development.
Pollen morphology has a unique structure determined by the genes of each species,
meaning it is reliable, quite stable, and generally unaffected by the external environ-
ment [
7
,
8
]. Accordingly, it is often used in plant taxonomic and palynological analyses for
identifying and classifying genera, species, or cultivars of horticultural plants [
9
–
11
]. In gen-
eral, the pollen grains with
H. syriacus
are apolar, spheroidal monads. Quantitative pollen
traits were employed to analyze the morphology of H. syriacus pollen grains in studies by
Sung et al. (pollen diameter, spine exine length, no. of spine exines, and distance between
spine exines) and Zhao et al. (spine length, width of spine base, spine distance, pollen diam-
eter, spine length/width of spine base, and pollen diameter/spine length), and both found
significant differences in spine features among cultivars [
3
,
5
]. However, taking this ap-
proach alone is inadequate; other relevant numerical traits must also be investigated, such
as the exact pollen shape and the pollen diameter in two directions in the two-dimensional
plane (X-axis and Y-axis), which are collectively needed to inform and analyze the variation
in pollen morphology among cultivars of
H. syriacus
. Quantitative morphological traits
of pollen grains are widely used in plant numerical classification [
11
,
12
]. Pollen grains
are also diverse in exine sculpturing. However, related qualitative traits, such as pollen
ornamentation and perforation, are mostly found in textual descriptions of general pollen
morphology and are rarely involved in the numerical taxonomy of pollen grains using
coding methods.
The same is possible for phenotypic characteristics of
H. syriacus
. We have already
performed a preliminary classification for
H. syriacus
based its quantitative and qualitative
floral morphological traits [
13
]; however, the contribution of its qualitative indicators to the
classification framework has not been evaluated yet. Accordingly, in this study, we aimed to
detect classification criteria by considering diverse morphological traits of a combination of
plant parts, namely flowers, leaves, and pollen grains, which could enhance the credibility
of the classification and improve its efficiency. Moreover, in general, research on reliable
qualitative indicators suitable for plant numerical classification is still limited. If they are
quantified and coded, a better classification framework can be produced than traditional
ones, which can facilitate the establishment of new taxonomic criteria, in turn [
14
]. In
earlier work, Fen et al. demonstrated that qualitative traits contributed substantially to
a robust classification within conifer pine Cathaya argyrophylla [
14
]. Hence, we sought to
apply the same methodology to H. syriacus in the present study.
Agronomy 2023,13, 828 3 of 18
Pollen export might help to form specific floral characteristics that attract pollina-
tors [
15
], thereby indirectly influencing plant fruiting and seed set rates. However, limited
knowledge is available on the co-varying relationships between the pollen morphology,
flower morphology, and the fruiting rate of
H. syriacus
. Insect pests might be among the
drivers of diversity in seed set levels among H. syriacus cultivars. Additionally, the effects
of pollen traits on the pollination mechanism or pollinator selection, or both, might be
another reason for it, yet these have not been extensively studied. The available evidence
suggests that the relationship between pollen and fertility is complex because the whole
developmental mechanism is jointly influenced by many factors, such as pollen’s grain size,
spine length, and spacing, and even the pollinator species or their ability to carry certain
amounts of pollen [16,17].
In this study, 24 morphological and palynological indicators were used together
to classify 24 cultivars of
H. syriacus
, revealing parsimonious criteria for the first time.
The variation in pollen morphology among cultivars was also assessed, as well as the
contribution of qualitative traits to the classification. We detected possible links between
pollen traits affecting the fruiting rate for
H. syriacus
. Altogether, these findings could fill a
knowledge gap concerning the classification of
H. syriacus
cultivars and provide further
insight into pollen features leading to the successful fertility enhancement of H. syriacus.
2. Materials and Methods
2.1. Plant Materials and Sources
A total of 24
H. syriacus
cultivars were collected from Hunan, Henan, Zhejiang, and
Shanghai, China, as well as from South Korea and the USA, and a resource garden was
established at the Central South University of Forestry and Technology (Table 1). From
selected healthy, clean, pest-free buds, and incompletely bloomed or blooming flowers, the
petals were separated from the anthers, with the pollen grains obtained from the anthers
using a laboratory blade. All these operations were carried out in a sterilized chamber and
on sulfate paper. Five individual plants per cultivar were taken and mixed together as one
composite sample. The collected pollen grains were dried under room conditions for 1 day,
placed in centrifuge tubes, and immediately subjected to a scanning electron microscope,
and remaining pollen materials were stored in a refrigerator at 4 ◦C.
Table 1. Information about the 24 Hibiscus syriacus cultivars.
No. Cultivar Source No. Cultivar Source
1 Marina US 13 Purple pillar US
2 White chiffon SH 14 Rubis KR
3 Pink giant SH 15 Chungmu KR
4 China chiffon SH 16 Suminokurahanagasa KR
5 Paeoniflorus HN 17 Pyonghwa KR
6 Woodbridge SH 18 Blue bird US
7 Diana SH 19 Akagionmamori KR
8 Lavender SH 20 Red heart SH
9 Hamabo SH 21 Qiancenghong HEN
10 Elegantissimus SH 22 Hongyun HEN
11 Arang HN 23 Huaban HEN
12 Lavandula chiffon SH 24 Naesarang KR
Note: SH: Shanghai, China; HN: Hunan Province, China; HEN: Henan Province, China; KR: South Korea; US: the
United States.
2.2. Scanning Electron Microscopy (SEM) and Coding the Pollen Qualitative Traits
The collected pollen grains were evenly spread on a sampling tray, then sprayed
with metal (SCD 500) and positioned under a scanning electron microscope (JSM 6360-LV)
operating at a voltage of 10.0 kV for observation, and electromicrographed at magnifications
of 150
×
, 700
×
, 1000
×
, and 1600
×
. For each cultivar, 10 mature, well-formed pollen grains
were randomly selected for measurement. In total, 240 pollen grains were measured.
Agronomy 2023,13, 828 4 of 18
A total of 12 pollen trait indicators were chosen for quantification and encoded using
the scalar quantifying method (Table 2) [
18
], including eight quantitative traits (“N”), which
are numerical and calculated in raw data form without coding. Of these, width of the spine
base (SW), spine index (SL/SW), and the average pollen grain radius and spine length
(D-spine) were evaluated for the first time for
H. syriacus
. Two binary traits (“B”), coded by
“0” or “1”, denoted negative and positive states, respectively. Two ordered multistate traits
(“O”) were each coded with consecutively arranged positive integers (“1”, “2”, “3”,
. . .
).
All four qualitative traits were new indicators (Table 2) never assessed before in
H. syriacus
cultivars, as were the quantified characteristics used to describe their degree of pollen
surface ruffling and the number of spines. The pollen description terminology used in this
study follows that developed by Erdtman et al. [19], Wang et al. [20], and Punt et al. [21].
Table 2. Twelve pollen traits and their codes for Hibiscus syriacus.
No. Characteristic Code Type Code Details
1 Pollen diameter parallel to the X-axis (D1) N /
2 Pollen diameter parallel to the Y-axis (D2) N /
3 Pollen shape ratio (D2/D1) N /
4 Length of the spine (SL) N /
5 Width of the spine base (SW) N /
6 Spine index (SL/SW) N /
7Radius of the pollen grain and spine length,
averaged (D-spine) N /
8 Spacing between spines (S-spine) N /
9Whether the pollen surface has micro-spines or
granular verrucae (B-GW) B Yes, 1; No, 0
10 Whether the pollen surface ruffles strongly (B-RS) B Yes, 1; No, 0
11 Number of pollen surface spines (O-SA) O Few (<40), 1; Medium (40–50), 2; Many (≥50), 3
12 Degree of pollen surface ruffling (O-DR) O Smooth and largely unruffled, 1; Light ruffles, 2;
Strong ruffles, 3
Note: D
1
refers to the diameter parallel to the X-axis in the observation view; D
2
refers to the diameter parallel
to the Y-axis in the observation view; the three classes of O-SA were determined by the average range values of
pollen spine numbers of 24 H. syriacus cultivars (30–60).
2.3. Flower and Leaf Morphological Indicators and Their Measurement
Eleven flower morphological indicators were employed in this study, consisting of
seven numerical (“N”) characteristics, two binary (“B”), and two ordered multistate (“O”)
characteristics (Table 3). These 11 floral morphological traits were used in our previous
morphological classification study of 27
H. syriacus
cultivars [
13
]. However, how they are
each defined, and consequently their contribution to the classification, are reported and
evaluated here for the first time. An indicator of the degree of leaf lobing was used as a
binary trait and applied here to
H. syriacus
(Table 3). The investigation methodology had
three components [
2
]: (1) MS: Survey of individual measurements of the subject plant and
its parts. (2) VG: Observation survey with one overall observation of the subject plant and
its parts. (3) VS: Observation survey with individual observations of the subject plant and
its parts.
2.4. Data Analysis
The mean, maximum, minimum, and coefficient of variation (CV) of eight numerical
pollen traits were analyzed for each cultivar (Table A1). Here, the pollen shape ratio
(D2/D1), which corresponds to the P/E (the length of the polar axis/equatorial diameter)
indicator for polar pollen grains, was classified according to the criteria proposed by
Erdtman [
22
]: oblate spheroidal (0.89–0.99), spheroidal (1.00), prolate spheroidal (1.01–1.14),
and subprolate (1.15–1.33). To compare the 24 cultivars in terms of the eight quantitative
pollen traits, a multivariate analysis of variance (MANOVA) based on the Shapiro–Wilk
Agronomy 2023,13, 828 5 of 18
normality test was used, followed by one-way analysis of variance (ANOVA), and with
Tukey’s HSD test to determine which of the 24 cultivars differed from each other.
Table 3. Twelve flower and leaf traits and their codes for Hibiscus syriacus.
No. Characteristic Investigation
Method Code Type Code Details
1Stalk length
(ST) MS N /
2Petal length
(PL) MS N /
3Petal width
(PW) MS N /
4Petals index
(PL/PW) MS N /
5Red center length
(RC) MS N /
6Length of red center line
(RCL) MS N /
7Red center index
(RCL/RC) MS N /
8Whether the stamens are heterogeneous in
terms of inner petals
(B-IP) VS B Yes, 1; No, 0
9Whether the petals have a red center
(B-RC) VS B Yes, 1; No, 0
10 Relationship of the calyx with the bract
(O-CB) VS O Shorter, 1; Near Equal Length, 2; Beyond, 3
11 Relationship of the red center line with the
red center (O-RC) VS O Near Equal Length, 1; Beyond, 2; Beyond
Obvious, 3
12 Whether the leaf lobing is strong
(B-LL) VG B Yes, 1; No, 0
Next, bivariate correlations among the 12 pollen traits were examined using Pear-
son’s r coefficient. To explore the developmental patterns between phenotypic features
of
H. syriacus
, the associations between pollen, flower, and leaf morphological indicators
were investigated. We also collected data on the fruiting rate of 11
H. syriacus
cultivars
(Table A2) [
2
], to test for pollen morphology effects on fruiting rates based on the
correlation analysis.
A classification framework based on the 24 combined morphological indicators
(Tables 2and 3) was applied to the 24
H. syriacus
cultivars. To do this, we first standardized
(STD) the raw data for each of the 24 traits to eliminate differences in their dimensions. To
this STD dataset, a principal component analysis (PCA) of 24 traits was applied, which
yielded new uncorrelated variables (PCs) [
23
–
25
]. Next, variables were compared in the
R-type clustering analysis (between-groups linkage as the clustering method and Pearson’s
r as a correlation measure), and each variable was placed as a unit in a cluster. In the
Q-type analysis (Ward’s method as the clustering method and squared Euclidean length
as the distance measure), samples were compared using uncorrelated or least correlated
variables (implemented in R-type clustering) [
26
]. Based on the clustering results, it was
possible to identify whether the qualitative indicators helped to distinguish
H. syriacus
cul-
tivars. All calculations and analyses described above were carried out using SPSS Statistics
19.0 software.
3. Results
3.1. Floral and Leaf Morphology of 24 H. syriacus Cultivars
The
H. syriacus
cultivars were evidently morphologically diverse. Two of the
24 cultivars
in this study lacked a red center (White chiffon and Diana). All seven numerical traits
varied considerably among the cultivars: ST (range: 4.00~36.30 mm; mean:
12.46 mm
), PL
(range: 39.60~70.60 mm; mean: 49.77 mm), PW (range: 22.20~52.30 mm; mean: 36.85 mm),
and PL/PW (range: 1.15~1.81; mean: 1.39). They also varied considerably among those
cultivars with a red center and red center line: RC (range: 7.30~19.60 mm; mean: 10.25 mm),
RCL (range: 7.40~37.90 mm; mean: 14.51 mm), and RCL/RC (range: 1.00~2.30 mm; mean:
1.28 mm). In addition, significant differences were found among cultivars with respect to
Agronomy 2023,13, 828 6 of 18
the presence or absence of inner petals (B-IP), the relationship of the calyx with the bract
(O-CB), the relationship of the red center line with the red center (O-RC), and the degree of
leaf lobing (B-LL) (Figure 1).
1
Figure 1.
The description and codes for five qualitative traits of Hibiscus syriacus flower and leaf
parts, for which the yellow arrow indicates the typical characteristic. Whether the stamens are
heterogeneous in terms of inner petals (B-IP): (
a
-
1
,
a
-
2
) stamens not differentiated into inner petals
(No, 0); (
b
-
1
,
b
-
2
) stamens differentiated into inner petals (Yes, 0). Whether the petals have the red
center (B-RC): (
c
-
1
,
c
-
2
) petals without the red center (No, 0); (
d
-
1
,
d
-
2
) petals with the red center
(Yes, 0). Relationship of the calyx with the bract (O-CB): (
e
-
1
) calyx shorter than bract (Shorter, 1);
(
e
-
2
) calyx almost equal to bract (Near equal length, 2); (
e
-
3
) calyx longer than bract (Beyond, 3).
Relationship of the red center line with the red center (O-RC): (
f
-
1
) red center line is almost equal to
red center (Near equal length, 1); (
f
-
2
) red center line is longer than red center (Beyond, 2); (
f
-
3
) red
center line is significantly longer than red center (Beyond obviously, 3). Whether leaf lobing is strong
(B-LL): (
g
-
1
,
g
-
2
,
g
-
3
) low degree of leaf lobing (No, 0); (
g
-
4
,
g
-
5
,
g
-
6
) high degree of leaf lobing (Yes, 1).
Agronomy 2023,13, 828 7 of 18
3.2. Overall Pollen Morphology of H. syriacus
The electromicrographs obtained via SEM were used to observe and illustrate the
pollen morphology of
H. syriacus
samples (Figure 2). Most
H. syriacus
pollen grains were
subspherical monads with a symmetrical structure, having a spiny pollen surface with
blunt spine ends. The germination pores were panpori (following the pollen terminology
of Erdtman et al. and Wang et al. [
19
,
20
]). The pollen surface appeared smooth or ruffled,
with varying degrees of ruffling, and the ornamentation was mostly striate, cerebroid, or a
mixture of striate and cerebroid. All studied samples differed markedly in terms of pollen
spines, while their pollen shapes were less variable.
2
Figure 2.
Hibiscus syriacus pollen grains electromicrographed by SEM. (
a
,
b
) An overall full view of
pollen grains as spheroidal monads with spines. (
c
) The germinal porus (panpori) on the pollen
surface. (
d
) A pollen grain with a smooth surface (pollen surface ornamentation is inconspicuous).
(
e
) A pollen grain with a ruffled surface (pollen surface with distinctive cerebroid ornamentation).
(f) A pollen grain with micro-spines or granulate on its surface.
3.2.1. Quantitative Traits of H. syriacus Pollen Grains
Details of the quantitative pollen traits of
H. syriacus
are presented in Table A1 and
Figure 3. For polar pollen grains, the equatorial diameter (E) is commonly used to evaluate
species’ pollen size [
27
,
28
]. Here, pollen diameter parallel to the X-axis (D
1
) is equivalent
to this indicator. For the 24 cultivars of
H. syriacus
, the D
1
and pollen diameter parallel
to the Y-axis (D
2
) had mean values of 124.19
µ
m and 124.85
µ
m, respectively, ranging
from
111.33 to 144.34 µm
and 112.39 to 146.03
µ
m. The D
1
(90.19–160.95
µ
m) and D
2
(90.90–158.22 µm)
values for the 240 measured pollen grains varied more significantly
(Figure 3a,b). The exact pollen shape (D
2
/D
1
) of
H. syriacus
was assessed here for the
first time, revealing less variation in this characteristic. For the 24 cultivars of
H. syriacus
,
the pollen shape (D
2
/D
1
) was frequently oblate spheroidal (38%) and prolate spheroidal
(46%) with a mean value of 1.01, ranging from 0.95 to 1.07. For the 240 measured pollen
grains, the pollen shape likewise mostly presented as oblate spheroidal (36%) and prolate
spheroidal (54%). The D
2
/D
1
reached its maximum in the cultivar White chiffon (1.18), and
its minimum in the cultivar Woodbridge (0.73) (Figure 3c).
Agronomy 2023,13, 828 8 of 18
2
Figure 3.
Histograms of eight quantitative traits of Hibiscus syriacus based on 240 measured pollen
grains. Shown is the normalized frequency of D
1
, D
2
, D
2
/D
1
, SL, SW, SL/SW, D-spine, and S-spine
for each sample of each
H. syriacus
cultivar. (
a
) The D
1
ranged from 90.19 to 160.95
µ
m, peaking at
120~130
µ
m. (
b
) The D
2
ranged from 90.90 to 158.22
µ
m, peaking at 120~130
µ
m. (
c
) The D
2
/D
1
ranged from 0.73 to 1.18, peaking at 1.0~1.05. (
d
) The SL ranged from 10.73 to 28.24
µ
m, peaking at
17.00~17.50
µ
m. (
e
) The range value of SW ranged from 6.91 to 18.61
µ
m, peaking at 9.00~10.00
µ
m.
(
f
) The SL/SW ranged from 0.84 to 3.09, peaking at 1.50~2.00. (
g
) The D-spine ranged from 1.92 to
6.00
µ
m, peaking at 3.50~4.00
µ
m. (
h
) The S-spine ranged from 9.98 to 32.75
µ
m, peaking at 20~25
µ
m.
Length of the spine (SL) was 17.40
µ
m, on average, and varied widely among the
24
H. syriacus
cultivars (13.42~25.04
µ
m), especially across the 240 measured samples
(10.73–28.24
µ
m) (Figure 3d). The width of the spine base (SW) is described here for
the first time. This trait measured 9.80
µ
m on average, ranging from 8.25 to 15.00
µ
m
among the
24 cultivars
, while showing a near four-fold difference across the 240 samples
(4.91~18.61
µ
m) (Figure 3e). The spine index (SL/SW) was 1.83, on average, ranging from
1.06 to 2.29 among the
24 cultivars
, while spanning from 0.84 to 3.09 among the 240 studied
pollen grains (Figure 3f).
The average of pollen grain radius and spine length (D-spine) was first used to describe
the pollen characteristic for
H. syriacus
. D-spine values ranged from 2.22 to 5.38, with a
mean of 3.67; for the 240 pollen grains of
H. syriacus
, the D-spine attained its maximum in
the cultivar Woodbridge (6.00) and its minimum in the cultivar Naesarang (1.92) (Figure 3g).
The spacing between spines (S-spine) was also determined in this study, having a mean
value of 21.14
µ
m (12.85~27.37
µ
m) among the 24 cultivars. Across the 240 pollen grains,
S-spine was largest in the cultivar Elegantissimus (32.75
µ
m) and smallest in the cultivar
Pink giant (9.98 µm) (Figure 3h).
3.2.2. Qualitative Traits of H. syriacus Pollen
Four new qualitative traits were employed to describe the characteristics of the pollen
surface for
H. syriacus
(Figure 4). We found relatively few cultivars with micro-spines or
granulate verrucae, these amounting to 20.83% of the samples (Figure 4(a-1,a-2,b-1,b-2)).
The spine numbers varied considerably between cultivars (30~54) (Figure 4c,(d-1,d-2),e),
being greatest in the cultivar Pink giant. We divided this trait into three classes; most of the
cultivars (70.83%) featured a lower number of spines. In addition, a significant difference in
pollen surface ruffling was found among the cultivars, with ruffled surfaces predominating.
Two qualitative indicators were used to code their ruffling features (
Figure 4f,g,(h-1,h-2)).
Agronomy 2023,13, 828 9 of 18
We used the apparent degree of surface ornamentation to describe the roughness or smooth-
ness of the pollen grain surface.
3
Figure 4.
The description and codes for four qualitative traits of Hibiscus syriacus pollen grains, where
the red arrow indicates the typical characteristic. Whether the pollen surface has granular verrucae
(B-GW): (
a
-
1
,
a
-
2
) pollen exine is smooth or ruffled without micro-spines or granular verrucae
(No, 0)
;
(
b
-
1
,
b
-
2
) pollen exine has micro-spines or granular verrucae (Yes, 0). Number of pollen surface
spines (O-SA): (
c
) pollen surface spines less than forty in number (<40, 1); (
d
-
1
,
d
-
2
) pollen surface
spines varying between forty and fifty (40~50, 2); (
e
) pollen surface spines greater than or equal to
fifty in number (
≥
50, 3). Whether the pollen surface ruffles strongly (B-RS): (
f
,
g
) pollen surface not
strongly ruffled, smooth, or slightly ruffled (pollen surface ornamentation is inconspicuous or with a
small number of ornaments) (No, 0); (
h
-
1
,
h
-
2
) pollen surface strongly ruffled (pollen surface with
distinctive ornamentation) (Yes, 1). Degree of pollen surface ruffling (O-DR): (
f
) pollen surface smooth
and free of ruffles (pollen surface ornamentation is inconspicuous) (smooth and largely unruffled, 1);
(
g
) pollen surface is slightly ruffled (pollen surface with a small number of ornaments) (light ruffles,
2); (
h
-
1
,
h
-
2
) pollen surface strongly ruffled with striate or a mixture of striate and cerebroid (pollen
surface with distinctive ornamentation) (strong ruffles, 3).
3.3. Variation and Correlation of Pollen Morphology between the 24 H. syriacus Cultivars
The range, mean, and cv for eight quantitative traits revealed pronounced differences
among the 24 cultivars of
H. syriacus
(Table A1). For example, Chungmu displayed a
high level of variation in the traits P and S-spine, while the cultivars Diana, Woodbridge,
White chiffon, Qiancenghong, Rubis, and Pyonghwa showed a high level of variation
in the traits D
1
, D
2
/D
1
, SL, SW, SL/SW, and D-spine, respectively. Eight quantitative
traits were simultaneously tested for a difference among cultivars. This MANOVA result
revealed a significant difference among 24 cultivars (Wilk’s
λ
= 0.01, F = 7.318, p< 0.01). The
Agronomy 2023,13, 828 10 of 18
follow-up ANOVA results for the eight quantitative traits were as follows: D
1
(F = 8.632),
D
2
(F = 8.434), D
2
/D
1
(F = 2.913), SL (F = 20.768), SW (F = 12.669), SL/SW (F = 12.228),
D-spine (F = 16.262), and S-spine (F = 7.152), which demonstrated there was significant
variation among the studied cultivars, as all the eight traits exhibited a high level of
statistical significance
(p< 0.01).
According to our post hoc study (Turkey’s HSD test) of
the 24 cultivars, Paeoniflorus and Naesarang were distinct from the other cultivars by a
separate subset of trait A. Other separate subsets were: Pink giant and Woodbridge for trait
SL/SW, Pink giant for trait S-spine, and Woodbridge for trait D-spine.
Correlations between pollen quantitative and qualitative traits of
H. syriacus
were also
determined. We found more correlations that were significant between quantitative traits
than qualitative traits (Figure 5). The trait D-spine was correlated with all six quantitative
traits except for SW, which had positive correlations with traits D
1
(0.64, p< 0.01) and
D
2
(0.46, p< 0.05) and negative correlations with traits D
2
/D
1
(–0.43, p< 0.05), SL (–0.91,
p< 0.01), SL/SW (–0.82, p< 0.01), and S-spine (–0.54, p< 0.01). Meanwhile, D-spine showed
a significant positive correlation with only one qualitative trait (O-SA, 0.59, p< 0.01). Trait
P/E had no significant correlation with any traits except for D-spine. Among the qualitative
traits, O-SA was correlated with all the quantitative traits except for P/E. In addition, O-DR
exhibited a significant positive correlation with B-RS, with both indicators describing the
surface ruffling characteristics of H. syriacus pollen grains.
4
Figure 5.
Heatmap of correlations between eight quantitative and four qualitative traits of pollen
grains of Hibiscus syriacus. The degree of a correlation is indicated by its shaded coloring, with
positive or negative numbers in the small square boxes indicating positive or negative correlations
between row and column traits, respectively. * p< 0.05, ** p< 0.01.
3.4. Correlations among Pollen, Flower, and Leaf Morphological Characteristics of
H. syriacus Cultivars
We tested for correlations between quantitative pollen traits and morphological traits
of flowers and leaves of
H. syriacus
(Table 4). The traits D
2
, D
2
/D
1
, SW, and S-spine did not
Agronomy 2023,13, 828 11 of 18
correlate with any flower or leaf traits. Trait E exhibited a significant positive correlation
with O-CB (0.550, p< 0.01). Trait SL showed positive correlation with both ST (0.496,
p< 0.05) and B-IP (0.434, p< 0.05), but negative correlation with PL (–0.410, p< 0.05) and
B-LL (–0.478, p< 0.05). Trait SL/SM was negatively correlated with PL (–0.565, p< 0.01)
and O-CB (–0.485, p< 0.05), yet positively correlated with B-IP (0.587, p< 0.01). Finally,
D-spine was positively correlated with three other traits: PL (0.483, p< 0.05), O-CB (0.561,
p< 0.01), and B-LL (0.506, p< 0.05), while negatively correlated with B-IP (–0.495, p< 0.05).
Table 4.
Correlation matrix of pollen vis-à-vis morphological traits of flower and leaf in
Hibiscus syriacus.
Morphological Traits Pollen Quantitative Traits
D1D2D2/D1SL SW SL/SW S-Spine D-Spine
ST
Flower traits
−0.008 0.111 0.247 0.496 * 0.137 0.265 0.336 −0.387
PL 0.279 0.171 −0.233 −0.410 * 0.330 −0.565 ** −0.383 0.483 *
PW 0.179 0.081 −0.197 −0.080 0.324 −0.292 −0.257 0.173
PL/PW −0.020 0.002 0.029 −0.342 −0.213 −0.120 0.025 0.246
RC 0.006 0.012 −0.128 0.060 0.379 −0.210 0.049 0.037
RCL −0.089 −
0.151
−0.115 0.094 0.309 −0.149 0.063 −0.053
RCL/RC −0.033 −
0.152
−0.256 0.121 0.077 0.060 −0.014 −0.072
B-IP −0.301 −
0.198
0.237 0.434 *−0.302 0.587 ** 0.204 −0.495 *
B-RC 0.086 0.018 −0.190 0.148 0.105 0.089 −0.004 −0.068
O-CB 0.550 ** 0.381 −0.376 −0.403 0.219 −0.485 *−0.194 0.561 **
O-RC −0.136 −
0.182
−0.038 −0.164 −0.001 −0.177 −0.094 0.123
B-LL Leaf trait 0.223 0.062 −0.338 −0.478 *−0.239 −0.220 −0.110 0.506 *
Note: Pearson’s linear correlation coefficient values between eight pollen quantitative traits and flower and leaf
traits. Corresponding details can be found in Tables 2and 3. The positive or negative numbers in bold indicate
positive or negative correlations between row and column traits, respectively. * p< 0.05, ** p< 0.01.
We also tested for correlations between the 12 pollen traits (Table 2) and the fruiting
rates based on data from 11 cultivars of
H. syriacus
(Table A1). These results showed that
only B-GW was positively correlated with fruiting rate (0.678, p< 0.05). This was an
encouraging finding and demonstrated that pollen surface spines and granulates were
intrinsically related to pollination.
3.5. Clustering Analysis of 24 H. syriacus Cultivars Based on the 24 Combined
Morphological Traits
The PCA results for the 24
H. syriacus
morphological traits showed a total contribution
of 84.55%, in which seven principal components (PCs) were derived. The crucial traits of
PC1 (25.81%) were D
2
and D
1
; likewise, for PC2 (15.02%), they were SL and D-spine; for
PC3 (11.89%), they were RC, RCL, RCL/RC, and B-RC; for PC4 (10.92%), it was PW; for
PC5 (10.56%), they were B-RS and O-DR; for PC6 (6.07%), it was O-CB; and for PC7 (4.27%),
it was B-GW. PC1, PC2, PC5, and PC7 were indicators related to pollen morphological char-
acteristics, while PC3, PC4, and PC6 were related to flower morphological characteristics.
R-type clustering was used to convey the rationality of indicator selection (Figure 6a).
The pairs of traits O-DR and B-RS, RC and RCL, PL and PW, and D
1
and D
2
were clustered
at close distances, indicating an equal contribution to the classification of
H. syriacus
. In
order to better interpret and compare the effects of traits on the classification, we retained
these indicators and used them in a follow-up Q-type clustering analysis.
The results obtained from Q-type clustering revealed that
H. syriacus
cultivars could
be divided into 2–6 clusters based on different indicators (Figure 6b):
(1) The 24 cultivars could be divided into two clusters at the grade bond line L1
(D = 20.00) based on the main indicators (D
2
, D
1
, SW, O-SA, D-spine, and SL). The first
cluster included two cultivars (Pink giant and Woodbridge), which presented larger values
for the traits D
2
, D
1
, SW, O-SA, and D-spine, but a smaller value for trait SL. The second
cluster comprised the remaining 22 cultivars.
Agronomy 2023,13, 828 12 of 18
(2) The 24 cultivars could be divided into three clusters at the grade bond line L2
(D = 18.43). The first cluster still consisted of Pink giant and Woodbridge. The remaining
22 cultivars were further classified into two more clusters based on their flower traits (B-RC,
RCL, RC, RCL/RC). The second cluster harbored two cultivars (White chiffon and Diana),
whose petals lack a red center. The remaining 20 cultivars with the red center constituted
the third cluster.
(3) The 24 cultivars could be divided into four clusters at the grade bond line L3
(D = 15.00). In the first cluster was Pink giant and Woodbridge, and in the second cluster
was White chiffon and Diana. Then, the other 20 cultivars were further classified into
two clusters based on the SL, SW, PW, D-spine, D
2
, D
1
, O-SA, and O-CB traits. The third
cluster had seven cultivars: Pyonghwa, Huaban, Paeoniflorus, Narsarang, Purple pillar,
Elegantissimus, and Arang; the fourth cluster entailed the remaining 15 cultivars. The
cultivars forming the third cluster presented larger values for SL (17.22~25.04
µ
m), SW
(8.33~12.00
µ
m), and PW (32.20~50.40 mm), and smaller values for D-spine (2.22~3.32), D
2
(112.39~129.51
µ
m), and D
1
(111.33~127.13
µ
m) when compared with those of the fourth
cluster. Moreover, the cultivars of the third cluster all had a small number of spines (O-SA,
code: 1), and their calyx length was shorter than that of the bract (O-CB, code: 1).
(4) The 24 cultivars were classified into five clusters based on the grade bond line
L4 (D = 10.00)—with the same first three clusters for L4 as for L3. The other 15 cultivars
were further split into two clusters based on two pollen grain traits (B-RS and O-DR). Two
cultivars (Marina and Qiancenghong) were included in the fourth cluster as their pollen
grains’ surface was weakly and slightly ruffled. The fifth cluster included the remaining
13 cultivars, all of which exhibited strong ruffles.
(5) The 24 cultivars were divided into six clusters based on the grade bond line L5
(D = 7.50). The clustering results at L5 were the same as those at L4, except for the latter’s
fourth cluster. However, based on two pollen traits (B-RS and O-DR), the fourth cluster
at L4 was further divided into two clusters, of which Elegantissimus and Arang grouped
together as the fifth cluster as their pollen grains’ surface was weakly ruffled. By contrast,
the remaining five cultivars were strongly ruffled and thus formed the sixth cluster.
4
Figure 6.
Tree plot of R-type cluster and Q-type cluster of Hibiscus syriacus. (
a
) R-type conducted on
24 combined morphological traits based on between-groups linkage method. (
b
) Q-type conducted
on Hibiscus syriacus 24 cultivars based on ward method.
Agronomy 2023,13, 828 13 of 18
4. Discussion
4.1. Pollen Morphological Variation of H. syriacus Cultivars and Novel Diagnostic Pollen Traits
Pollen variation diversity can contribute to taxonomic and phylogenetic analyses
within the Malvaceae family, whose species can be identified by their spine
characteristics [
3
,
29
–
31
]. The pollen of Malvaceae plants is large, being 50~242
µ
m in diam-
eter, with spines and panpori on the pollen surface, while Hibiscus pollen generally varies in
size, from 118 to 252.5
µ
m in diameter [
20
]. Based on the Palynological Database,
H. syriacus
pollen grains were recorded as a spheroidal shape of large size (>100
µ
m) [
32
], and accord-
ing to Sung et al. and Zhao et al. [
3
,
5
], the
H. syriacus
pollen grain diameters ranged from
111.33 to 144.34
µ
m and 111.65 to 148.98
µ
m, respectively, while both found high variabil-
ity in its spine features. In contrast, two indicators of pollen diameter were introduced
in this study, D
1
and D
2
, with ranges from 111.33 to 144.34
µ
m and
112.39 to 146.03 µm
,
respectively, while the ratio of the two indicators (D
2
/D
1
) described the pollen grain
shape. We observed little variation in the pollen shape (D
2
/D
1
), with that being principally
oblate spheroidal (38%) and prolate spheroidal (46%). Four quantitative traits (D
2
, D
1
,
SW, SL, and D-spine) and three new qualitative traits (O-SA, O-DR, and B-RS) examined
here could thus be considered as useful diagnostic pollen traits for the classification of
H. syriacus cultivars.
The trait D–spine takes pollen diameter parallel to the X-axis (D
1
) and spine length
(SL) into account. Andrade et al. applied this index to distinguish mature pollen grains
of H. rosa-sinensis, and it was sufficiently accurate to distinguish the species from others
in the same family [
30
]. Here, we employed the D-spine trait for the first time to detect
variation between
H. syriacus
cultivars, finding that it made a prominent contribution to
the diagnosis and classification of the 24 cultivars, as it showed significant variation among
them (range: 2.22~5.38). We suggest the D-spine trait may be an essential pollen trait that
can assist in distinguishing species or cultivars of Hibiscus. Furthermore, the SW trait,
as a new quantitative trait, differed markedly between
H. syriacus
cultivars. At the same
time, SW exhibited a positive correlation with D
2
, D
1
, and O-SA, indicating that cultivars
with larger pollen sizes or more spines on their pollen surfaces tend to be accompanied
by a broader spine base. The spine index (SL/SW) was also applied here for the first time;
however, a weaker contribution to the classification was found for it.
The contribution of qualitative indicators to plant classification can be easily over-
looked when they are not quantified. A few qualitative indicators are typically used to
describe pollen morphology, usually the pollen surface germinal colpus or porus, and exine
ornamentation [
20
,
33
]. Spine abundance is often used as a numerical trait for description,
as done for
H. syriacus
and H. rosa-sinensis [
3
,
30
]. Meo et al. found that the number of
spine rows between colpi was also the taxonomically important characteristic of Parthenium
hysterophorus [
34
]; however, it is weak in its ability to distinguish between plant species
or cultivars. Instead, we employed a qualitative trait (O-SA) here to describe the spine
abundance, which played a significant role in the classification, with its use as the primary
basis for classification at L1 and L3. Furthermore, because the trait O-SA correlated with
most quantitative pollen traits, we could assume that pollen grains with large sizes, short
spines, or wide spine bases generally also have a higher number of spines. In addition, we
employed two new qualitative indicators to describe pollen surface ruffling in two ways.
The first is the trait B-RS, for which a smooth or slightly ruffled surface was defined as
not strongly ruffled. Most cultivars had a ruffled pollen surface, the five exceptions being
Marina, Woodbridge, Elegantissimus, Arang, and Red heart. The second is O-DR, which
has three levels of pollen surface ruffling (smooth, lightly ruffled, and extremely ruffled).
This trait could further help to distinguish between smooth and slightly ruffled as key
features, so that the five cultivars mentioned above could be further clustered into two
clusters (one for the first three and one for the last two).
Agronomy 2023,13, 828 14 of 18
4.2. Combining Morphological Indicators Helps to Better Distinguish Cultivars of H. syriacus and
the Contribution of Qualitative Indicators to Clustering
From the results for the Q-type clustering of
H. syriacus
cultivars, the main criteria
classification was based on pollen traits at the L1 grade line: D
2
, D
1
, SW, D-spine, SL, and
O-SA. At the L2 grade line, the traits related to the red center of petals (B-RC, RC, RCL,
and RCL/RC) enabled us to further classify the remaining 22 cultivars: in this way, two
cultivars without the red center were identified (White chiffon and Diana). Prior evidence
suggested these (B-RC, RC, RCL, and RCL/RC) indicators could effectively distinguish
H. syriacus
cultivars as PC3 (11.89%) in a classification based on floral morphological traits
alone [
13
]. The present study’s results lend further support to using these traits as an
independent basis for
H. syriacus
cultivars in a combined classification, in that they were
uncorrelated with pollen indicators. At grade line L3, those cultivars with a red center could
be further classified into two clusters based on their pollen (SL, SW, D-spine, D
2
, D
1
, and
O-SA) and floral traits (PW and O-CB). Given that O-CB was found positively correlated
with E, we may presume those H. syriacus cultivars with smaller pollen grains are usually
accompanied by a calyx shorter than the bract. Knowledge of this correlation can enable
horticulturists to promptly identify
H. syriacus
cultivars, but admittedly more samples are
still needed to support this finding. In addition, at grade lines L4 and L5, the qualitative
pollen traits related to ruffle features (B-RS and O-DR) were the main classification criteria
elucidated. Although the R-type cluster results indicated these two traits are closely related,
we kept both since O-DR provided a more in-depth distinction of results generated via
B–RS. Lastly, a contribution of the leaf morphological trait (B-LL) to
H. syriacus
classification
was not found.
Overall, the results of this study demonstrate that a number of
H. syriacus
cultivars
are distinguishable using only quantitative traits. Accordingly, coding and quantifying
quantitative traits could contribute to the identification of a greater number of cultivars.
Similar results and patterns were reported for Cathaya argyrophylla [
14
]. We propose
that the integrated use of morphological indicators of species could generate a broader
taxonomic basis.
4.3. Effects of H. syriacus Pollen Traits on Fruiting
Pollen morphological characteristics can influence pollination and breeding, as demon-
strated by studies by Mccallum et al., Mendoza et al., and Xia et al., as reported for Ipomoea
purpurea,Orius laevigatus, and Ottelia acuminata [
27
,
35
,
36
]. Pollen, seed, and fruit charac-
teristics often have positive correlations [
37
–
39
]. The mechanisms by which pollen spines
affect fruiting success are complex and challenging to elucidate; they may be related to
the spine distribution pattern, spine length, spine density, or even the space distance be-
tween spines [
17
,
40
]. It would be helpful if we could provide breeding guidance based
on the relationships between pollen morphology and pollination or fruit set. We found
that the trait B-GW is positively correlated with fecundity (0.678, p< 0.05), which indicates
that high fruiting rates occur in cultivars that have micro-spine or granular verrucae on
their pollen surface; e.g., Blue bird (26.2%) and Red heart (26.0%). Of the 24 studied culti-
vars, just five harbored this feature (Arang, Rubis, Suminokurahanagasa, Akagionmamori,
and Qiancenghong).
Pollen grains with spines and granulate verrucae may adhere to the long hairs of
bees for transport, as was previously found for an Pavonia sp. (Malvaceae) [
41
]. How-
ever, either pollen size or echinate exine structure alone was not an excellent factor for
pollen collectability [
40
] since the pollination mechanism is complex and related to pollen-
collecting bee species, with different genera of bees showing divergence in their collecting
behavior [
40
,
42
–
44
]. Fruit set is likely also affected by insect pests [
45
,
46
], and whether the
incidence of insect pests correlates with pollen morphology or a specific substance warrants
further investigation.
Furthermore, floral pollinators’ behaviors reflect a selection of pollen traits. Evidence
from a study by Lynn et al. of Taraxacum ceratophorum revealed that bumblebee pollinators
Agronomy 2023,13, 828 15 of 18
were prone to picking up pollen grains within a narrow distribution of spine distance [
17
],
indicating that a certain spine distance can favor the likelihood of pollination (i.e., a
trait selected because it enhances plant fitness vis-à-vis pollinator community). In this
respect,
H. syriacus
, a species pollinated chiefly by bumblebees [
47
], probably has the
same selection pattern as described above, and we observed that those cultivars with
high fruiting rates also featured a smaller value for spine spacing (S-spine) (Blue bird,
20.78
µ
m; Red heart, 20.86
µ
m) than the average for all 24 cultivars (21.14
µ
m). In addition
to pollinators’ selection mechanism, which concerns pollen size, spines, and spacing, a
pollinator’s body size also determines the amount of pollen it can carry, as does its degree
of hairiness [
16
,
48
]. A model showed that the poor interaction bond between pollenkitt-free
spines and pollenkitt-covered exine might weaken the compacting within pollen storage
organs; this pattern was prominent on large pollen grains as they reduced the contact
surface and thus affected pollen collection [
49
]. Spine length might also influence pollen
adherence to a pollinator’s body, but though proven, it is not a significant trait for Taraxacum
ceratophorum during its pollen pickup [
17
]. In this study, we obtained an interesting finding
that those cultivars with high fruiting rates mainly had smaller values for both E and spine
length (SL), e.g., Blue bird and Red heart, which had high fruit sets, whereas their D1was
123.36 or 124.52
µ
m, respectively. By contrast, cultivars with higher D
1
values, namely
Pink giant (141.09
µ
m) and Woodbridge (144.34
µ
m), had very low fruit sets, at 4.4% and
1.5%, respectively. The traits S-spine and SL did not directly correlate with fruiting rate
in our correlation analysis, but they are worth discussing, as effective explanations for
this phenomenon are still limited. In this respect, according to the mechanical-defense
hypothesis, bumblebees do not collect pollen with bent spines [
49
]. We observed that some
H. syriacus
cultivars produce pollen with bent spines, but the limited data available in this
study prevented us from verifying this hypothesis. Therefore, more
H. syriacus
samples
are needed for further testing, to better discern and interpret the relationships between the
pollination mechanism and breeding system of H. syriacus.
5. Conclusions
Significant variation among studied
H. syriacus
cultivars was demonstrated, especially
in their pollen spine features. The derived classification scheme based on flowers and pollen
morphological indicators let us classify 24
H. syriacus
cultivars into 2–6 clusters. The main
diagnostic quantitative traits are D
2
, D
1
, SW, SL, D-spine, RC, RCL, and RCL/RC, while
the main diagnostic qualitative traits are O-SA, B-RC, B-RS, O-DR, and O-CB. Among all of
those, two new quantitative traits (SW and D-spine) and five new qualitative traits (O-SA,
B-RC, B-RS, O-DR, and O-CB) made a robust contribution to the classification of
H. syriacus
cultivars. The number of pollen spines (O-SA) of
H. syriacus
is strongly correlated with
its quantitative pollen traits, and three floral (B-IP and O-CB) and leaf (B-LL) phenotypic
traits are correlated with certain quantitative pollen traits. The trait B-GW is correlated
with fruiting rate, and pollen diameter parallel to the X-axis (D
1
), spine length (SL), and
spine spacing (S-spine) might all be potential factors that lead to successful breeding in
H. syriacus.
Author Contributions:
Conceptualization, X.W. and F.X.; methodology, F.X.; software, F.X., J.C. and
J.Z.; formal analysis, F.X.; investigation, F.X. and Y.J.; resources, X.W. and Y.W; writing—original draft
preparation, F.X.; writing—review and editing, X.W.; funding acquisition, X.W., Y.W. and C.C. All
authors have read and agreed to the published version of the manuscript.
Funding:
This research was funded by the Key Discipline of the State Forestry Administration
(LinRenFa [2016] No. 21), “Double First-Class” Cultivation Discipline of Hunan Province (Xi-
angJiaoTong (2018) No. 469), and Scientific Research Project of Hunan Provincial Department
of Education (21B0224).
Institutional Review Board Statement: The study did not require ethical approval.
Informed Consent Statement: The study did not involve humans.
Agronomy 2023,13, 828 16 of 18
Data Availability Statement: Data available from the author.
Conflicts of Interest: The authors declare no conflict of interest.
Appendix A
Table A1.
The minimal, maximal, mean values and coefficient of variation (cv) for eight quantitative
pollen traits of Hibiscus syriacus.
Samples D1D2P/E SL
Mean Range cv Mean Range cv Mean Range cv Mean Range cv
1 130.70 110.89–145.87 7.67% 124.04 104.11–131.79 6.47% 0.95 0.82–1.13 8.75% 15.07 13.32–17.91 8.94%
2 123.03 110.35–136.40 9.52% 131.06 120.92–139.25 5.04% 1.07 0.93–1.18 7.50% 16.66 13.14–22.76 17.09%
3 141.09 132.97–145.58 2.45% 146.03 132.60–158.22 6.32% 1.03 0.97–1.13 4.91% 15.65 13.25–17.80 10.45%
4 127.95 122.82–134.50 3.31% 129.78 123.55–135.09 2.88% 1.01 1.00–1.04 1.06% 16.81 12.48–20.26 14.83%
5 127.13 119.37–133.47 3.60% 129.30 110.46–138.07 6.04% 1.02 0.85–1.06 6.31% 23.70 20.15–27.10 8.82%
6 144.34 133.46–160.95 5.64% 139.12 112.37–148.10 9.58% 0.97 0.73–1.05 13.09% 13.42 12.09–15.49 8.25%
7 120.83 98.91–135.14 13.77% 117.76 103.68–128.76 9.53% 0.98 0.93–1.05 4.77% 15.31 13.84–17.55 8.02%
8 123.44 112.96–140.53 8.66% 121.44 116.96–126.63 3.09% 0.99 0.90–1.04 5.43% 15.55 12.73–19.05 12.33%
9 126.29 119.89–129.83 2.88% 125.44 120.65–129.62 2.11% 0.99 0.97–1.03 1.90% 19.56 16.64–22.36 9.50%
10 125.19 105.58–133.40 9.44% 129.51 105.82–145.29 10.70% 1.03 0.99–1.10 3.71% 20.15 15.08–23.40 11.81%
11 119.08 90.19–131.66 10.38% 123.24 105.56–138.39 9.84% 1.04 0.99–1.17 5.21% 19.73 17.73–21.13 4.98%
12 118.66 112.55–125.56 4.22% 118.32 104.18–127.02 5.91% 1.00 0.92–1.07 5.27% 16.73 14.02–19.65 11.38%
13 114.59 104.39–128.85 5.31% 119.76 111.69–129.57 4.43% 1.05 0.97–1.15 5.04% 19.33 14.68–23.21 13.45%
14 123.16 113.81–135.99 5.95% 126.94 119.62–134.17 4.04% 1.03 0.96–1.10 4.17% 14.45 11.50–18.30 15.58%
15 122.76 90.72–137.44 12.08% 119.16 90.9–135.96 12.10% 0.97 0.90–1.05 4.50% 14.20 11.32–17.95 14.25%
16 116.54 109.08–127.62 5.93% 119.18 111.99–127.62 4.35% 1.02 0.92–1.12 5.35% 15.06 12.14–19.45 16.80%
17 117.64 108.77–128.85 4.93% 116.95 113.16–121.18 1.99% 1.00 0.91–1.06 4.53% 20.24 13.58–24.71 15.39%
18 121.06 114.61–130.31 3.80% 123.36 113.45–138.66 4.15% 1.02 0.95–1.10 5.40% 16.13 12.71–19.12 13.25%
19 126.71 102.54–144.72 8.79% 126.80 103.63–131.22 7.64% 1.00 0.95–1.04 2.75% 18.54 17.05–22.53 8.84%
20 125.33 117.65–138.44 5.55% 124.52 111.99–137.85 5.34% 0.99 0.94–1.04 3.75% 16.08 10.73–19.42 15.35%
21 138.20 132.71–143.62 2.54% 134.37 131.24–138.45 1.80% 0.97 0.94–1.00 2.00% 18.47 16.41–20.07 4.99%
22 121.18 103.63–125.44 5.26% 121.79 99.99–138.45 7.65% 1.00 0.96–1.11 4.22% 14.54 12.00–19.55 16.41%
23 114.31 102.90–123.26 5.79% 112.39 102.9–118.68 5.38% 0.98 0.93–1.02 3.45% 17.22 14.38–18.61 8.40%
24 111.33 98.55–120.09 7.61% 116.22 97.27–126.29 9.04% 1.04 0.99–1.10 3.58% 25.04 22.89–28.24 6.78%
Samples SW SL/SW D–spine S–spine
Mean Range cv Mean Range cv Mean Range cv Mean Range cv
1 8.59 6.61–10.15 13.85% 1.79 1.31–2.29 18.30% 4.36 3.50–5.01 10.76% 20.36 16.26–23.51 11.20%
2 9.01 7.13–10.67 12.35% 1.85 1.53–2.37 11.96% 3.77 2.93–5.19 17.18% 22.09 16.52–27.74 16.03%
3 15.00 10.99–18.61 16.54% 1.06 0.90–1.37 16.21% 4.56 3.78–5.43 11.45% 12.85 9.98–19.73 20.57%
4 8.37 6.73–9.86 10.84% 2.01 1.78–2.42 8.71% 3.88 3.12–4.99 15.57% 19.19 14.80–22.74 12.66%
5 10.43 8.99–12.14 10.53% 2.29 1.97–2.61 10.20% 2.70 2.23–3.24 10.59% 20.49 16.14–26.44 16.78%
6 12.73 11.54–14.36 6.43% 1.06 0.84–1.28 11.54% 5.40 4.72–6.00 7.56% 18.75 16.20–23.38 12.21%
7 9.47 8.13–11.56 11.07% 1.63 1.36–1.83 9.18% 3.96 3.33–4.83 13.62% 20.28 14.57–27.85 18.96%
8 8.96 6.94–10.51 12.32% 1.75 1.57–2.11 11.10% 4.00 3.62–4.62 8.81% 18.98 15.15–22.57 15.51%
9 9.93 8.12–12.70 17.00% 2.02 1.38–2.63 18.93% 3.26 2.82–3.76 10.69% 25.63 20.22–31.54 14.08%
10 9.66 8.73–11.28 8.16% 2.09 1.64–2.36 9.77% 3.15 2.48–4.38 17.25% 27.37 20.57–32.75 13.79%
11 10.66 9.32–11.72 7.34% 1.86 1.60–2.12 8.49% 3.03 2.23–3.46 12.69% 24.14 15.73–30.56 19.24%
12 9.18 6.57–11.33 14.99% 1.84 1.40–2.22 12.72% 3.59 2.93–4.26 11.79% 18.04 11.51–21.48 16.09%
13 12.00 9.19–14.48 13.54% 1.66 1.01–2.24 23.53% 3.01 2.41–3.56 12.51% 23.74 16.28–30.04 20.59%
14 9.64 6.50–12.36 19.02% 1.56 0.93–2.42 28.34% 4.36 3.17–5.65 17.97% 18.57 14.37–26.65 21.80%
15 9.24 7.28–12.56 15.69% 1.56 1.21–1.88 15.62% 4.44 2.86–5.85 22.93% 22.70 11.66–29.09 22.96%
16 8.25 7.27–10.35 10.88% 1.84 1.43–2.32 16.82% 3.97 3.01–5.26 18.22% 21.90 17.93–28.00 17.12%
17 9.19 7.25–11.59 15.83% 2.22 1.87–2.72 13.02% 3.00 2.20–4.74 23.33% 24.65 18.59–31.32 15.44%
18 8.74 5.31–11.31 22.37% 1.91 1.40–2.51 20.75% 3.81 3.08–4.75 13.89% 20.78 17.90–28.74 17.10%
19 8.50 7.02–9.84 9.72% 2.19 1.93–2.53 8.75% 3.44 2.68–3.87 11.56% 23.45 20.04–27.43 10.08%
20 8.48 4.91–10.04 17.15% 1.91 1.70–2.19 8.55% 3.99 3.26–5.74 17.63% 20.86 18.13–23.08 8.60%
21 10.88 6.92–14.54 22.89% 1.78 1.38–2.64 23.71% 3.75 3.46–4.30 5.91% 20.92 14.03–27.63 21.24%
22 8.43 6.57–10.94 16.13% 1.73 1.50–2.05 10.94% 4.26 3.20–5.13 16.02% 20.23 15.00–24.85 14.00%
23 8.34 6.50–10.60 17.78% 2.11 1.67–2.58 14.32% 3.34 3.00–4.06 8.87% 20.40 18.57–23.90 7.57%
24 11.59 9.15–14.28 17.70% 2.23 1.76–3.09 21.53% 2.23 1.92–2.48 8.93% 21.11 14.46–28.10 17.76%
Table A2. The fruiting rates of 11 Hibiscus syriacus cultivars.
No. Cultivar Fruiting Rate (%)
1 ‘Pink giant’ 4.4
2 ‘Woodbridge’ 1.5
3 ‘Elegantissimus’ 0.1
4 ‘Arang’ 15.8
5 ‘Rubis’ 13.2
6 ‘Chungmu’ 0.1
7 ‘Suminokurahanagasa’ 2.3
8 ‘Blue bird’ 26.2
9 ‘Akagionmamori’ 0.1
10 ‘Red heart’ 26.0
11 ‘Naesarang’ 1.0
Agronomy 2023,13, 828 17 of 18
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