Comparative Anatomical and Palynological Studies on Rumex L. species (Polygonaceae) in NE Iran

In present study, anatomical and palynological characters of Rumex species in NE Iran were examined. For anatomical study, manual cross sections of fresh and dried stems and leaves were prepared and stained by differential staining. In palynological study, the pollen were extracted from anther, acetolysed and observed by SEM. The results of anatomical research showed, annular collenchymous tissue below the epidermis layer, different shape of vascular bundle, sclerenchymous strands around the vascular bundles in stem, dorsi-ventral mesophyll and continuous and discontinuous vascular bundles in leaves. The palynological findings indicated, panporate, tricolporate, tetracolporate, granulate, microechinate and punctate pollen in studied species.


INTRODUCTION
The genus Rumex L. belongs to the family Polygonaceae and the order Caryophyllales which has about 200 species distributed in temperate regions, European countries, America, Asia and Australia continents (Heywood, 1978;Qaiser, 2001;Sanchez and Kron, 2008;Chase and Reveal, 2009).This genus comprises around 28 and 8 species in Iran and Khorassan Razavi Province (NE Iran) respectively (Rechinger, 1968).Rumex has antioxidant, antimicrobial and viral, anti-cancer activities and decreases blood sugar and cholesterol level (Jildirim et al., 2001;Cos et al., 2002;Ferreres et al., 2006;Sedaghat et al., 2010;Harshaw et al., 2010).Despite many reports about medicinal characteristics of this genus, internal structure and pollen studies have been done on a few Rumex species e.g.The anatomy of Rumex with special reference to the morphology of the internal bundles and the origin of the internal phloem in Polygonaceae (Joschi, 1936),Comparative anatomy of the leaves of Rumex in Jiangxi ( Li et al., 2008), anatomical study of some medicinal plants of family Polygonaceae (Hameed et al., 2010), Stem anatomy of medicinally important Rumex hastatus D. Don (Polygonaceae) (Sahney and Vibhasa, 2012) and Taxonomic implications of pollen morphology of seven species of Rumex L. from Pakistan (Yasmin et al., 2010).Whereas, comparative anatomical and palynological studies have not been done on Iranian Rumex species, this report focused on the above research to recognize variation in internal structure and pollen micromorphological features among the five Rumex species in NE Iran.

MATERIALS AND METHODS
For anatomical study of vegetative organs, the base of stem and mature leaves from five species i.e.R. crispus L., R. dentatus L., R. tuberosus L., R. chalapensis Mill.and R. conglomeratus Murr.were fixed in FAA solution, then manually cross sections were made and stained by green methyl and carmine then observed by different magnification of light microscopy Labomed model CMZ4.The localities of studied specimens were presented in Table .1.Some anatomical characters such as the number of collenchymous layer below the epidermis tissue, the position of secretory ducts in cortex, the shape of phloem bundles, the number and type of sclerenchymous bundles above the phloem were analyzed.

R. conglomeratus
For palynological study, the pollen grains of R. crispus, R. conglomeratus and R. tuberosus were extracted from the anther and dehydrated by Glacial Acetic Acid, then acetolised, and studied by SEM and LM (Erdtman, 1960;Moore et al., 1991).Pollen characters of R. dentatus and R. chalapensis was adapted from Yasmin's et al. ( 2010) report.Some characteristics like: P (polar axis length), E (equatorial axis length) and the P/E ratio were measured at magnification 5000, 20000.The pollen terminology in general followed Punt et al. (2007).

RESULTS
Anatomical results of stem, revealed uni-seriate epidermis layer, collenchymous tissue, parenchymous tissue including secretory duct, sclerenchymous bundles which surround vascular bundles and vascular bundles with different shapes.The shape of sclerenchymous strands and phloem bundles, the number of collenchymous and sclerenchymous tissues varied among the studied species (Table 2, Figures . 1A-E).All the studied species had parenchymous pith except R. dentatus had hollow pith in stem.Also, internal vascular bundles were observed in R. crispus.Druse crystals were perceived in the cortex and pith of R. dentatus stem.In the leaf cross section of studied species, dorsi-ventral mesophyll was noticed.The palisadic parenchyma in R. tuberosus and R. crispus was biseriate while in the rest of species was uni-seriate.Also the ratio of palisadic to spongy parenchyma was variable among the species (Table 2, Figure 1F).1G -L).The P/E ratio and quantitative features were presented in Table 3.

DISCUSSION
In anatomical analysis of stem, the minimum and maximum number of collenchymous layer below the epidermis tissue were noticed in R. crispus (2-3 layered) and R. dentatus ( 4-5 layered).Also the minimum and maximum number of sclerenchymous strands around the vascular bundles were observed in R. dentatus (2-3 layered) and R. crispus (6-8 layered) respectively.Oblong elliptical bundles was just perceived in R. crispus while, in the rest of studied species triangular-obovate bundles were observed.Just R. tuberosus had sclereid above the phloem and no secretary ducts.Just R. dentatus had hollow pith and R. crispus had internal vascular bundle.Joshi pointed that most of the species of Rumex showed presence of internal bundles.Also, absence of internal bundles has been reported in R. dentatus by Joshi (1936).Joshi believes "the perennial species of Rumex which are without internal bundles are the oldest forms of the genus.From these have arisen the perennial forms with internal bundles and the annual forms without internal bundles".Thus all studied species in this research except R. dentatus represented the oldest forms of Rumex.Sahney and Vibhasa (2012) in anatomical studies on Rumex hastatus, mentioned "Rumex hastatus is similar to R. dentatus but differs from it due to presence of chlorenchyma below the furrows, tannin filled cells below the chlorenchyma and large druses in particular position in the cortex and absence of hollow pith.In R. hastatus, internal vascular bundles were formed below the collenchymous between the first formed bundles, though in R. crispus internal vascular bundles forms exactly below the older ones.In leaf anatomy of seven species of Rumex L. of Jiangxi, the shape of epidermal cells, stomata apparatus, glands arrangement, the shape and the number of vascular bundle of midrib were assessed by Li et al. (2008).They explained "comparative anatomy study of leaves might provide some helps to the study of phylogeny and classification of Rumex L."The palynological results of present research, showed tricolporate, tetracolporate, panporate type while the shape of pollen in polar view was the same in three species i.e. spherical-triangular. Also, the exine ornamentation was observed there is quite considerable variation in pollen morphology especially; exine and its sculpturing under SEM is of real systematic value and make the pollen grains decidedly distinct structures which can be employed for the delimitation of closely related species" (Yasmin et al., 2010).But in present research, differences in anatomical and pollen structures are not significant features to identify studied species.

Table 2 :
The stem and leaf anatomical characters in studied species of Rumex Palynological results showed tricolporate, tetracolporate, panporate and spherical-triangular pollen in polar view.The ornamentation was observed porate-punctuate to granulate (Figures

Table 3 :
Pollen characters in studied species in Rumex microechinate, porate-punctuate to granulate.Yasmin et al. (2010) reported, "tricolporate and tetracolporate, radially symmetrical and isopolar pollen in Rumex species.Moreover, tectum was granular and perforate-punctate in R. dentatus and R. chalepensis respectively".In Yasmin's et al. research, three distinct pollen types were introduced on the basis of exine ornamentation i.e.Chalepensis type (Perforate-punctate tectum), Dentatus type (granulate tectum) and Acetosa type (coarsely reticulate tectum).According to the above types, R. crispus and R. conglomeratus fall into Dentatus type and R. tuberosus fall into Chalepensis type.Based on Yasmin's et al. study, "