Comparative Pollen Morphology of Selected Species of Blumea DC. and Cyathocline Cass. and Its Taxonomic Significance

The pollen morphology of 20 species from Blumea and Cyathocline Cass. was investigated using a light microscope (LM) and scanning electron microscopy (SEM) to explore their taxonomic significance. This study showed that pollen grains of these species were usually tricolporate, rarely tetracolporate (B. sinuata). Nine pollen types were distinguishable through the exine sculpture characters and the number of apertures. It was easily distinguished Cyathocline from species of Blumea s. str. by its much smaller size (15.04 μm × 15.07 μm) and sparse and longer spines (24 spines, spine length 4.23 μm) with acute apex, which suggest that C. purpurea might not belong to the genus Blumea s. str. The palynological characteristics indicated that Section Macrophllae and Section Paniculatae of Blumea were not monophyletic groups. The pollen morphology differentiation of B. lacera clade is consistent with the interspecific relationship revealed by the molecular phylogenetic tree. However, the pollen morphology of the Blumea densiflora clade is inconsistent with the interspecific relationship based on molecular phylogenetic analysis. This palynology research can only partly support the previously published molecular phylogeny of Blumea s. str.

Blumea duclouxii is different from other species of Blumea in that they possess the capitula with only one layer of phyllary. Its system position is still uncertain. The genus Blumea was established by De Candolle (1833) [16]. The species boundaries are still difficult to delimit. For example, B. riparia and B. megacephala were treated as two variants under the same species by Randeria [2] and Pornpongrungrueng et al. [4]. While Zhang & Yu [17]. Chen & Anderberg [3] divided them into two different species. De Candolle [5] divided the species of Blumea into two series. Randeria [2] interpreted the proposed groups of De Candolle [5] as the sectional rank of Blumea. Then sectional classifications were applied

Pollen Shape
The results showed that the pollen grains of the studied species were radially symmetrical and isopolar, most of these pollen grains were subprolate to spherical and occasionally triangular (Figures 1-5), and the ratio of polar axis length to equatorial axis length (P/E) was ranging from 0.97 to 1.11. The measured morphological characteristics of pollen are shown in Table S1. Pollen grains of Blumea were 15.07-23.09 µm in equatorial diameter (Table S1). Significant differences existed in the P, E and P/E of pollen between different species (p < 0.01).

Apertures
In this study, pollen apertures were tricolporate and tetracolporate [ Figures 1,2 and 5] [ Table S2]. The tricolporate was easily observed under both light and scanning electron microscopy. Under SEM, more subtle characters of apertures could be observed. Colpus was usually rather long, not curved, with a narrower end and a wider middle. Excluding pollen grains of B. sinuata (Loureiro), Merrill was tetracolporate [ Figure 5(C-1,C-2)], and the pollen grains of the other species were tricolporate.

Apertures
In this study, pollen apertures were tricolporate and tetracolporate [Figures 1, 2 and 5] [ Table S2]. The tricolporate was easily observed under both light and scanning electron microscopy. Under SEM, more subtle characters of apertures could be observed. Colpus was usually rather long, not curved, with a narrower end and a wider middle. Excluding pollen grains of B. sinuata (Loureiro), Merrill was tetracolporate [ Figure 5(C-1,C-2)], and the pollen grains of the other species were tricolporate.

Pollen Exine Ornamentation
All the pollens were echinate. Exines of the pollen grain were microperforate and spinose. Spines were attenuated or contracted in acute or blunt apex. The spines were roughly conical or distinctly broader at their bases and ranged from 2.78-5.23 μm in length (Table S2). There were significant differences in the quantity and length of spines among different species (p < 0.01). The exines of pollen grains were microperforate, with round or irregular perforations of varying sizes. Pollens could be roughly divided into two types according to the different densities of interspinular microperforations. The first type was microperforations sparsely distributed in the gap among the base of spines, such as B. balsamifera [ Figure 3

Multivariate Analysis
For cluster analysis (UPGMA), the pollen characteristics of 20 taxa were analyzed, and their infrageneric and interspecific relationships were observed based on these sampling (Tables S1 and S2, Figure 6). The delimitation of these groups was mainly based on the number of spines, spine length, polar diameter and equator diameter, tip shape of spines and density of interspinular microperforations (p < 0.001). In the dendrogram tree ( Figure 6), the 20 taxa were divided into four major groups. Except for Cythocline purpurea

Pollen Exine Ornamentation
All the pollens were echinate. Exines of the pollen grain were microperforate and spinose. Spines were attenuated or contracted in acute or blunt apex. The spines were roughly conical or distinctly broader at their bases and ranged from 2.78-5.23 µm in length (Table S2). There were significant differences in the quantity and length of spines among different species (p < 0.01). The exines of pollen grains were microperforate, with round or irregular perforations of varying sizes. Pollens could be roughly divided into two types according to the different densities of interspinular microperforations. The first type was microperforations sparsely distributed in the gap among the base of spines, such as B. balsamifera [ Figure 3

Multivariate Analysis
For cluster analysis (UPGMA), the pollen characteristics of 20 taxa were analyzed, and their infrageneric and interspecific relationships were observed based on these sampling (Tables S1 and S2, Figure 6). The delimitation of these groups was mainly based on the number of spines, spine length, polar diameter and equator diameter, tip shape of spines and density of interspinular microperforations (p < 0.001). In the dendrogram tree ( Figure 6), the 20 taxa were divided into four major groups. Except for Cythocline purpurea and Blumea sinuata, the other groups were divided into four subclusters. Blumea paniculata and B. napifolia and B. fistulosa formed a subcluster ( Figure 6).
and Blumea sinuata, the other groups were divided into four subclusters. Blumea paniculata and B. napifolia and B. fistulosa formed a subcluster ( Figure 6).   (Table S1). The spines  (Table S1). The spines were roughly conical or distinctly broader at their bases, ranging from 3.21 µm to 5.75 µm in length, acute or blunt.

Cythocline Purpurea
The equatorial length of pollen grains ranged from 13.15 µm to 16.33 µm, and the polar axis length ranged from 14.14 µm to 17.13 µm. Pollen grains of this species were spherical, tricolporate and echinate. The polar areas were large, each with 21-28 spines, an average of 24. Spines are conical, acute, 3.46-4.56 µm in length, average 4.23 µm. The tips of the spines were sharp.

Pollen Types Description
According to the comparative study of pollen morphology, the pollen of all these species could be divided into nine categories, i.e., Blumea sinuata pollen type, Cythocline purpurea pollen type, B. densiflora pollen type, B. napifolia pollen type, B. aromatica pollen type, B. virens pollen type, B. repanda pollen type, B. balsamifera pollen type and B. clarkei pollen type. A brief description of each pollen type was as follows: Type A Blumea sinuata pollen type The typical characteristics of this kind of pollen were tetracolporate, spherical, with denser and longer spines. The pollen grain size was large, 19.52-23.90 µm in equatorial diameter, and the polar axis was 19.72-24.30 µm. This pollen was only found in B. sinuata [ Figure 5(C-1,C-2)].
Type C Blumea densiflora pollen type This type of pollen was triangular or spherical, with sparse (the number of spines < 33) and short spines (the length of spines < 3 µm). This type of pollen was only found in B. densiflora ( Figure 4A).

Type D Blumea napifolia pollen type
The typical characteristics of this kind of pollen were tricolporate, spherical, with denser and longer spines. This type was similar to B. sinuata type, except it was tricolporate. This kind of pollen was found in B. napifolia [ Figure 4(D-1,D-2)], B. fistulosa [ Figure 3(H-1,H-2)] and B. paniculata [ Figure 5(B-1,B-2)]. The pollen grain size of B. paniculata was smaller than that of B. napifolia and B. fistulosa.

Type E Blumea virens type
This type was similar to B. aromatica type, but the grooves of apertures were deeper than those of B. aromatica type. This pollen was only found in B. virens [ Figure 5(D-1,D-2)].

Type F Blumea repanda pollen type
The typical characteristics of this kind of pollen were tricolporate and spherical, with longer and sharp spines and relatively dense inter-spinular perforations. This type of pollen was found in B. repanda [ Figure 4(H-1,H-2)] and B. lacera [ Figure 4(E-1,E-2)].

Type G Blumea balsamifera pollen type
This type of pollen was spherical, tricolporate, with a medium number of spines. Pollen grains were trifid in polar view. Spines were short, with a blunt apex. Exines were relatively sparse microperforate.

Type H Blumea clarkei pollen type
This type of pollen was spherical, tricolporate, with a medium number of spines. Pollen grains were trifid in polar view. Spines were long, with acute apex. Exines were relatively sparse microperforate. This type of pollen was found in B. hieraciifolia Type I Blumea aromatica pollen type This type of pollen was spherical, tricolporate, with a medium number of spines. Pollen grains were trifid in polar view. Exines were relatively dense microperforate, with sparse spines. Spines were short and blunt. Most species had this pollen type. We found this type of pollen in B. hookeri [ Figure 4 See the following key for a comparison of different pollen types.

Discussion
All the studied pollen grains had common characteristics such as spines and interspinular perforations on their exines, which were nearly spherical and were typical types of pollen grains of the tribe Inuleae, some similar to that of the other entomophilous Inuleae species such as Pulicaria and Inula [31][32][33], indicating a close genetic relationship between these species. The pollen morphology of Blumea was similar to those of Blumea species of Wittenbach [30]. In this study, pollen showed significant interspecific variation in size and exine spines.

Size Range
Among all the studied species, the pollen grains were highly variable in size. Although pollen size of Inuleae species had not been used as a primary factor for delimitation of pollen types alone, because of pollen grain size possibly related to polyploidy within a species of Asteraceae [37][38][39][40]. The environmental and nutritional conditions, as well as the processing methods of pollen, also affected the size of the pollen grain [41]. The Blumea sinuata type pollen was quite different from the other species. This species was also significantly different from the allied species in that the stem of B. sinuata was noticeably stouter. It was possibly related to the polyploidy of this species, according to the chromosome number counts. But under similar environmental conditions and the same treatment method for pollen grains, there were still significant differences in pollen size among some species. Of all the species studied, Cythocline purpurea has the smallest pollen grains.

Apertures
The aperture structure also contributed to differentiating Blumea sinuata from the other investigated taxa, which was characterized by both 3-zonocolporate and 4-zonocolporate pollen grains, while the other taxa exhibited only 3-zonocolporate pollen grains. The tetracolporate form pollen was unusual in the Asteraceae, although this type had been reported by Wodehouse [25], Wittenbach [30], Osman [31], and Wortley [20]. Wodehouse [25] believed that tetracolporate aperture, perhaps, was related to the irregular distribution of chromosomes or as the result of hybridity. Among all studied species, only B. sinuata was tetracolporate. This was also possibly related to its polyploidy.

Exine Sculpturing
The pollen sculpture was somewhat uniform in most investigated species. The pollen grains were spiny with perforate sculpture. In this study, the pollens examined were highly variable regarding the number and shape of exine spines. Although the tendency toward spine reduction of Inuleae was considered an advancement character in the Asteraceae family [23], we believed that the high number of spines in Blumea sinuata pollen was possibly related to its polyploidy. Spine length and density were stable morphological characters for generic differentiation [23,25]. Among all investigated species, spine length, density and shape could also be used as species delimitation. The inter-spinular perforation density varied among different species (Table S2). Compared to other species, the pollen of Cythocline purpurea had the least number of spines and longer spines.

Taxonomic Significance of Pollen Features
Three pollen types could be found in Blumea Section Macrophllae DC., i.e. B. densiflora type, B. balsamifera type and B. aromatica type. The pollen grains of B. densiflora were smaller, triangular or spherical, with the shortest and least spines, and were quite easily distinguished from the other species in this section. The pollen grains of most species in this section were spherical, occasionally, with few triangular pollen in B. martiniana. The palynological characteristics of other species in the Section Macrophllae had little variation, except in pollen grain size and the number and length of spines and microperforations of exines. The pollen grains of B. balsamifera were the smallest, with the most exine spines in this section and sparsely inter-spinular microperforations. The pollen grain of B. hookeri had the longest spines in this section, followed by B. aromatica. Although B. hookeri and B. densiflora were treated as one species by some botanist, the pollen morphological characteristics were significantly different.
The palynological characteristics of species in Section Paniculatae DC. varied greatly. The pollen grains were tricolporate or tetracolporate. The pollen of Blumea sinuata was different from that of the other species, with four germination holes, the greatest number of spines and the longest spines. The pollen grains of B. napifolia and B. fistulosa were larger, with more and longer spines. The pollen morphological characters of B. paniculata were similar to those of B. napifolia in that polar region fissures were not obvious, and spines were denser. But the pollen grain size was smaller than that of B. napifolia. Blumea duclouxii had the smallest pollen grains in this section, followed by B. lacera. The palynological characteristics of B. duclouxii were very similar to those of B. lacera. It was difficult to decide its taxonomic position only by pollen morphology.
The morphological characteristics of all the species studied in Blumea Section Semivestitae were relatively consistent, except for slightly larger pollen grains and the relatively sparse microperforate exines with longer acute spines in B. repanda. Pollen morphological characteristics of B. riparia and B. megacephala in Section Semivestitae were similar to that of B. aromatica, B. hookeri, B. martiniana in Section Macrophllae.
From the pollen characteristics of all studied species, we can find that the palynological characteristics of species do not support the taxonomic classification of the sections of Blumea De Candole [5]. The pollen morphology of different sections may be consistent. On the other side, the division of the sections of Blumea was artificial by various authors [2,5,18].
Blumea Section Macrophllae and Section Paniculatae were probably not monophyletic groups, with different evolutionary directions and variable pollen morphology. However, pollen characteristics have a certain significance in the species identification of Blumea. For example, pollen grains of B. sinuata were tetracolporate, while pollen grains of B. densiflora pollen were triangular, sparsely microperforate. However, species with similar pollen types were difficult to segregate.
Cythocline purpurea had the smallest pollen grains, with the least spines, of all the species studied. Spines were long, with acute apices in C. purpurea. This combination of characters was quite different from the other studied species. The pollen morphological characters of C. purpurea were easily distinguished from other species of Blumea s. str., which suggested that C. purpurea might not belong to the genus Blumea s. str.
Two major well-supported clades of Blumea s. str. were recognized based on a molecular phylogenic tree by Pornpongrungrueng et al. [12], including Blumea densiflora clade and B. lacera clade. Our study showed polymorphism in the pollen morphology of the B. densiflora clade and the B. lacera clade. The pollen morphology differentiation of the B. lacera clade was consistent with the interspecific relationship revealed by the molecular phylogenetic tree. For example, B. napifolia and B. paniculata were closely related in the molecular tree and showed similar pollen characteristics. But pollen morphology of B. densiflora clade, including B. densiflora, B. aromatica, B. balsamifera and B. martiniana displayed significant variation, which is inconsistent with the interspecific relationship suggested by the molecular morphology tree [12]. Therefore, our palynology research can only partly support the previously published molecular phylogeny of Blumea s. str.

Plant Materials
Mature pollen samples were obtained by removing one or two florets from dried specimens. Voucher specimens were deposited in the Herbarium of Chengdu Institute of Biology (CDBI). The herbarium voucher details were included in the specimens investigated list ( Table 2).

Micromorphological Examination
Pollen samples were prepared by acetolysis as described by Erdtman [42] and viewed with a light microscope (LM). Pollen mounted in neutral gum was examined under transmission light using Olympus microscopes BX43. Pollen samples were prepared for the scanning electron microscope (SEM). Pollen grains were dried in the air, then directly mounted on stubs and sputter-coated with gold-palladium for five minutes. SEM examination was carried out by Phenom Pro microscope, operating at 10 kV. Images were digitally processed, and the final plates were prepared using Adobe PhotoShop 7.0. Pollen characteristics, including polar length of pollen grains (P), and equatorial length (E), were measured using digital light microscopy images based on 20 pollen grains from every investigated species by software Image J [43]. Due to the small size of the pollen in the genus Blumea, the length and quantity of spines were observed and measured more accurately under scanning electron microscopy. The ratio of polar length to equatorial length (P/E) of pollen was calculated. Pollen morphology was described according to the standards of Erdtman [44] and Wang [45]. If the pollen shape index (P/E) > 2, the pollen was considered perprolate. If 1.32 < P/E ≤ 2, the pollen was considered prolate. If 1.14 < P/E ≤ 1.32, the pollen was considered as subprolate. If 0.88 < P/E ≤ 1.14, the pollen was considered spherical. Descriptive terminology follows Punt et al. [46].