Plant Systematics and Evolution (2020) 306:15

https://doi.org/10.1007/s00606-020-01641-3

ORIGINAL ARTICLE

Pollen morphology of Leptodermis (Rubiaceae) and its systematic

implications
Xiaoming Guo1,2 · Qi Liao1,2 · Xing Guo1,2 · Shan Zhou1,2 · Yanan Guo1 · Ying Zhang1,2 · Ruijiang Wang1

Received: 9 April 2019 / Accepted: 10 January 2020

© Springer-Verlag GmbH Austria, part of Springer Nature 2020

Abstract
Leptodermis is a taxonomically problematic genus, and little information about its palynology was known previously. In
order to find taxonomic informative characters for species delimitation and their systematic implications, 105 pollen samples,
representing 28 species and four varieties, were observed and analyzed. Phylogenetic inference was also conducted based on
six chloroplast fragments from 32 taxa to trace the evolutionary trend of pollen characters in Leptodermis. Results showed that
two types of pollen can be identified based on the aperture number and position, viz. zonoaperturate pollen with 3 or 4 colpi
situated equatorially and pantoaperturate pollen with 6 to 15 apertures evenly spread over the surface forming a polygonal
platelike pattern. Pollen polymorphism was rarely observed in a few Leptodermis species. Pollen dimorphism also occurs in
Leptodermis, in which the muri are smooth in pollen from pin flowers whereas have minute granules in pollen from thrum
flowers. Phylogenetic mapping demonstrated that each of the nine clades matched well with aperture number and position
and the 3-colpate condition was plesiomorphic. In Leptodermis, pantoaperturate grains evolved three times independently and
were apomorphic. In addition, the frequent occurrence of pollen polymorphism in some species might be explained by shifts
among 3-colpate, 4-colpate, and pantoaperturate pollen grains. The pollen-type mapping to the phylogenetic tree revealed
that the aperture number and position could well match different phylogenetic clades, suggesting that pollen morphology
could provide useful information for taxonomic and systematic studies in Leptodermis.

Keywords Leptodermis · Palynology · Rubiaceae · Systematics · Taxonomy

Introduction recently published species, Leptodermis hechiensis (Guo

et al. 2018) and two unpublished taxa. Leptodermis is
The genus Leptodermis (Rubiaceae) consists about 43 spe- characterized by shrubby habit, heterostylous flowers with
cies from Western Himalaya to Japan, of which 37 species fused membranous bracteoles, salverform to infundibuliform
(33 endemics) occur in China (Chen 2011), including a corolla, 5 corolla lobes with valvate estivation, 3- to 5-lobed
style, 5-celled ovary with only one erect ovule in each loc-
ule, ellipsoid to ovoid capsules with persistent calyx limbs,
Handling Editor: Thais N. C. Vasconcelos. and medium-sized seeds with obvious reticulate arils (Guo
et al. 2018). Molecular phylogenetic analysis (Backlund
Electronic supplementary material The online version of this
et al. 2007; Rydin and Bremer 2009) revealed that Lepto-
article (https​://doi.org/10.1007/s0060​6-020-01641​-3) contains
supplementary material, which is available to authorized users. dermis, represented only by L. potaninii var. potaninii, is
sister to Serissa and then to Spermadictyon, Paederia, and
* Ruijiang Wang Saprosma. These genera constitute the tribe Paederieae.
wangrj@scbg.ac.cn Species taxonomy of Leptodermis is currently based on
1
Key Laboratory of Plant Resources Conservation habit, leaves, bracteoles, flowers, inflorescence, fruits, and
and Sustainable Utilization, Guangdong Provincial Key seed arils (Chen 2011). These characters, however, are much
Laboratory of Digital Botanical Garden, South China variable and difficult for species delimitation. Field obser-
Botanical Garden, Chinese Academy of Sciences, vation showed that leaf size, indumentum, and texture of
Guangzhou 510650, Guangdong, China
leaf blades vary greatly with the habitats within or among
2
University of Chinese Academy of Sciences, Beijing 10049, populations, e.g., the leaves of Leptodermis ovata are usually
China

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 15 Page 2 of 16 X. Guo et al.

large, thin and with sparse hairs in wet and moderate habitat, for 2 h or more, and then washed in phosphate buffer, dehy-
while they are small, thick and with dense hairs in dry and drated in an acetone series, embedded in Spurr’s resin, and
extreme conditions. Pollen characters are stable and reliable cured at 70 °C. Ultrathin sections (80 nm) were cut using
in Rubiaceae and are often useful for species delimitation a Leica-Ultracut S ultramicrotome (Leitz Inc., Wiesbaden,
(Dessein et al. 2005b), e.g., in Psychotria (Johansson 1993) Germany) and stained with uranyl acetate and lead citrate.
and Spermacoce (Dessein et al. 2002). Pollen morphol- Transmission electron micrographs were taken with JEM-
ogy was proved to be of great significance as a valid tool 1010 TEM (JEOL Ltd, Tokyo, Japan). Measurements of the
in elucidating the systematics and phylogeny of a specific polar axis (P) and equatorial diameter (E) were conducted
tribe, such as Spermacoceae (previously as Hedyotideae) on digital SEM images with JEOL’s SmileView 2.2.6.1
(Lewis 1965a), Anthospermeae, and Paederieae (Robbrecht software (JEOL Ltd., Tokyo, Japan) based on 20 grains per
1982), Morindeae (Johansson 1987), or certain genera, such sample.
as Hedyotis (Lewis 1965b) and Coffea (Chinnappa 1981; Totally, 384 chloroplast fragments (matK, rbcL, rpl32-
Chinnappa and Warner 1981, 1982). Palynological charac- trnL, rpoB-trnC, 5′trnL-trnF, and trnS-5′trnG), representing
ters of only three Leptodermis species were observed previ- 32 Leptodermis taxa, were applied for phylogenetic simula-
ously (Robbrecht 1982; Anjum and Muhammad 2007), and tion by maximum likelihood (ML) analyses (Liao 2015).
the fragmentary understanding of the pollen morphological Four samples from Serissa and Paederia were selected
characters resulted in considering the genus as 3-colpate, a as outgroup, based on the phylogenetic results mentioned
very common and plesiomorphic state in the family Rubi- above. Their locality information and GenBank numbers
aceae (Dessein et al. 2005b). are listed in Online Resource 1. Alignment used to produce
In order to provide a comprehensive documentation of phylogeny is listed in Online Resource 2.
the pollen morphology of Leptodermis, samples from 32
taxa were examined. A combined analysis involving mor-
phology, molecular, and geographical information on the Results
genus is performed. The objectives of the present study are
to reveal the diversity of pollen variation, investigate the Pollen description
taxonomic significance of the pollen characters, and analyze
their systematic implications. The genus Leptodermis is eurypalynous. The grains are gen-
erally monads, isopolar (Figs. 1, 2, 3, and 4), and radially
symmetric.
Materials and methods
Size
We collected pollen grains from 105 Leptodermis speci-
mens, representing 28 species and four varieties for scanning In zonoaperturate pollen grains, the size varies from medium
electron microscopy (SEM) observation (some samples were to large, with polar axis (P) ranging within 28.0–61.3 μm
repeatedly collected from the same population for phyloge- and equatorial diameter (E) within 23.8–51.9 μm. The small-
netic analysis but with different field collecting number). est grains from Leptodermis gracilis var. longiflora (R.J.
One taxon Leptodermis pilosa var. pilosa was observed by Wang et al. 2865) have P = 28.0 μm and E = 27.5 μm, the
transmission electron microscopy (TEM). Serissa japonica largest grains from L. buxifolia (P. Yang et al. 45D) having
was also selected for SEM. Pollen material was obtained P = 55.0 μm and E = 41.4 μm in mean value. Pantoapertu-
from anthers in pre-anthesis buds (Table 1). The palynologi- rate pollen grains are medium sized, with diameter rang-
cal terminology follows Hesse et al. (2009) and Halbritter ing within 26.8–50.4 μm. The smallest grains are from L.
et al. (2018). potaninii var. potaninii (R.J. Wang et al. 2571) with diame-
Preparation for observing pollen grains follows Ma et al. ter = 30.6 μm and the largest grains from L. forrestii (T.T. Yu
(2005). For SEM, anthers were dropped and triturated in eth- 5978) with diameter = 45.9 μm in mean value. There is little
anol 70%, vibrated 30 min in ultrasonic-wave cleaner, cen- regular variation of the size of pollen grains. However, pol-
trifugalized 4 min in 6000 rpm. The samples were mounted len grains from thrum flowers are usually larger than those
on copper stubs, air-dried, and coated with gold in a JFC- from pin flowers in the same species (Table 1).
1600 sputter coater (JEOL Ltd, Tokyo, Japan). Pollen grains
were examined and photographed with JSM-6360LV SEM Shape
(JEOL Ltd., Tokyo, Japan). For TEM, the fresh material was
hydrated firstly. Then, the unacetolysed pollen was fixed in The pollen grains of Leptodermis are prolate (Fig. 1a), sub-
2.5% glutaraldehyde at pH 7.2, rinsed in 0.1 mol/L phos- prolate (Figs. 1g, 2d, f), or spheroidal (Figs. 1d, j, 2a, j,
phate buffer for 2 h, then postfixed in 1% osmium tetroxide 3d, j, m). Additionally, the pollen shape may be variable in

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Table 1  Main pollen characters of Leptodermis and Serissa species

Clade/species/ Vouchers Flower type P (μm) E (μm) P/E Shape Aperture Ektexine Figures
locality ornamenta-
tion

I
L. buxifolia, Wen- R.J. Wang, Q. Liao, Pin 35.2 (31.1–40.2) 34.7 (32.7–36.1) 1.01 SP 3-ZC MR-PE-P 1b
chuan, Sichuan, S.S.Zhu 2878
China (IBSC)
R.J. Wang, Q. Liao, Thrum 36.2 (31.9–39.5) 35.8 (32.9–37.2) 1.01 SP 3-ZC MR-G
S.S. Zhu 2879
(IBSC)
L. buxifolia, Maox- X. Guo, P. Yang Pin 48.5 (43.6–54.3) 35.6 (31.0–40.7) 1.36 PR 3-ZC MR-P 1a, c
ian, Sichuan, 45C (IBSC)
China X. Guo, P. Yang Thrum 55.0 (49.3–61.3) 41.4 (37.6–46.7) 1.33 SU 3-ZC MR-G
45D (IBSC)
L. diffusa, Wenxian, J.X. Li 2361 Thrum 49.8 (47.9–53.4) 42.0 (38.7–44.3) 1.19 SU 3-ZC MR-G
Gansu, China (HNWP)
R.J. Wang 1493 Pin 39.4 (37.0–42.9) 35.6 (33.9–37.0) 1.11 SP 3-ZC MR-PE-P
(IBSC)
R.J. Wang 1494 Thrum 37.7 (36.3–38.5) 38.6 (35.9–39.5) 0.98 SP 3-ZC MR-G
(IBSC)
R.J. Wang 1498 Thrum 42.6 (35.5–46.6) 35.8 (35.2–36.5) 1.19 SU 3-ZC MR-G 1f
(IBSC)
R.J. Wang 1499 Pin 39.1 (34.6–41.9) 37.5 (35.1–39.5) 1.04 SP 3-ZC MR-P
(IBSC)
X.M. Guo, Y. Thrum 36.1 (33.0–38.0) 36.0 (34.0–41.0) 1.00 SP 3-ZC MR-G
Zhang, G.T. Wang
572 (IBSC)
X.M. Guo, Y. Pin 31.9 (29.0–34.0) 31.6 (29.0–35.0) 1.01 SP 3-ZC MR-P
Zhang, G.T. Wang
574 (IBSC)
X.M. Guo, Y. Pin 32.8 (31.0–37.0) 31.3 (30.0–32.0) 1.05 SP 3-ZC MR-P
Zhang, G.T. Wang
574a (IBSC)
Z.X. Peng 5752 Thrum 40.1 (35.1–43.3) 43.3 (39.4–46.0) 0.93 SP 3-ZC MR-G 1d, e
(LZU)
II
L. limprichtii, R.J. Wang, Q. Liao, Pin 32.8 (30.8–35.7) 30.0 (23.8–36.2) 1.09 SP 3-ZC PE-P 1i
Leshan, Sichuan, S.S. Zhu 2811
China (IBSC)
R.J. Wang, Q. Liao, Thrum 34.3 (29.2–38.7) 31.8 (29.5–35.3) 1.08 SP 3-ZC MR-G
S.S. Zhu 2812
(IBSC)
L. oblonga, Huayin, R.J. Wang 1487 Pin 39.8 (37.6–43.9) 31.0 (29.0–33.6) 1.29 SU 3-ZC MR-P
Shaanxi, China (IBSC)
T.N. Liou 10652 Thrum 34.7 (31.8–38.1) 38.6 (35.7–40.8) 0.9 SP 3-ZC MR-G
(IBSC)
L. oblonga, Huix- R.J. Wang 1898 Pin 39.3 (38.0–41.6) 34.7 (34.0–35.3) 1.13 SP 3-ZC MR-P
ian, Henan, China (IBSC)
R.J. Wang 1899 Thrum 37.8 (32.6–44.7) 35.5 (32.0–38.3) 1.06 SP 3-ZC MR-G 1j, k, l
(IBSC)
L. ordosica, R.J. Wang 1485 Pin 44.0 (42.4–44.9) 33.8 (29.6–35.2) 1.3 SU 3-ZC MR-P
Qipanjin, Inner (IBSC)
Mongolia, China R.J. Wang 1486 Thrum 42.3 (37.8–46.8) 32.4 (29.4–34.1) 1.3 SU 3-ZC MR-G
(IBSC)
Z.J. Xu 169 Pin 48.8 (44.1–55.6) 35.2 (31.3–39.0) 1.39 PR 3-ZC MR-P
(HIMC)

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Table 1  (continued)
Clade/species/ Vouchers Flower type P (μm) E (μm) P/E Shape Aperture Ektexine Figures
locality ornamenta-
tion

L. purdomii, Wenx- R.J. Wang 1502 Thrum 43.7 (38.9–50.0) 34.5 (31.7–36.6) 1.27 SU 3-ZC MR-G
ian, Gansu, China (IBSC)
R.J. Wang 1504 Pin 37.1 (34.8–35.3) 30.0 (27.8–37.4) 1.23 SU 3-ZC MR-P 1g, h
(IBSC)
III
L. dielsiana, X.M. Guo, Y. Thrum 37.4 (35.0–40.0) 37.2 (36.0–41.0) 1.00 SP 3-ZC MR-G
Mangkang, Tibet, Zhang, G.T. Wang
China 592 (IBSC)
L. gracilis var. R.J. Wang, Q. Liao, Pin 33.9 (32.2–37.1) 35.3 (32.5–37.0) 0.96 SP 3-ZC MR-P 2b
longiflora, Mae- S.S. Zhu 2864
rkang, Sichuan, (IBSC)
China R.J. Wang, Q. Liao, Thrum 28.0 (27.7–28.3) 27.5 (26.5–28.6) 1.02 SP 3-ZC MR-G
S.S. Zhu 2865
(IBSC)
L. handeliana, R.J. Wang, S. Zhou Thrum 36.4 (35.3–38.0) 34.9 (31.3–37.2) 1.04 SP 3-ZC MR-G 2a
Yanyuan, 1754 (IBSC)
Sichuan, China
L. pilosa var. R.J. Wang, Q. Liao Pin 33.0 (28.0–36.2) 31.0 (26.6–33.9) 1.06 SP 3-ZC MR-P
pilosa, Gongshan, 2547 (IBSC)
Yunnan, China R.J. Wang, Q. Liao Thrum 35.1 (31.7–38.1) SP PC MR-G
2548 (IBSC)
R.J. Wang, Q. Liao Thrum 34.8 (32.7–36.1) 33.8 (29.3–35.6) 1.03 SP – MR-G 2c
2551 (IBSC)
R.J. Wang, Q. Liao Pin 31.7 (30.3–33.1) 35.9 (35.0–37.1) 0.88 SP 4(3)-ZC MR-P
2552 (IBSC)
X.M. Guo, Y. Thrum 40.8 (39.0–43.0) 37.9 (36.0–41.0) 1.08 SP 3-ZC MR-G
Zhang, G.T. Wang
523 (IBSC)
L. pilosa var. R.J. Wang, Q. Liao Pin – – 4-ZC MR-P 1o
pilosa, Dulong, 2553 (IBSC)
Yunnan, China R.J. Wang, Q. Liao Pin – – 3(4)-ZC MR-P 1n
2555 (IBSC)
R.J. Wang, Q. Liao Thrum – – 3-ZC MR-G 1m
2556 (IBSC)
R.J. Wang, Q. Liao Pin 30.7 (27.5–32.8) 33.4 (30.2–35.8) 0.92 SP – MR-P
2557 (IBSC)
R.J. Wang, Q. Liao Thrum 33.5 (28.8–37.9) 37.0 (33.1–45.6) 0.91 SP 3-ZC MR-G
2558 (IBSC)
L. pilosa var. X. Guo, P. Yang 2C Pin 37.0 (34.8–39.4) 35.1 (31.9–38.5) 1.06 SP 3-ZC MR-P 4e, f
pilosa, Kunming, (IBSC)
Yunnan, China
L. pilosa var. gla- P. Yang, H.Z. Wen Thrum 40.3 (34.0–41.2) SP PC MR-G
brescens, Lijiang, 64 (IBSC)
Yunnan, China
IV
L. gracilis var. gra- X. Guo, P. Yang Pin 39.9 (37.1–42.9) 39.1 (36.1–41.8) 1.02 SP 4-ZC MR-P 2e
cilis, Bomi, Tibet, 39C (IBSC)
China X. Guo, P. Yang Thrum 50.6 (46.6–53.0) 41.3 (38.3–44.8) 1.22 SU 4-ZC MR-G 2d
39D (IBSC)
L. gracilis var. gra- X. Guo, P. Yang Pin 32.8 (30.8–35.7) 30.0 (23.8–36.2) 1.09 SP 4-ZC MR-P
cilis, Milin, Tibet, 43C (IBSC)
China X. Guo, P. Yang Thrum 34.3 (29.2–38.7) 31.8 (29.5–35.3) 1.08 SP 4-ZC MR-G 2f
43D (IBSC)

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Table 1  (continued)
Clade/species/ Vouchers Flower type P (μm) E (μm) P/E Shape Aperture Ektexine Figures
locality ornamenta-
tion

L. hirsutiflora var. Tibet Exp. 4570 Thrum 38.8 (37.3–41.2) 37.8 (34.8–42.0) 1.03 SP 4-ZC MR-G
hirsutiflora, Jia- (HNWP)
cha, Tibet, China
L. hirsutiflora var. X. Guo, P. Yang Thrum 44.7 (38.0–54.4) 40.7 (35.1–51.9) 1.10 SP 4-ZC MR-G
ciliata, Jiacha, 44D (IBSC)
Tibet, China
L. hirsutiflora var. X.M. Guo, Y. Pin 33.3 (32.0–34.0) 34.3 (33.0–36.0) 0.97 SP 4-ZC MR-P
ciliata, Milin, Zhang, G.T. Wang
Tibet, China 604 (IBSC)
L. kumaonensis, R.J. Wang, S. Zhou Pin 39.2 (34.0–42.1) 37.4 (33.3–40.5) 1.05 SP 4(3)-ZC, PC MR-P
Nielamu, Tibet, 1861 (IBSC)
China
L. kumaonensis Tibet Exp. 50003 Pin 43.4 (40.5–44.8) 42.3 (40.3–43.9) 1.03 SP 4-ZC MR-P
(HNWP)
L. scabrida, Bomi, X. Guo, P. Yang Pin 45.6 (40.9–48.4) 38.3 (33.4–41.4) 1.18 SU 4-ZC MR-P 2g, h
Tibet, China 38C (IBSC)
X. Guo, P. Yang Thrum 50.0 (45.5–55.0) 41.3 (36.7–46.3) 1.21 SU 4-ZC MR-G
38D (IBSC)
L. scabrida, Milin, X. Guo, P. Yang Pin 46.9 (43.8–51.9) 38.1 (36.3–41.7) 1.23 SU 4-ZC MR-P 2i
Tibet, China 41C (IBSC)
L. sp., Linzhi, Tibet, X.X. Huang 16152 Thrum 39.0 (34.4–40.1) 38.5 (34.0–40.0) 1.01 SP 4-ZC MR-G
China (IBSC)
V
L. forrestii, Chayu, R.J. Wang, S. Zhou Thrum 42.9 (40.3–46.4) SP PC? MR-G
Tibet, China 1807 (IBSC)
L. forrestii, Muli, T.T. Yu 5978 Pin 45.9 (39.9–45.9) SP PC MR-P
Sichuan, China (IBSC)
L. ludlowii, Linzhi, Tibet EXP. 3518 Thrum 43.8 (41.4–46.7) SP PC MR-G 2j, l
Tibet, China (HNWP)
L. yui, Muli, X.M. Guo, Y. Thrum 37.9 (36.0–41.0) SP PC MR-G 2k
Sichuan, China Zhang, G.T. Wang
548 (IBSC)
VI
L. brevisepala, R.J. Wang, Q. Liao, Pin 40.1 (38.1–42.8) SP PC, 3-ZC MR-P
Huize, Yunnan, S.S. Zhu 2755
China (IBSC)
R.J. Wang, Q. Liao, Thrum 42.0 (40.0–44.0) SP PC MR-G
S.S. Zhu 2756
(IBSC)
L. brevisepala, R.J. Wang, S. Zhou Pin 40.3 (38.2–43.4) SP PC MR-P
Miyi, Sichuan, 1727 (IBSC)
China R.J. Wang, S. Zhou Thrum 37.9 (35.6–40.0) SP PC MR-G
1728 (IBSC)
L. glomerata, R.J. Wang, Q. Liao Pin – PC MR-P 2m
Nanhua, Yunnan, 2573 (IBSC)
China
L. glomerata, X.M. Guo, Y. Pin 34.1 (33.0–35.0) SP PC MR-P
Gongshan, Yun- Zhang, G.T. Wang
nan, China 517 (IBSC)
X.M. Guo, Y. Thrum 41.8 (37.0–46.0) SP PC MR-G
Zhang, G.T. Wang
518 (IBSC)

13
 15 Page 6 of 16 X. Guo et al.

Table 1  (continued)
Clade/species/ Vouchers Flower type P (μm) E (μm) P/E Shape Aperture Ektexine Figures
locality ornamenta-
tion

L. potaninii var. R.J. Wang, Q. Liao Pin – PC MR-P 3c

glauca, Xingyi, 2532 (IBSC)
Guizhou, China
R.J. Wang, Q. Liao Pin – PC MR-P 3b
2533 (IBSC)
L. potaninii var. R.J. Wang, Q. Liao Pin 38.3 (37.3–40.3) SP PC MR-P 2n
potaninii, Yun- 2538 (IBSC)
long, Yunnan, R.J. Wang, Q. Liao Pin – PC MR-P
China 2539 (IBSC)
L. potaninii var. R.J. Wang, Q. Liao Pin – PC MR-P
potaninii, Dulong, 2562 (IBSC)
Yunnan, China R.J. Wang, Q. Liao Thrum 36.5 (34.3–38.8) SP PC MR-G 2o
2563 (IBSC)
L. potaninii var. R.J. Wang, Q. Liao Pin 30.6 (26.8–32.5) SP PC MR-P
potaninii, Dali, 2571 (IBSC)
Yunnan, China
L. potaninii R.J. Wang, Q. Liao Thrum – PC MR-G
var. potaninii, 2589 (IBSC)
Chengjiang, Yun-
nan, China
L. potaninii R.J. Wang, Q. Liao Pin – PC, 4-ZC MR-P
var. potaninii, 2606 (IBSC)
Xingshan, Hubei,
China
L. scissa, Yanyuan, R.J. Wang, S. Zhou Pin – PC MR-P 3a
Sichuan, China 1759 (IBSC)
X.M. Guo, Y. Thrum 41.6 (36.0–44.0) SP PC MR-G
Zhang, G.T. Wang
545 (IBSC)
X.M. Guo, Y. Thrum 39.9 (37.0–44.0) SP PC MR-G
Zhang, G.T. Wang
545_ (IBSC)
X.M. Guo, Y. Thrum 41.0 (38.0–44.0) SP PC MR-G
Zhang, G.T. Wang
545b (IBSC)
VII
L. beichuanen- R.J. Wang, Q. Liao, Thrum – – 4-ZC MR-G
sis, Maoxian, S.S. Zhu 2883
Sichuan, China (IBSC)
L. beichuanen- R.J. Wang, Q. Liao, Thrum 38.9 (37.2–39.7) SP PC MR-G 3e, f
sis, Beichuan, S.S. Zhu 2885
Sichuan, China (IBSC)
L. sp. 1, Tianquan, R.J. Wang, Q. Liao, Thrum 40.4 (38.8–41.7) SP PC MR-G 3d
Sichuan, China S.S. Zhu 2829
(IBSC)
R.J. Wang, Q. Liao, Pin 38.2 (37.9–38.5) SP PC MR-P
S.S. Zhu 2830
(IBSC)
L. sp. 2, Yanyuan, R.J. Wang, S. Zhou Pin 44.9 (40.0–50.4) SU PC MR-P
Sichuan, China 1745 (IBSC)
R.J. Wang, S. Zhou Thrum 43.8 (40.5–47.1) SP PC MR-G
1746 (IBSC)

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Table 1  (continued)
Clade/species/ Vouchers Flower type P (μm) E (μm) P/E Shape Aperture Ektexine Figures
locality ornamenta-
tion

VIII
L. coriaceifo- R.J. Wang, Q. Liao Pin 35.9 (32.8–40.6) 35.4 (32.9–38.2) 1.01 SP 4-ZC, PC MR-P 3i
lia, Fengshan, 2522 (IBSC)
Guangxi, China R.J. Wang, Q. Liao Thrum 39.3 (37.2–41.3) SP PC, 4-ZC MR-G
2523 (IBSC)
L. coriaceifo- R.J. Wang, Q. Liao Pin – – 3-ZC MR-P 3g
lia, Lingyun, 2524 (IBSC)
Guangxi, China R.J. Wang, Q. Liao Thrum – – 4-ZC MR-G
2525 (IBSC)
R.J. Wang, Q. Liao Pin – – 4-ZC MR-P
2526 (IBSC)
R.J. Wang, Q. Liao Pin – – ? MR-P
2527 (IBSC)
R.J. Wang, Q. Liao Pin 37.8 (36.6–39.3) 39.4 (38.3–41.9) 0.96 SP 4-ZC MR-P
2530 (IBSC)
R.J. Wang, Q. Liao Thrum 38.3 (36.2–40.3) 38.2 (32.3–41.7) 1 SP 4(3)-ZC MR-G 3h
2531 (IBSC)
X.M. Guo, Y. Thrum 40.5 (39.0–42.0) SP PC MR-G
Zhang, G.T. Wang
507 (IBSC)
L. coriaceifo- R.J. Wang, S. Zhou, Pin – – 4-ZC MR-P
lia, Debao, S.J. Deng, Q.
Guangxi,China Liao 2179 (IBSC)
L. coriaceifolia, S.G. Chen 115 Thrum – – 4-ZC MR-G
Xichou, Yunnan, (IBSC)
China
L. hechiensis, R.J. Wang, Q. Liao Pin – – 4(3)-ZC, PC MR-P
Yizhou, Guangxi, 2491 (IBSC)
China
L. hechiensis, R.J. Wang, Q. Liao Pin 32.2 (29.4–34.7) 32.2 (28.3–37.6) 1 SP 4-ZC MR-P
Jinchengjiang, 2500 (IBSC)
Guangxi, China R.J. Wang, Q. Liao Thrum 40.4 (31.2–46.0) 38.5 (36.3–41.4) 1.05 SP 4-ZC MR-G
2501 (IBSC)
R.J. Wang, Q. Liao Pin 38.7 (36.5–42.6) 40.3 (37.2–43.6) 0.96 SP 4-ZC MR-P
2502 (IBSC)
R.J. Wang, Q. Liao Thrum 41.7 (36.7–46.0) 42.2 (40.1–45.5) 0.99 SP 4-ZC MR-G
2503 (IBSC)
IX
L. ovata, Ruyuan, R.J. Wang 1106 Pin 34.6 (33.8–35.4) SP None MR-P 3n
Guangdong, (IBSC)
China R.J. Wang 2253 Thrum 39.6 (37.8–41.5) SP None MR-G 3o
(IBSC)
L. vestita, Yang- X.M. Guo, Y. Pin 34.6 (29.0–40.0) SP PC MR-P 3m
shan, Guangdong, Zhang, G.T. Wang
China 498 (IBSC)
X.M. Guo, Y. Thrum 38.9 (34.0–41.0) SP PC MR-G
Zhang, G.T. Wang
499 (IBSC)
L. yangshuoen- R.J. Wang, Q. Liao Pin 35.1 (31.8–38.9) SP PC MR-P 3j, k
sis, Yangshuo, 2473 (IBSC)
Guangxi, China R.J. Wang, Q. Liao Thrum 36.2 (33.4–39.5) SP PC MR-G 3l
2474 (IBSC)

13
 15 Page 8 of 16 X. Guo et al.

Table 1  (continued)
Clade/species/ Vouchers Flower type P (μm) E (μm) P/E Shape Aperture Ektexine Figures
locality ornamenta-
tion

Outgroup
Serissa japonica, S. Zhou 110 (IBSC) Pin 33.8 (31.0–35.3) 24.5 (20.9–27.0) 1.38 PR 3-ZC MR-P 4a, b, c
Lueyang, Shaanxi.
China

I–IX corresponds to the identified clade number in Fig. 5

P polar diameter, E equatorial diameter, P/E ratio of polar to equatorial diameter, ZC zonoaperturate, PC pantoaperturate, SP spheroidal, PR pro-
late, SU subprolate, MR microreticulate, PE perforate, P psilate, G nanoechinate sculpture, – no data

the same flower. The outline of pollen grains in polar view columellae, and an endexine layer (Fig. 4d–f). Columellae
(amb) is mostly circular (Fig. 1b), but slightly lobed due to of the mesocolpia are obviously thicker than those closely
sunken colpi (Fig. 1b, e, h, k). surrounding the colpi.

Apertures
Mapping pollen types to the phylogenetic tree
Two types of pollen grains in Leptodermis can be recognized
The simulated phylogenetic tree revealed that Leptodermis
based on aperture number and position, viz. zonoaperture
is monophyletic (Fig. 5, BS = 100); nine robustly supported
with 3 or 4 colpi and pantoaperture with 6 to 15 apertures.
clades can be recognized. Pollen of Clades I, II, and III is
In zonoaperturate pollen, the colpi are situated only equa-
generally characterized by three colpi, except for Leptoder-
torially and have a psilate surface (Figs. 1b, n, 2b, n, 3b), or
mis pilosa var. pilosa (R.J. Wang et al. 2548, 2552, 2553,
coarsely verrucate membrane (Figs. 1e, h, 2h, m); the colpus
and 2555) and L. pilosa var. glabrescens (P. Yang et al. 64)
membrane sometimes is embedded in the sunken, slit-like,
(Table 1). Pollen grains of Clades IV and VIII are 4-col-
and infolded colpi (Figs. 1k, 3a, d). In pantoaperturate pollen
pate, except for L. kumaonensis (R.J. Wang et al. 1861),
grains, the apertures evenly spread over the pollen surface,
L. coriaceifolia (R.J. Wang et al. 2523, 2524, and X.M.
which is divided into several regular and polygonal platelike
Guo et al. 507), and L. hechiensis (R.J. Wang et al. 2491)
areas due to the apertures converging at the ends (Figs. 2j, n,
(Table 1). Pollen of Clades V, VI, VII, and IX is gener-
3a). The surface of colpi is the same for zonoaperturate and
ally pantoaperture, except for L. beichuanensis (Wang et al.
pantoaperturate grains.
2883) (Table 1). The different pollen characters scatter over
In addition, the types of pollen were also observed in
five clades (Clades III, IV, VI, VII, and VIII). Phylogenetic
the same species, such as Leptodermis pilosa var. pilosa,
mapping demonstrated that all the nodes in the outgroup lack
L. kumaonensis, L. brevisepala, L. potaninii var. potaninii,
pantoaperturate pollen grains, while the ingroup basal Clade
L. beichuanensis, L. coriaceifolia, and L. hechiensis, either
IX (Fig. 52, BS = 100), involving L. yangshuoensis, L. ovata,
dominance of 3-colpate grains but rarely with 4-colpate
and L. vestita, from Karst habitat in Nanling Mountainous
and pantoaperturate ones, or dominance of 4-colpate grains
area (Table 1), is characterized by having pantoaperturate
but rarely with pantoaperturate ones, as in L. kumaonen-
pollen.
sis, L. coriaceifolia, and L. hechiensis. In L. brevisepala, L.
potaninii var. potaninii, and L. beichuanensis, pantoapertu-
rate pollen grains are dominant, coexisting with a few 3- or
4-colpate grains. Discussion

Ektexine ornamentation and stratification Eurypalynous phenomenon in Paederieae

Pollen muri are densely covered with minute granules in Leptodermis is not the first genus reported to have pantoap-
grains from thrum flowers, whereas smooth in grains from erturate grains in Rubiaceae, such as in Coptosapelta (Verel-
pin flowers. The ektexine of Leptodermis is mostly microre- len et al. 2004), Gardenia (Persson 1993), Geophila (aper-
ticulate, sometimes perforate (Fig. 1i). A perforate ektexine tures evenly spread over the pollen surface) (Dessein et al.
was only observed in Leptodermis diffusa and L. limprichtii. 2005b), Psychotria (Johansson 1993; Jansen et al. 1996),
TEM observation showed that the pollen wall of Lep- and Spermacoce (the colpi arranged in a looplike pattern
todermis is composed of a discontinuous tectum, a row of (Dessein et al. 2002, 2005a), three parallel rows of colpi

13
Pollen morphology of Leptodermis (Rubiaceae) and its systematic implications Page 9 of 16 15

Fig. 1  a–c, Leptodermis

buxifolia; d–f, L. diffusa; g, h,
L. purdomii; i, L. limprichtii;
j–l, L. oblonga; m–o, L. pilosa
var. pilosa; a, d, g, j, m, in
equatorial view; b, e, h, k, n,
in polar view; a–c, g–i, n, o,
surface views of grain from pin
flower; d–f, j–m, surface views
of grain from thrum flower; c, f,
l, o, showing a microreticulate
ektexine; i, showing a perforate
ektexine

13
 15 Page 10 of 16 X. Guo et al.

Fig. 2  a, Leptodermis hande-

liana; b, L. gracilis var. longi-
flora; c, L. pilosa var. pilosa;
d–f, L. gracilis var. gracilis; g–i,
L. scabrida; j, l, L. ludlowii; k,
L. yui; m, L. glomerata; n, o, L.
potaninii var. potaninii; a, d, g,
equatorial view; b, e, h, in polar
view; j, k, m, n, general view of
pollen grain; c, f, i, l, o, show-
ing a microreticulate ektexine.
b, e, g, h, i, m, n, Surface views
of grain from pin flower; a, c, d,
f, j–l, o, surface views of grain
from thrum flower

13
Pollen morphology of Leptodermis (Rubiaceae) and its systematic implications Page 11 of 16 15

Fig. 3  a, Leptodermis scissa;

b, c, L. potaninii var. glauca; d,
L. sp.1; e, f, L. beichuanensis;
g–i, L. coriaceifolia; j–l, L.
yangshuoensis; m, L. vestita; n,
o, L. ovata; g, equatorial view;
h, polar view; a, b, d, e, j, k, m,
n, general view of pollen grain;
c, f, i, l, o, showing a microre-
ticulate ektexine; a–c, g, i–n,
surface views of grain from pin
flower; d–f, h, o, surface views
of grain from thrum flower

13
 15 Page 12 of 16 X. Guo et al.

Igersheim 1991; Dessein et al. 2005b). The present study

reveals that Leptodermis is eurypalynous, with both zono-
aperturate and pantoaperturate grains, which differs from
the other stenopalynous genera in the tribe Paederieae, viz.
Serissa (Robbrecht 1982), Paederia (Robbrecht 1982; Iger-
sheim 1991; Lu et al. 2010), and Spermadictyon (Robbre-
cht 1982; Anjum and Muhammad 2007), all only having
3-(4)-colpate pollen. In Rubiaceae, this kind of eurypalynous
phenomenon was also reported in Coffeeae, Gardenieae,
Morindeae, Psychotrieae, Sabiceeae, and Spermacoceae
s.str. (Dessein et al. 2005b).

Pantoaperturate derived from zonoaperturate

Walker and Doyle (1975) reported that pollen grains with

three colpi are primitive and with four or more colpi are
derived in many dicotyledonous families. A similar conclu-
sion was made for the South Indian Rubiaceae (Mathew and
Philip 1983). Phylogenetic mapping of pollen types demon-
strated that the 3-colpate pollen type was in the outgroup
taxa and may represent the plesiomorphic character and the
pantoaperturate pollen may be apomorphic in Leptodermis
(Fig. 5). The plesiomorphic 3-colpate character persisted in
the Clades I, II, III and outgroup; however, the very basal
Clade IX had apomorphic pantoaperturate pollen. This may
indicate at least one shift from zonoaperture to pantoaper-
ture happened in this clade. In Leptodermis, pantoapertu-
rate character as apomorphy evolved three times indepen-
dently. All species in Clades IV and VIII characterized by
four colpi are isolated by huge geographical distance and
mountains. This demonstrates that 4-colpate grains evolved
independently at least two times in Leptodermis (Fig. 5).
In Leptodermis, the pollen evolution from zonoaperture to
pantoaperturate grains might be related to some unknown
pollination mechanism and needs further study.

Pollen type may be a useful tool to distinguish some

problematic taxa

Fig. 4  Pollen grains of Serissa and pollen wall stratification of Lepto- Phylogenetic study of Leptodermis revealed nine distinct
dermis. a–c, The type of 3-colpate pollen grains of Serissa japonica;
a, in equatorial view; b, in polar view of 3-colpate grain; c, ektexine
groups, which are supported robustly by pollen characters.
ornamentation from pin flower, showing interwoven muri forming a The number of apertures and their position on pollen grains
microreticulate pattern; d, cross section of a pollen wall of Leptoder- play a very important role on highlighting the different phy-
mis parvifolia, showing tectum, columellae, and endexine; e, f, pollen logenetic clades, suggesting that pollen morphology could
wall stratification of L. pilosa var. pilosa, showing aperture (A), tec-
tum (T), columellae (C), endexine (N), and intine (I)
provide useful information for taxonomic studies in Lep-
todermis. Winkler (1922) distinguished Sect. Glomeratae
and Sect. Pauciflorae rudely in Leptodermis according to
(Dessein et al. 2002), or spiral pattern). But, in Leptodermis, the number of flowers per inflorescence. Our palynological
the arrangement of apertures and the regular and polygonal evidence is neither in agreement with this oversimple classi-
platelike surface of the pantoaperturate pollen is obviously fication nor provides informative support for the subgeneric
different from that of the genera mentioned above. delimitations based on aperture number and position. But
The previous literatures reported that the tribe Paederieae at species level, the aperture number and position can be
is stenopalynous, with 3-(4)-colpate pollen (Robbrecht 1982; useful for species identification in Leptodermis. An example

13
Pollen morphology of Leptodermis (Rubiaceae) and its systematic implications Page 13 of 16 15

Fig. 5  Chloroplast maximum

likelihood best-scoring tree with
likelihood BS values ≥ 50%
above the branches. Numbers
I to IX over key nodes report
the nine clades identified. Shift
sites are showed by allows.
Down allows mean shifts from
3-colpate grains to 4-colpate.
Up allows mean shifts from
zonoaperture grains to pantoap-
erture

13
 15 Page 14 of 16 X. Guo et al.

appertains to the delimitation of Leptodermis beichuanensis s.l., the taxonomic value of pollen morphology is limited in
and L. buxifolia. Both taxa have very similar leaves, flow- Clematis at species level (Xie and Li 2012). The weak cor-
ers, and inflorescence, but palynological evidence shows that relation between the taxonomy and pollen was also found
L. buxifolia has 3-colpate pollen, while pantoaperturate or in Leptodermis. In Papaver nudicaule, the polymorphism
4-colpate pollen characterizes L. beichuanensis. Another rate is related to the contents of toxic elements on the pollen
case is the merge of L. pilosa var. pilosa and L. pilosa var. (Reshetova et al. 2015). Although the polymorphism was
glabrescens. They were wrongly separated based on indu- found in Leptodermis, the exploration to the mechanism of
mentum features, L. pilosa var. glabrescens being thought to transition between apertures types needs further study. It
be sparsely pilose (Lo 1999). Our field examination showed may reveal the factors, either endogenous or environmental,
the indumentum continuously variable in the two taxa. Pol- trigging this morphological variation, and provide a typi-
len observation also revealed that they both have medium- cal model for the species evolution in the Eastern Hima-
sized, spheroidal, and pantoaperturate pollen grains, and laya–Hengduan Mountains. The frequent occurrence of pol-
microreticulate tectum. It is here proved that palynological len polymorphism in some species of Leptodermis might
evidence has taxonomic significance in Leptodermis and it be explained by the shifts among 3-colpate, 4-colpate, and
will be applied in the ongoing taxonomic revision of this pantoaperturate grains. The occurrence of infraspecific plas-
genus. ticity certainly lowers the systematic value of pollen features
in some cases.
Pollen dimorphism firstly found in Leptodermis The evolutionary trend of the pollen types is complicated.
Some pollen types evolved parallel in several clades, e.g., in
Rubiaceae has the largest number of distylous genera within Clade VIII both 4-colpate and pantoaperturate grains, occur
angiosperms (Ganders 1979), and pollen dimorphism in the in Leptodermis coriaceifolia and L. hechiensis. Reversal of
Rubiaceae has been observed since twentieth century (Baker pollen character states is also commonly found in the genus,
1956; Bremekamp 1966). In distylous flowers, pollen grains e.g., within Clades III and IV that have zonoaperturate pol-
from thrum flowers are larger than that from pin flowers len; here reversal resulted in the occurrence of pantoapertu-
(Vuilleumier 1967; Naiki and Nagamasu 2003; Wolff and rate pollen in L. pilosa var. pilosa, L. pilosa var. glabrescens,
Liede-Schumann 2007), although there are also some excep- and L. kumaonensis. Similarly, the zonoaperturate pollen in
tions (Dessein et al. 2005b). In Leptodermis, pollen grains L. brevisepala, L. potaninii var. potaninii, and L. beichuan-
from thrum flowers are usually larger than those from pin ensis occurred in the pantoaperturate Clades VI and VII due
flowers in the same species. It is generally considered that to the reversal evolution.
differences in pollen size could be attributed to increase in
seed number (Lloyd 1979). In Leptodermis, pollen muri are
densely covered with minute granules in grains from thrum
flowers, whereas smooth in grains from pin flowers, as in Conclusion
another Asian Rubiaceae genus, Damnacanthus (Naiki and
Nagamasu 2003). Leptodermis is an eurypalynous genus. The pollen grains of
Leptodermis show morphological variation in shape, size,
Both parallel and reversal evolution occurs number and position of apertures, and ektexine ornamenta-
in Leptodermis pollen tion, which may contribute to the investigation of problem-
atic taxa in Leptodermis. Pollen dimorphism and polymor-
Pollen variation within species was frequently observed phism occur in Leptodermis. Tricolpate pollen grains are
in Rubiaceae (Stoffelen et al. 1997). The best-documented plesiomorphic in the genus, and the pantoaperturate char-
case of pollen polymorphy within species was observed acter state evolved three times independently as apomorphy.
in Coffea (Chinnappa and Warner 1982). Pollen polymor- Phylogenetic mapping demonstrated that each of the nine
phism was also observed in Leptodermis pilosa var. pilosa, clades matched well with aperture number and position on
L. kumaonensis, L. brevisepala, L. potaninii var. potaninii, pollen grains. The aperture number and position could well
L. beichuanensis, L. coriaceifolia, and L. hechiensis. This match the different phylogenetic clades, suggesting that pol-
phenomenon was also found in the genus Clematis sect. len morphology could provide useful information for taxo-
Tubulosae, sect. Viorna, and sect. Viticella, in which the nomic and systematic studies in Leptodermis.
tricolpate pollen grains are predominant and pantoporate
Acknowledgements We would like to thank Ping Yang and Gangtao
pollen grains can be found too, and confirmed that the pan- Wang from SCBG for help in the field. We are grateful to Ms. Xiaoy-
toporate type is derived and has evolved several times in ing Hu and Ms. Xinlan Xu from SCBG for technical assistance with
the genus. Although evolutionary trends can be inferred electron microscopy, the curators in HIMC, HNWP, IBSC, LZU for
permission to remove pollen from herbarium specimens. We thank Dr.
from palynological characters in some groups of Clematis

13
Pollen morphology of Leptodermis (Rubiaceae) and its systematic implications Page 15 of 16 15

Elmar Robbrecht from Meise Botanic Garden, Dr. Bine Xue, and Prof. Hesse M, Halbritter H, Weber M, Buchner R, Frosch-Radivo A, Ulrich
Fengxia Xu from SCBG for their helpful comments on earlier versions S, Zetter R (2009) Pollen terminology: an illustrated handbook.
of the manuscript. This work was financially supported by the National Springer, Wien
Nature Science Foundation of China (30770156) and Strategic Priority Igersheim A (1991) Palynological investigations of Paederia L.
Research Program of Chinese Academy of Sciences (XDA13020602). (Rubiaceae-Paederieae). In: Puff C (ed) Opera Botanica Belgica.
National Botanic Garden of Belgium, Meise, pp 135–149
Jansen S, Robbrecht E, Smets E (1996) The systematic value of
Compliance with ethical standards endexine ornamentation in some Psychotrieae pollen (Rubi-
aceae-Rubioideae). Grana Palynol 35:129–137. https​: //doi.
Conflict of interest All authors declare that they have no conflict of org/10.1080/00173​13960​94290​74
interest. Johansson JT (1987) Pollen morphology of the tribe Morindeae (Rubi-
aceae). Grana Palynol 26:134–150. https:​ //doi.org/10.1080/00173​
Information on Electronic Supplementary Mate- 13870​94299​43
Johansson JT (1993) Pollen morphology in Psychotria (Rubi-
rial aceae, Rubioideae, Psychotrieae) and its taxonomic signifi-
cance. A preliminary survey. Nordic J Bot 13:32. https​://doi.
Online Resource 1. Voucher information: Location, voucher No., her-
org/10.1111/j.1756-1051.1993.tb000​04.x
barium, GenBank accession numbers.
Lewis WH (1965a) Cytopalynological study of African Hedyotideae
Online Resource 2. Alignment used to generate the phylogenetic tree.
(Rubiaceae). Ann Missouri Bot Gard 52:182–211. https​://doi.
org/10.2307/23948​68
Lewis WH (1965b) Pollen morphology and evolution in Hedyotis sub-
genus Edrisia (Rubiaceae). Amer J Bot 52:257–264. https​://doi.
org/10.2307/24399​37
References Liao Q (2015) Biogeography of Leptodermis (Rubiaceae). MSc Thesis,
University of Chinese Academy of Sciences, Guangzhou
Anjum P, Muhammad Q (2007) Pollen flora of Pakstan-Liv. Rubiaceae. Lloyd DG (1979) Evolution towards dioecy in heterostylous popula-
Pakistan J Bot 39:999–1015 tions. Pl Syst Evol 131:71–80. https​://doi.org/10.1007/BF009​
Backlund M, Bremer B, Thulin M (2007) Paraphyly of Paederieae, 84123​
recognition of Putorieae and expansion of Plocama (Rubiaceae- Lo HS (1999) Leptodermis wall. In: Chen WQ (ed) Flora Reipublicae
Rubioideae). Taxon 56:315–328 Popularis Sinicae. Science Press, Beijing, pp 120–153
Baker HG (1956) Correction: pollen dimorphism in the Rubiaceae. Lu JL, Kuang YF, Liao JP (2010) Pollen morphology of Paederia L.
Evolution 10:23–31. https​://doi.org/10.2307/24060​20 (Rubiaceae) from China. J Trop Subtrop Bot 18:491–496
Bremekamp CEB (1966) Remarks on the position, delimitation and Ma QX, Wang RJ, Chen BH (2005) Pollen morphology of Spiradiclis
subdivision of the Rubiaceae. Acta Bot Neerl 15:1–33. https​:// BI. (Rubiaceae). J Trop Subtrop Bot 13:159–166
doi.org/10.1111/j.1438-8677.1966.tb002​07.x Mathew PM, Philip O (1983) Studies in the pollen morphology of
Chen T (2011) Leptodermis. In: Wu ZY, Raven P, Hong DY (eds) Flora South Indian Rubiaceae. In: Nair PKK (ed) Advances in pollen
of China. Science Press, Beijing, pp 198–211 spore research. Tomorrow’s Prints & Publishing, New Delhi
Chinnappa CC (1981) Palynology and systematics of the genus Coffea. Naiki A, Nagamasu H (2003) Distyly and pollen dimorphism in
J Coffee Res 11:55–69 Damnacanthus (Rubiaceae). J Pl Res 116:105–113. https​://doi.
Chinnappa CC, Warner BG (1981) Pollen morphology in the genus org/10.1007/s1026​5-002-0075-1
Coffea (Rubiaceae) and its taxonomic significance. Bot J Linn Soc Persson C (1993) Pollen morphology of the Gardenieae-Garde-
83:221–236. https:​ //doi.org/10.1111/j.1095-8339.1981.tb0034​ 7.x niinae (Rubiaceae). Nordic J Bot 13:561–582. https ​ : //doi.
Chinnappa CC, Warner BG (1982) Pollen morphology in the genus org/10.1111/j.1756-1051.1993.tb001​01.x
Coffea (Rubiaceae): ii. pollen polymorphism. Grana Palynol Reshetova SA, Solodukhina MA, Yurgenson GA (2015) The inter-
21:29–37. https​://doi.org/10.1080/00173​13820​94276​77 relation between pollen abnormalities and polymorphism and the
Dessein S, Huysmans S, Robbrecht E, Smets E (2002) Pollen of African increased contents of toxic elements in flowers and flower buds in
Spermacoce species (Rubiaceae) morphology and evolutionary Aconogonon angustifolium (Pall.) Hara. and Papaver nudicaule
aspects. Grana Palynol 41:69–89. https​://doi.org/10.1080/00173​ L. Russ J Ecol 46:36–42. https​://doi.org/10.1134/s1067​41361​
13027​60156​882 50101​42
Dessein S, Harwood R, Smets E, Robbrecht E (2005a) Pollen of the Robbrecht E (1982) Pollen morphology of the tribes Anthospermeae
Spermacoce (Rubiaceae) species from the Northern Territory of and Paederieae (Rubiaceae) in relation to taxonomy. Bull Jard Bot
Australia: morphology and taxonomic significance. Austral Syst Natl Belg 52:349–366. https​://doi.org/10.2307/36678​88
Bot 18:367–382. https​://doi.org/10.1071/SB030​25 Rydin C, Bremer B (2009) Evolutionary relationships in the Sperma-
Dessein S, Ochoterena H, De Block P, Lens F, Robbrecht E, Schols P, coceae alliance (Rubiaceae) using information from six molecular
Smets E, Vinckier S, Huysmans S (2005b) Palynological charac- loci: insights into systematic affinities of Neohymenopogon and
ters and their phylogenetic signal in Rubiaceae. Bot Rev 71:354– Mouretia. Taxon 58:793–810
414. https​://doi.org/10.1663/0006-8101 Stoffelen P, Robbrecht E, Smets E (1997) Pollen morphology of Coffea
Ganders F (1979) The biology of heterostyly. New Zealand J Bot and Psilanthus (Rubiaceae-Coffeeae), mainly from Africa. Grana
17:607–635. https​://doi.org/10.1080/00288​25X.1979.10432​574 36:313–327. https​://doi.org/10.1080/00173​13970​93626​25
Guo XM, Liao Q, Zhou S, Wang RJ (2018) Leptodermis hechiensis Verellen J, Smets E, Huysmans S (2004) The remarkable genus Cop-
(Rubiaceae), a new species from Guangxi, China. Phytotaxa tosapelta (Rubiaceae): pollen and orbicule morphology and sys-
349:67–72. https​://doi.org/10.11646​/phyto​taxa.349.1.8 tematic implications. J Pl Res 117:57. https​://doi.org/10.1007/
Halbritter H, Ulrich S, Grímsson F, Weber M, Zetter R, Hesse M, s1026​5-003-0128-0
Buchner R, Svojtka M, Frosch-Radivo A (2018) Illustrated pol- Vuilleumier BS (1967) The origin and evolutionary development of
len terminology, 2nd edn. SpringerOpen, Switzerland. https:​ //doi. heterostyly in the angiosperms. Evolution 21:210–226. https​://
org/10.1007/978-3-319-71365​-6 doi.org/10.2307/24066​70

13
 15 Page 16 of 16 X. Guo et al.

Walker JW, Doyle JA (1975) The bases of angiosperm phylogeny: Xie L, Li LQ (2012) Variation of pollen morphology, and its implica-
palynology. Ann Missouri Bot Gard 62:664–723. https://doi. tions in the phylogeny of Clematis (Ranunculaceae). Pl Syst Evol
org/10.2307/2395271 298:1437–1453. https​://doi.org/10.1007/s0060​6-012-0648-y
Winkler H (1922) Monographische Übersicht der Gattung Leptoder-
mis. Repert Nov Spec Regni Veg 18:145–166 Publisher’s Note Springer Nature remains neutral with regard to
Wolff D, Liede-Schumann S (2007) Evolution of flower morphology, jurisdictional claims in published maps and institutional affiliations.
pollen dimorphism, and nectar composition in Arcytophyllum, a
distylous genus of Rubiaceae. Organisms Diversity Evol 7:106–
123. https​://doi.org/10.1016/j.ode.2006.02.005

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