Studying the morphological diversity of Bunium paucifolium and some Elwendia species (Apiaceae)

Abstract The genus Bunium L. (Apiaceae) has about 50 species in the world, distributed in Asia, Europe and North Africa. Recently, Bunium has been divided into two separate genera Elwendia and Bunium. Kerman province is the largest province in Iran and has a wide distribution of these genera especially E. persica, as the most important medicinal and spice species. There are a few published studies on the morphology of these genera, especially on flower and fruit morphology. 20 morphological traits were studied to analyze the differences between species. The most important traits in the first principal component analysis (PCA1) were the number of bracts and umbellets per umbel, stylopodial length, corolla shape, empty fruits and the number of flowers and fruits per umbellet. Corolla shape was raised and vertical in B. paucifolium while horizontal and wide in three other species (E. cylindrica, E. persica and E. wolffii). There are 3 to 4 male flowers in the center of umbellet in E. cylindrica, E. wolffii and B. paucifolium while in E. Persica, no male flowers were observed. This study revealed that morphological traits particularly corolla shape are consistent with the molecular phylogenetic results reported by other researchers.


PUBLIC INTEREST STATEMENT
The genus Bunium L. (Apiaceae) has about 50 species in the world and has a wide distribution in Iran especially in Kerman province. Recently, Bunium has been divided into two separate genera Elwendia and Bunium. Due to the absence of a comprehensive study on quantitative and qualitative traits, in this survey, morphological traits of flower, pollen and fruit in two genera Elwendia (E. cylindrica, E. persica and E. wolffii) and Bunium (B. paucifolium) were investigated. The most important traits in the principal component analysis (PCA) were the number of bracts and umbellets per umbel, stylopodial length, corolla shape, empty fruits and the number of flowers and fruits per umbellet. Our study revealed that morphological traits, particularly corolla shape, are consistent with the molecular phylogenetic results reported by other researchers and confirms the studies in the separation of Buinum to two genera Bunium and Elwendia.

Introduction
Bunium genus is an important medicinal and spice plant belonging to Apiaceae family, Apioideae subfamily and Apieae tribe (Mozaffarian, 2007). The Apiaceae family is classified into three subfamilies that the largest of them is the Apioideae (Plunkett & Downie, 1999). Apiaceae family includes about 450 genera and 3700 species in all around the world that are rich in secondary metabolites including many valuable genera in terms of economic and medical importance (Duran et al., 2010;Olle & Bender, 2010). The plants belonging to the Apiaceae family have its own distinctive features such as umbel inflorescences, actionomorphic and five merous corolla, inferior ovary with two locules and the schisocarp fruits that are separated from each other by carpophor. In fact, these characters in the Apiaceae family are in the late evolutionary stages of the series of dialypetalous (Ghahreman, 1993;Mozaffarian, 2007;Plunkett & Downie, 1999). Morphological traits are important as a source of taxonomic features. Different species are identified based on characteristics such as the number umbel per plants, shape and number of bracts and bracteoles, sexuality and shape of flowers, shape and number of fruits, the length of the mericarp, the number and shape of the furrows of the mericarp, secretory ducts, commissural width, carpophore and stylopodium (Chahota et al., 2017;Ghahremaninejad et al., 2014). Although some researches have presented stenopalynous type of pollen in this family (Erdtman, 1952;Klimko et al., 2012) but other studies have stated significant importance of pollen in family taxonomy. Cerceau Larrival and Roland Heydacker (1976), Van Zeist and Bottema (1977) have grouped this family into several types based on the pollen features. Studies of the pollen structure and size, state and size of pores, mesocolpium and apocolpium structure are considered as important parameters in determining of phylogenetic relationships of different species of this family (De Leonardis et al., 2009). The Bunium L. has about 50 species in the world, distributed in Asia, Europe and North Africa (Degtjareva et al., 2009). Recently, Bunium genus has been divided into two separate genera Elwendia and Bunium genus; so some species were transferred from Bunium to Elwendia (Degtjareva et al., 2013;Pimenov, 2017). One of the most important species is Kermani black zira or Elwendia persica (Boiss.). Kerman province is the largest province in Iran, and has the highest distribution of E. persica (synonym B. persica). Although E. persica is a well-known species that is used in medicinal and nutrition industries, there are a few data on morphological traits of flower, fruit and pollen in these two genera in Iran. Initially, the studies of Degtjareva et al. (2009) on some Bunium species distributed in ancient Mediterranean based on ITS and psbA-trnH sequences and morphological characters (the number of cotyledons, the width of mericarp commissure) showed that Bunium species are divided into two main Clade including Bunium-I and Bunium-II. According their results, B. persica, B. wolffii and B. cylindrica are placed in first calde (Bunium-I) whereas B. paucifolium is placed in Bunium-II) clade (Degtjareva et al., 2009). Recently, these researchers transferred Bunium I clade to the genus Elwendia Boiss. and Bunium II Clade to the genus Bunium (Degtjareva et al., 2013;Pimenov, 2017). Morphological characteristics, along with molecular characteristics can be useful and effective in taxonomy and understanding the evolutionary relationships. There are no published studies on inflorescence, flower and fruit traits these genera except for a few basic level studies. The reproductive features tend to be much less variable at least in the basic structure. Therefore, reproductive characters are regarded as more useful for plant classification and understanding the evolutionary relationships than vegetative characters. Therefore, due to the absence of a comprehensive study on quantitative and qualitative traits of reproductive feature, in this survey, morphological traits of flower, pollen and fruit in two genera Elwendia (E. cylindrica, E. persica and E. wolffii) and Bunium (B. paucifolium) were studied.

Plant material
Several field collections showed that two genera Elwendia and Bunium have been distributed in different parts of Kerman province. Species identification was performed using conventional keys and reports from previous researchers. Table 1 shows different species and their location in this study.

Morphological evaluation
Nineteen morphological traits cited were recorded during the growing season at different populations of studied genera. These characteristics were studied using naked eye, stereo microscope)OLYMPUS TL2 (and light microscopy (OLYMPUS BH-2) connected to camera. Mature pollen grains at dehiscence stage were dusted onto a drop of water on a microscope slide. The size of pollen was compared by measuring the length of polar (P) and equatorial (E) axes of 10 pollen grains on each slide randomly. The measurements were made with an ocular micrometer (Rezanejad, 2007;Rezanejad & Majd, 2012). The shape category is determined by dividing the equatorial width into the polar length of the pollen grain (P/E) and according to the classification of Punt et al. (2007). Of the Nineteen traits, thirteen were quantitatively measured, while six were qualitative. The quantitatively measured traits were Umbels per plant (

Statistical analysis
Data for each quantitative morphological trait were randomly recorded on ten replications. The mean values of these replications were used for data analysis. For qualitative variables, mode values were considered and coded for statistical analysis. Different measures such as variances, correlation and principal component analysis (PCA) were determined using the software SPSS Ver.24 by PCA method as well as NT SYS Ver.2.11a software by the unweighted pair-group (UPGM) method (Moghaddam & Pirbalouti, 2017).

Results and discussion
Morphological and anatomical fruit characteristics, as well as molecular and phylogenetic studies are importance and useful for plant identification and classification (Liu et al., 2016). Sampling studies showed that in some regions, all studied species were seen together ( Table 1). The inflorescences in all species are compound umbels with a long tail, the rays of the umbels are unequal and extensive (Figures 1 and 2). Underground parts are irregularly spherical, the small to large depending to age; leaves are basal or alternately arranged along the stem, and leaf blades are often pinnate (Figure 1). The color of bush in three species of E. cylindrica, E. persica and B. paucifolium is green, while various populations of E. wolffii are purple specially during flower and fruit development (Figure 1). Each compound umbel in maturity stage has been composed of 7-10 unequal umbellets depends on the species (Figure 2, Table 2). They have 2-6 involucral bracts which are linear, sharp, herbaceous and caduceus; however, most of umbels are without bracts in B. persica (Figure 2). Each umbellet has 10-20 flowers depending on the species type. The involucels have 5-7 bracteoles that are smaller than bracts. Bracteoles length in each involucel is different (heteromorphy) (Figure 3, Table 2). There are 3 to 4 male flowers in the center of umbellet in E. cylindrica, E. wolffii, E. persica and B. paucifolium. However, some populations of E. persica like the DehBala population had no male flowers (Figure 3). Patterns of flower development in andromonoecious species of Apiaceae have been discussed by Ajani et al. (2016). They reported functionally male flowers show the same development pattern as hermaphroditic flowers except for the gynoecium, which gets inhibited at a late stage of development (Ajani et al., 2016). The accumulation and density of flowers in the inflorescences is an important biological trait that is dependent to the species type. The accumulation of flowers in large umbel inflorescences attracts insects from long distance; and this matter is helpful for pollination (Linder, 1998).
No discernible and grown calyx was observed in the flowers studied populations (Figures 3  and 4). Ajani et al. (2016) reported that from the functional point of view, flowers in Apioideae do not necessarily need large sepals. The outer flowers are usually protected by involucral and/or involucellar bracts and the inner ones are densely packed and protected by neighboring meristems (Ajani et al., 2016). The corolla shape showed obvious differences between species so that it was raised and vertical in B. paucifolium, while horizontal and wide in three other species (Figures 3 and 4, Table 3). The corolla in all studied populations had five valvate petals with bent vertices into the flower which has similarly been reported in Pycnocyciea (Sheikhbahaei et al., 2014). The mid part of each petal is purple with white edge; the density of purple color in the dorsal surface of the petal is more than its ventral part. The androecium has five stamens with dorsifix anthers; gynoecium is syncarpous with stylopodium at apex of ovary. During fruit development, bracts and bracteols fall ( Figure 5). The stylopodium is conical in E. cylindrica, path conical in E. persica and E. wolffii and flat in B. paucifolium ( Figure 6).
The structure and size of fruit showed differences in various species. They were narrow and cylindrical with two narrowed ends in E. cylindrica, ovate to broad in E. persica, cylindrical in B. wolffii and elliptic to ovate in B. paucifolium ( Figure 6). There are five similar ribs in each fruit which are undulate in E. cylindrica and E. persica, wavy in B. wolffii and ovate in B. paucifolium ( Figure 6). The fruit length is 5.14, 2.83, 3.89 and 2.53 mm in E. cylindrica, E. persica, E. wolffii and B. paucifolium, respectively (Figures 5 and 6, Table 2). Therefore, E. cylindrica and B. paucifolium show the highest and lowest size in length respectively. Some morphological, genetic and phytochemical variations have been reported for some populations of B. persica in India (Chahota et al., 2017;Dar et al., 2011). However, no comprehensive study was found on the features of inflorescence, flower, pollen and fruit in studied species.
The correlation coefficients quantitative traits of different populations Elwendia and Bunium were presented in Table 4. The high positive and significant correlation value was obtained between filled fruits and bracteolates per umbellet. The stylopodia length was positively and significantly associated with bracts per umbel and empty fruits per umbellet. The principal component analysis (PCA) of species indicated five principal components. The high positive correlation coefficients were obtained for 23.76% of total variance and the important traits included in the first principal component (PC1) were bracts per umbel, stylopodia length, empty fruits, flowers per umbellet, fruits per umbellet and umbellets per umbel. The second principal component (PC2) had high contributing 22.08% variance and traits included in this group were filled fruits, bracteolates per umbellet, fruit length, fruits per umbellet and flowers per umbellet. The third principal component (PC3) was accounted for 10.24% of the total variation, with high factor loading for polar axis and pedicel length. Similarly, The fourth principal component) PC4(and fifth principal component)PC5(are showing 8.97% and 7.34% variance with major traits of umbel per plant and equatorial axis (Table 5). Chahota et al. (2017) reported in the fourteen populations of B. persica in Hymalaya, inflorescence, seed and branch traits were most significant in detecting variation. Also, the studies of Dar et al. (2011) showed that wide range of variation was found between six ecotypes of this species growing in Kashmir (India) for various morphological traits. Phenotypic coefficient of variation for all the traits was higher than the genotypic coefficient of variation, indicating that all these characters interacted with the       environment (Dar et al., 2011). The studies on morphological, genetic traits and phytochemical composition of Bunium persica populations showed that inflorescence, seed and branch traits were most significant in detecting variation (Chahota et al., 2017).
The variability in both qualitative and quantitative traits was found in B. paucifolium and Elwendia species. The distribution plot of species based on quantitative traits showed that E. cylindrica and E. wolffii overlap somewhat and E. persica is close to them (without overlap); however, B. paucifolium is different from the three other species (Figure 8). Therefore, according this plot, B. paucifolium is placed in a group separate from the others. The obtained results of the study on the qualitative characteristics of the examined samples in the NTSYS software confirm the results obtained of quantitative traits and show that B. paucifolium is separated from other species (Figure 9).
In conclusion, the results of this study showed that corolla shape obviously shows the difference between Bunium and Elwendia species and confirms molecular studies performed by Degtjareva et al. (2013) and Pimenov (2017) in the separation of Buinum to two genera Bunium and Elwendia. However, B. paucifolium in some traits such as plant color, presence/absence of male flowers and bracts, is similar to Elwendia species.