Pollen Morphology of Indian Species of Saraca L. (Leguminosae)-A Threatened and Legendary Medicinal Tree

The genus Saraca L. (Leguminosae) is a universal panacea in herbal medicine. The present study investigates the comparative pollen morphology of four species of Saraca viz. S. asoca (Roxb.) de Wilde, S. declinata (Jack) Miq., S. indica L., and S. thaipingensis Cantley ex Prain growing in India to reveal differences of their pollen structures to aid taxonomic and evolutionary values. The detailed morphology and surface structure of pollen grains were studied and described using light microscopy and scanning electron microscopy. The pollen grains of Saraca showed isopolar, para-syncolporate, tricolporate, with radially symmetric, prolate and prolate-spheroidal structure. The surface of pollen of S. indica is rugulate with large lirae but in S. declinata, the surface is micro-rugulate to vermiculate with relatively thin lirae and that of S. thaipingensis is indistinct as the psilate surface with a frequent protuberance and fewer perforations were observed along with the gemmae like structure. Exine ornamentation helped to separate S. indica and S. asoca. Exine thickness varies from 3-4 μm. Presence of protuberance and exine thickness varies among individuals of the species spread over different locations. Present work also provides a unique palynological identity and interrelationship of these four species based on cluster analysis taking 23 pollen characters with the help of statistical method like the plotting of ternary graph. Ternary plots also helped to calculate the level of plasticity of each character in the intraand inter-specific level.


Introduction
The genus Saraca L. of the family-Leguminosae, subfamily-Detarioideae [1] and the tribe-Detarieae [2], is a small to medium-sized evergreen rainforest trees growing to a maximum height of nine meters. 11 species are distributed in tropical and subtropical rain forests of southeastern and southern Asia [3]. Four species of Saraca are distributed in India; i.e., S. asoca (Roxb.) de Wilde ( Fig. 1(A)) is a small tree distributed in different places of India. S. declinata (Jack) Miq. is distributed in India and other countries like Burma, Malaysia, Myanmar, Sri Lanka and commonly known as Red Ashok ( Fig. 1(D)). S.indica L. is a medium-sized tree and commonly known as Sita Ashok ( Fig. 1(G)), frequently planted in association with Buddhist temples [2]. S. thaipingensis Cantley ex Prain is present in India under captivating and frequent in Myanmar, Thailand, Malaysia and Indonesia having yellow flowers ( Fig. 1(J)) and thus commonly known as Yellow Saraca or Yellow Ashok. S. asoca having close morphological resemblance with S. indica and co-exists in India with S. indica [3]. Central and eastern Himalayan foothills, northern plains and western coasts are reported to have a rich distribution of S.indica [4]. All of these plants are considered to contain medicinal properties. The crude extract of flowers of S.asoca is considered the best uterine tonic [5,6]. Due to the decline in population S. asoca is listed as 'globally vulnerable' in the Red List of Threatened Species of International Union for Conservation of Nature and Natural Resources [7] (http://dx.doi.org/10.2305/IUCN.UK.1998.RLTS.T34623A9879360.en).
Little is known about the pollen morphology of the species of Saraca [8][9][10] and it is important to know the detailed structural evaluation of pollen as it can help the research on gene flow, aerobiology, reproductive biology, and systematics. Morphology of the pollen grain is often a pertinent taxonomic marker in the identification of higher plants [11] and is not influenced by environmental conditions. Palynology is unique to provide a tremendous amount of information and the variation of pollen morphological characters have importance for phylogenetic structuring [12] and having phylogenetic significance [13] it was applied to evaluate intergeneric relationships [14] and classify the eudicots [15].
Ternary plots were used to evaluate the correlation of floral characters related to pollination which helped to establish the evolutionary relationships among pollinators [16]. Selection of non-variable characters among the large set of morphological features of Saraca was also done with the help of ternary plots [10].
The present study was undertaken to specify the pollen morphology of four species of Saraca and the phenetic relationship among them, evaluating the stability and plasticity of characters with the help of ternary plots.

Materials and Methods
Partially opened matured fresh flowers of four species of Saraca viz. S. asoca, S. declinata, S. indica, and, S. thaipingensis were collected at 9 AM from the month of April 2015 to March 2018 for palynological studies from the plants grown in Acharya Jagadish Chandra Bose Indian Botanic Garden, Shibpur, West Bengal, India and other localities mentioned in Tab. 1. Polliniferous samples were collected from different places of India and all the voucher specimens of the studied species deposited at Herbarium of the University of Burdwan (Acronym BURD [17]).

Sample Preparation and Light Microscopy
Pollen grains were isolated from the anther of each species separately and acetolysis was done following the method of Erdtman [18,19]. For the light microscopic study, the acetolysed pollen grains were centrifuged to decant for 3-5 minutes. The pollen material was mounted on the slide with glycerin jelly without any staining. The pollen grains were examined under oil immersion objective using a 10 × eyepiece of a compound microscope (Olympus CH20i microscope) fitted with CMOS Camera (IS 500, 5.0 MP) and its attachment with a computer. Both the polar axis and the equatorial axis of pollen grains (5 samples for each voucher), aperture size, and exine thickness were measured. Quantitative measurements (Tab.

SEM Study
For scanning electron microscopic (SEM) study, the previously acetolysed pollen material was mounted on a metallic stub, coated with gold, in a sputtering chamber (Ion-sputter JFC-1100). The coating was restricted to 150Å and SEM examination was carried out with a Carl Zeiss EVO 40 (EDX Oxford Instrument No-7636) system and its relevant software, Carl Zeiss Smart SEM 0.5.03. The terminology for the description of pollen grains is used after different literatures [18,20,21]. SEM images of pollens are represented in Fig. 2

S. thaipingensis
(TH-1) * PA = polar axis, ǂ ED = equatorial diameter, § L/B ratio = ratio between length and breadth of colpus, † R = range, ʃ CL/PA = Ratio between the length of the colpus and polar axis and A = average

Cluster Analysis and Preparation of Dendrogram
A dendrogram (Fig. 3) was obtained using PAST3 software to conduct multivariate cluster analysis (Correlation outgroup) applying 23 character states of pollen morphological features and data matrix presented in Supplementary Tabs. 2 and 3. Four species of Saraca and their different individuals were considered as IN-1 to TH-2 (Tab. 1) and character states were oriented with four character codes i.e., 0, 1, 2 and 3.

Measuring the Mean Proportion Percentage and Preparation of Ternary Plots
Mean of Proportion Percentage = ℎ ℎ ℎ 3 × 100% Values of character states were converted to proportion percentage with the formula obtained (Supplementary Tab. [4][5][6][7][8][9][10]. Then the data were plotted in the datasheet of PAST 3 software and using the toolkit, transposition of arrangement was done. Then entering into the plot option and applying Ternary plot option, specific plot-diagramme was obtained. Application of ternary plots done here in two ways; firstly, the combination of four species of Saraca in four ternary plots ( Supplementary Fig. 2), one species at a time helped to select identifying characters of pollen and secondly, combination of each species with their closely related individuals in the ternary plot ( Supplementary Fig. 3) helped to characterize plasticity and stability of characters of pollen. Colored space within the ternary shows the range of characters. The affinity of dots towards the epicenter of the triangle shows the stability of characters whereas the increase of the distance of a dot from the epicenter represents the increase of plasticity of the character represented by the dot.
The variability of pollen aperture is higher in different species of Saraca. as well as the shape and size. The surface ornamentation also varies considerably like reticulate, macro-rugulate, micro-rugulate, psilate. The presence of gemmae like structure is also a very important character.

Apertures
The number and type apertures are similar among four species of Saraca. Most of the species have angulaperture. The compound type of aperture is present in all cases as endoaperture is present as pore at the equator of each of the three colpi (Figs. 2(C), 2(G), 2(K), 2(O)). The length and breadth ratio of the aperture (colpi) is greater than 2 in all of the four species. Each endoaperture contain distinct ring-like thickening or annulus and the height of it varies in four species. The annulus of S. indica has the maximum height ( Fig. 2(K)) and that of S. thaipingensis (Fig. 2(O)) is apparently very minute and the annulus is medium in height in S. declinata ( Fig. 2(G)). The height of the annulus of S. asoca is also remarkably high ( Fig. 2(C)). The amb (ambitus or contour or circumference of the pollen grains in polar view) is peritreme to angulaperturate. Three colpi fuse or anastomose with each other at each pole producing apocolpial region and that area varies in four species (Figs. 1(C), 1(F), 1(I), 1(L), Figs. 2(B), 2(F), 2(J), 2(N)). However, in S. thaipingensis the area and nature of the apocolpial field itself vary in different samples as the colpa becomes anastomose ( Supplementary Fig. 1(R), (T)) in some and remains separated in others (Fig. 2(N)).

Exine Morphology and Surface Ornamentation
The surface ornamentation varies as rugulate with large lirae in S.indica (Fig. 2(L)), micro-rugulate with relatively thin lirae but in S. declinata (Fig. 2(H)) and in S. thaipingensis pollen surface is indistinct but psilate with gemmae like structure ( Fig. 2(P)). Pollen grains of S. asoca is relatively large in size in respect to other species but the surface ornamentation ( Fig. 2(D)); i.e., reticulate type, produces the remarkable difference with other pollen grains. Exine thickness varies from 3-4 µm. S. asoca has thicker exine than S. indica. Tectum ornamentation is present but somewhat indistinct. Detailed qualitative features of four species and their other individuals are presented in Tab. 3. Qualitative characters of each type of pollen grains are represented in Supplementary Tab. 1.
Pollen grains of another sample of the same species (Voucher BURD12049) showed similar characters inrespect to shape and size ( Supplementary Figs. 1(B), 1(C), 1(D)). The apocolpium has a larger area (24.17 μm 2 ). The thickness of sexine and nexine is relatively broader than others.
Pollen grains from the voucher BURD12009 ( Supplementary Figs. 1(F), 1(G), 1(H)) and voucher BURD12012 (Supplementary Figs. 1(J), 1(K), 1(L)) have a larger size (57.73 μm and 61.4 μm respectively). The length of colpi is longer in them and the breadth of it is surprisingly high in the pollen grains of voucher BURD12012 (7.8 μm). The area of endoaperture is less variable. The apocolpium space is relatively greater. The surface ornamentation is also non-variable.

S. indica
The size of the pollen grains of S. indica also varies considerably. Pollen grains are tricolporate with sub-triangular amb (Figs. 1(H), 1(I), Fig. 2(I)). Length of colpi is longer but the depth is relatively shallow. The thickness of the sexine and nexine also varies considerably. Pores (Fig. 2(K)) are relatively larger and the height of the annulus is relatively higher. Amb is sub-triangular in shape. The exine is observed to have rugulate surface (Fig. 2(L)).
The thickness of pollen grains is variable. Pollen grains of voucher BURD12004 has thin exine but that of voucher BURD12010 is with thick exine (Supplementary Figs. 1(N), 1(O), 1(P)). Pollen grains contain larger pores and the apocolpium index is relatively small. The surface is macro-rugulate like other samples.

S. thaipingensis
The size of the pollen varies from medium to large but the basic morphology is very similar among all of the samples Endoaperturates are relatively small (18.32 µm 2 ) and the annulus is also low in height (Fig. 2(O)). Apocolpium index is also relatively high. Thickness of sexine is greater than that of nexine. The surface of the pollen grains is psilate but the presence of gemmae like structure ( Fig. 2(P)) is specific to the species.
The variability of morphological features is low. The L/B ratio and thickness of nexine vary considerably in the pollen grains collected from another sample, voucher BURD12050 (Supplementary Figs. 1(R), 1(S), 1(T)).

Description of Dendrogram
Qualitative characters of each type of pollen grains are represented in Supplementary Tab. 1. The dendrogram (Fig. 3) obtained from the cluster analysis applying the data matrix (Supplementary Tab. 3) showed two distinct clusters, where cluster 1 contains relatively higher similarity among DE-1, DE-3 and DE-2, and TH-1 and TH-2 showed a relative difference. In cluster 2 AS-1, IN-1 and IN-2 showed higher similarity but AS-2 is separated. Three separate ternary plots were prepared including DE-1, DE-2, and DE-3 in the first, AS-1, IN-1 and IN-2 in the second and TH-1, TH-2 and AS-2 in the third ternary plot.

Selection of Characters Using Ternary Plots
Triangular or ternary diagram obtained from proportion percentage of each character for three of these four species of Saraca shows that 100% of each character is represented by the apex of the angles. Lateral sides of the triangle indicate the scale of species-specific character in percentage. From the ternary plot in the Supplementary Fig. 2(A), it is obtained that the height of annulus character (Tab. 2 character no. 14, Yellow color in the ternary plot) value in the ternary diagram is highest (68%) in case of S. indica representing its unique pollen characters. Similarly, the area of apocolpium space (Tab. 2 character no. 17, dark green color in the ternary plot) is the unique pollen character for S. declinata representing nearly 66% of correlation. In case of S.thaipingensis exine ornamentation, containing psilate surface with gemmae like structure and few perforations (Tab. 2. character no. 20, red coloring in the ternary plot) is unique and the value is maximum, representing 100%. Supplementary Fig. 2(B) represents the combination of S. indica, S. declinata, and S. asoca and only the characters represented by the character no. 17 (Tab. 2); i.e., area of apocolpium space is unique for S. declinata. Supplementary Fig. 2(C) represents the combination of S. indica, S. thaipingensis and S. asoca and the character of the surface of pollen, i.e. presence of exine protuberance represented by the character no. 20 (Tab. 2), is unique for S. thaipingensis and characters like the area of the pore (Tab. 2 character no. 12) and height of annulus (Tab. 2 character no. 14) are equally important for the identification of S. indica and S. asoca. Ternary plot with the combination of characters of S. declinata, S. thaipingensis and S. asoca ( Supplementary Fig. 2(D)) represent the presence of protuberance on the surface of pollen is important for S. thaipingensis, large area of apocolpium space (Tab. 2 character no. 17) is important identifying character of pollen of S. declinata and the greater height of annulus (Tab. 2 character no. 14) is definitive for pollen of S. asoca. From this analysis, it is represented that the selection of definite key character for the identification of pollen of S. declinata and S. thaipingensis from the set of quantitive characters is possible but that is difficult to separate S. indica and S. asoca.
Ternary plots like supplementary Figs. 3(A) to 3(D) are the combination of pollen characters of three specimens from each cluster of the dendrogram (Fig. 3). Supplementary Fig. 3(B) showed that pollen of S. indica (voucher BURD12010) has exine protuberance (Tab. 2 character no. 20) unlike any other representative of S. indica and perimeter of the pollen is much higher than S. asoca (voucher BURD12008). Supplementary Fig. 3, C showed that S. declinata (voucher BURD12012) has the pollen with the protuberance in its surface but its abundance is rare. Supplementary Fig. 3(D) showed that both S. thaipingensis (voucher BURD12005 and voucher BURD12050) has protuberance on the surface of their pollen but the abundance is more in the pollen grains of voucher BURD12050. Presence of protuberance showed inter-specific variations.

Discussion
The shape of pollen, apertural types and exine ornamentation were found to be more significant pollen characters in Onobrychis of Fabaceae [22]. Size of the pollen plays a greater role in the sub-generic grouping of Polygonaceae [23]. The present investigation revealed that the shape of the pollen grains of the studied species of Saraca varies from prolate to prolate-spheroidal. The prolate-spheroidal shape of the pollen grain is found in S. indica, but in S. declinata pollen grains are sub-prolate and in S. thaipingensis the shape varies from prolate to prolate spheroidal. It was reported that P/E values and morphological features in polar view are relatively stable in each group in polygonate [24]. Pollen grains of different samples collected from different localities of Saraca also showed relative stability of P/E ratio but morphological features obtained from polar view varies considerably. According to Matamoro-Vidal et al. [25], exine thickness is an important character for the taxon delimination. Exine thickness is most variable character shown in the analysis in both intra-and inter-specific taxa of Saraca.
Pollen grains are usually categorized largely on the basis of their shape, size, aperture types, symmetry, polarity and exine sculpturing [14,26]. The type of aperture varies from tricolporate to syntricolporate and the size varies from medium (25-50 μm) to large (above 50 μm). Present study reflects that the variation in pollen size and the thickness of exine are less effective for the specification of Indian Saraca. Pollen grains were categorized into four groups based on the surface ornamentation as i) reticulate ii) macro-rugulate iii) micro-rugulate and iv) psilate. Present study also revealed that some of the pollen characters like annulus height (comparatively higher), narrowest apocolpium space, macrorugulate surface ornamentation, etc. are unique in S. indica; annulus is relatively lower in height, higher apocolpium space and micro-rugulate surface ornamentation are in S. declinata; and annulus lowest in height, narrow apocolpium space, psilate ornamentation and gemmae like protuberance and few perforations are present in S. thaipingensis. The ternary plot (Supplementary Fig. 2) revealed that character of the annulus in S. indica, apocolpium space in S. declinata and surface ornamentation in S. thaipingensis and S. asoca are potent marker characters turned to be useful traits for species identification.
Palynological characters provide very important information for the phylogenetic reconstructions [12]. Moreover, from a phylogenetic as well as evolutionary point of views, the polarity, symmetry, aperture types and exine sculpturing are the most important pollen characters [13]. It was concluded that the latitudinal gradient including moisture and temperature is important in the evolution of size and shape of pollens due to harmomegathy [27]. The diversity of shape and size of pollen of Saraca may also carry such influence. Harmomegathic stress produced due to loss of water caused the decrease in size and change of shape of pollen to avoid dehydration [28]. Pollen of S. asoca has relatively greater size as the abundance of this species can be observed near the forests having higher rainfall. The shape of the pollen often causes reproductive barriers among related sub-species [29]. The difference in the size of pollen of S. indica and S. asoca is not very much pronounced but the reproductive barrier may be caused due to the difference of exine sculpturing. Pollen grains with greater apocolpium space is an advanced feature [30][31][32]. Pollen grains of S. thaipingensis with higher apocolpium space is advanced though that of S. declinata is higher because S. declinata produce heteropolar pollen grains. Ornamentation pattern of pollen surface is important in the study of plant evolution and systematic taxonomy [33]. According to the concept of evolution of pollen grains stated by Yang et al. [34], it can be found that the surface morphology of pollen of S. asoca having the reticulate type of exine is primitive among all. Both coarse rugulate (psilate) and rugulate-perforate exine sculpture were evolved independently from (micro) reticulate sculpturing of exine [35]. Walker [13] Concluded that evolution of pollen depends upon the gradual reduction of exine sculpture. From above concept, it can be concluded that the macro-rugulate surface of pollen of S. indica evolved from reticulate ( Fig. 2(D)) type of S. asoca and micro-rugulate ( Fig.  2(H)) type of surface of pollen of S. declinata is derived from macro-rugulate type ( Fig. 2(L)). Most advanced type of pollen surface is psilate ( Fig. 2(P)) with perforated tectum and gemmae like structure which is found in the pollen of S. thaipingensis which is derived from the micro-rugulate type of surface. The proposed evolutionary trends of pollen of four species of Saraca are given in Fig. 4.

Palynology vs. Systematics of S. indica and S. asoca
In the present palynological investigation, pollen size, shape, aperture of both the species (i.e., S. asoca and S. indica) are of the same type as described for two other species of Saraca [10]. Though, two distinctly different types of exine ornamentation were observed; S. asoca showed perforate to reticulate surface ( Fig. 2(D)) and S. indica showed micro rugulate surface ornamentation (Fig. 2(L)). Therefore, the palynological study confirms the existence of two different sets of taxa comparable to two different species as treated by Zuijderhoudt [36] and Ding et al. [3].
Variation of quantitative data of pollen grains makes difficulty for morphology-based taxonomic identification of different species of Saraca. Samples collected from different geographical locations for the same species showed a considerable degree of character variation which leads to the conclusion that most of the considered characters of Saraca are plastic in nature. Though, the presence of protuberance in all the studied individuals is a strong identifying character for the species S. thaipingensis. Plasticity of shape, size and surface ornamentation may be regulated by harmomegathic stress produced due to changing humidity. Pollen of S. asoca is primitive due to the presence of reticulate exine ornamentation and from which pollens of S. indica evolved which has a rugulate type of ornamentation. Gemmae like exine ornamentation present in the pollen of S. tahipingensis is the most advanced type. Exine ornamentation of S. indica, i.e., macro-rugulate, differs from S. asoca which has the reticulate type of ornamentation indicates the existence of two different taxa. It can also be concluded that the qualitative characters of pollen (Tab. 3) are more applicable than quantitative characters (Tab. 2) to distinguish different species of Saraca, taxonomically.