Abstract
Abstract -Pollen morphology of 17 species of Viola representing five sections, Melanium, Plagiostigma, 'Spathulidium' ined., Sclerosium, and Viola, was studied using light and scanning electron microscope. Pollen grains were usually symmetrical, tetrazonocolporate to pentazonocolporate in section Melanium and trizonocolporate to tetrazonocolporate in the other four sections. Pollen shape was circular to subtriangular, tetragonal or pentagonal in polar view and prolate to oblate, spheroidal or pyramidal in equatorial view. Exine ornamentation was granulate, psilate and mostly perforate. The psilate type was only observed in V. modesta. We found heteromorphy in aperture number in V. caspia of section Viola, V. occulta of section Melanium and V. behboudiana of section Sclerosium, which corroborates their higher ploidy than in related species (octoploid versus tetraploid).
Introduction
Viola L. is the largest genus of Violaceae, with ca 600 species and is the only one widely distributed in the Northern Hemisphere (BALLARD et al. 1999) . Infrageneric classification has varied, but recent phylogenetic analysis indicates that the genus can be subdivided into two subgenera and 16 sections worldwide (YOUSEFI et al. 2012) . Five sections belonging to subgenus Viola are present in Iran: section Melanium Ging., section Plagiostigma Godr., section Viola, section Sclerosium W. Becker, and the undescribed section 'Spathulidium' ined. The three first sections are widespread in the Northern Hemisphere and together comprise a few hundred species, while the two last sections are restricted to parts of southwestern Asia with fewer than a dozen species. The sections are ancient allotetraploid except for section 'Spathulidium' which is inferred to be allooctoploid . Recently, molecular phylogenetic analyses of sect. Melanium (including 25 species) were performed using ITS sequences and ISSR markers (YOCKTENG et al. 2003) ; they have shown that sect. Melanium forms a derived and monophyletic group. Section Melanium, the pansies, is represented in Iran by five annual-ephemeral species, V. arvensis, V. kitaibeliana, V. modesta, V. occulta, and V. tricolor . The only species of section Plagiostigma occurring in Iran, V. somchetica, belongs to subsect. Patellares (Boiss.) Rouy and Foucaud. Section Viola is restricted to northernmost Iran, where it occurs with six species, V. alba (subsp. alba), V. odorata, and V. sintenisii in subsect. Viola and V. caspia, V. reichenbachiana, and V. rupestris in subsect. Rostratae. Section Sclerosium is distributed in the South of Iran with three putative species, V. behboudiana, V. cinerea and V. stocksii. Section 'Spathulidium' ined. refers to a small group of three central Asian species, in Iran represented by V. spathulata and V. pachyrrhiza; V. maymanica occurs in northern Afghanistan (SCHMIDT 1992) . 17 species of Viola are distributed in different parts of Iran, mainly in the north. Of these, only V. spathulata is endemic to the territory (Elbrus mountains), but another five species are endemic to the region.
The delimitation of Viola species within sections and subsections can be problematic in many regions of the world, owing partly to phenotypic plasticity, partly to hybridisation and polyploidy, and partly to the few morphological characters separating taxa. Introgression appears, however, to be rare and restricted to certain groups (VALENTINE 1962 , KRAHUL-KOVÁ et al. 1996 , MARCUSSEN et al. 2001 , HILDEBRANDT et al. 2006 .
Pollen morphology of Violaceae in general has been examined by some authors (ERDTMAN 1952 , PETTET 1964 , WALKER and DOYLE 1975 . A few additional studies have been published on pollen morphology in Viola. GORB (1994) studied 12 Ukrainian species of Viola sect. Melanium (including V. arvensis, V. kitaibeliana, and V. tricolor) using both light and scanning electron microscopy. PERVEEN and QAISER (2009) examined five Viola species (including V. stocksii) from Pakistan. DINÇ (2009) studied two species of subsection Viola (V. sandrasea, V. kizildaghensis) endemic to Turkey. Pollen heteromorphism is defined as the production by the same plant of pollen grains differing in aperture number (TILL-BOTTRAUD et al. 1995) . This phenomenon occurs in over 30% of angiosperm species (MIGNOT 1994) . In Viola pollen heteromorphism occurs in about a third of the studied species and appears to be associated with polyploidy (NADOT et al. 2000) .
The main aim of this study is to provide a descriptive investigation of pollen grains in the genus Viola and also of pollen heteromorphism in the species distributed in Iran, using light and scanning electron microscopy. In light of the nomenclatural confusion and paucity of morphological characters for discrimination of Viola taxa in Iran (YOUSEFI et al. 2012) , pollen morphology and heteromorphism may provide new characters useful for the delimitation of these taxa. In this study, pollen morphology of 13 species of the genus Viola with the exception of V. arvensis, V. kitaibeliana, V. tricolor and V. stocksii are described for the first time.
Material and methods
Pollen of 24 taxa representing the 17 species of Viola occurring in Iran was studied by means of light microscopy (LM) and scanning electron microscopy (SEM). Pollen samples were obtained from herbarium specimens collected the same year. In order to ensure the constancy of pollen characters among different populations of a certain species, 2-3 specimens per population were included in the analysis; otherwise each species was represented by only one population. The voucher specimens were deposited in Guilan University Her-barium. A list of the voucher specimens is given in table 1. Pollen grains were stained with basic-fuchsine, then mounted in glycerin jelly and prepared for LM observation. Some measurements i.e. polar axis (P), equatorial axis (E), colpus length and exine thickness were made by LM (Nikon microscope model Optiphot-Z optic) for 30 pollen grains under a magnification of 1000x. For analysing pollen heteromorphism, the number of apertures were counted for 100 pollen grains, then the percentage of each aperture-class -three, four and five-were calculated and the percentage lower than 95 was reported as heteromorphic pollen (MIGNOT 1994) .
For SEM observations, pollen grains soaked in absolute ethanol were pipetted directly on to 12.5 mm diameter stubs and air-dried at room temperature; they were then coated in a sputter coater with approximately 25 nm of Gold-Palladium. The micrographs were ob- tained from a LEO1430 VP (England) at an accelerating voltage of 10-15 kV under magnifications of 2040× to 11190×. Pollen terminology according to WALKER and DOYLE (1975) , PUNT (2007) and HALBRITTER et al. (2008) has been followed.
Results

General pollen characters of the genus Viola
The pollen grains were shed in monads, symmetrical, tetrazonocolporate to pentazonocolporate in section Melanium and trizonocolporate to tetrazonocolporate in the other four sections (Figs. 1-2, Tab. 2). The apertures were colporate, equatorially elongated and reached the poles of the pollen with rounded or acute ends.
The smallest pollen grains belonged to V. spathulata (P = 29 mm, E = 26.08 mm) and the largest ones to V. arvensis (P = 69.09 mm, E = 68.23 mm). The P/E ratio varied between 0.94 and 1.18 (Tab. 2). The exine thickness varied between 0.6 µm (V. spathulata) and 1 µm (V. occulta).
Exine ornamentation proved to be a valuable taxonomic character in Viola. It varies from perforate, granulate, to psilate (Tab. 2). Among these patterns the simple psilate type represented by V. modesta is very characteristic.
Section Melanium (Figs. 1A-G Iran: Guilan, Lashtenesha, the rice field, GUH-13296-2
Tab. 1. -continued
Fig. 1. Micrographs of pollen grains in Viola arvensis
Scale bar: 10 µm. (Figs. 3A-B) . At species level, V. kitaibeliana with pyramidal shape in equatorial view (Fig. 1C) can be distinguished from the other species, which are prolate to spheroidal in shape.
Fig. 2. Micrographs of pollen grains in Viola pachyrrhiza
Exine ornamentation does not provide valuable characters in delimitation of section Melanium from other sections, but it is useful for species recognition within this section. For instance, two morphologically close species, V. tricolor and V. modesta, have different types of exine sculpturing, i.e. granulate and psilate, respectively (Figs. 3C, F-G) . Furthermore, V. arvensis, which exhibits a perforate to granulate exine, can be separated from its relative V. occulta, which has perforate to subpsilate exine (Figs. 3D- 
E).
Section Plagiostigma (Figs. 1H, 3H-I) Viola somchetica is the only Iranian species of this section and its pollen grain in equatorial view and exine ornamentation is prolate-spherodial (Fig. 1H ) and perforate-subpsilate (Figs. 3H-I), respectively. From a palynological point of view, this species does not show any obvious morphological apomorphies.
Section Sclerosium (Figs. 1I-L, 3J-L, 4A-C)
Viola behboudiana is easily separated from the other Iranian species of the section by two palynological characters: the shape of pollen grains, which is oblate-spheroidal in V.
behboudiana (Figs. 1I-J) and prolate-spheroidal in V. cinerea and V. stocksii (Figs. 1K-L), POLLEN STUDIES IN THE GENUS VIOLA (VIOLACEAE) FROM IRAN

Fig. 3. SEM micrographs of pollen grains in Viola arvensis
and the presence of pollen heteromorphism in V. behboudiana. All of the three species have perforate to perforate-subpsilate exine ornamentation (Figs. 3J-L, 4A-C).
Section 'Spathulidium' ined. (Figs. 2A-B, 4D-F)
The two Iranian species of Section Spathulidium are morphologically similar but can be easily separated using palynological characters: V. pachyrrhiza has perforate exine (Fig.  4D) , whilst V. spathulata has granulate exine ornamentation (Figs. 4E-F) . Pollen morphology shows a basic similarity between section Spathulidium and section Plagiostigma; however these two sections can be differentiated according to exine ornamentation (Tab. 2).
Section Viola
No palynological characters separate this section from the others.
Subsection Rostratae (Figs. 2C-F, 4G-L, 5A)
This subsection has three species in Iran, V. caspia, V. reichenbachiana and V. rupestris. The morphological recognition of the first two species is very problematic due to the variable morphological characters; however palynological studies provide useful characters for delimiting these taxa. Pollen grains of V. caspia are prolate-spheroidal to subprolate in equatorial view (Fig. 2C ) with a granulate exine (Figs. 4G-J) , while V. reichenbachiana has oblate-spheroidal pollen grains (Figs. 2D-E) with a perforate-subgranulate exine (Fig.  4K) . Furthermore, V. caspia shows pollen heteromorphism, in which the dominant pollen shape has three apertures and 6 % of pollen grains have four apertures. The characteristics of pollen sculpturing of V. rupestris are similar to V. caspia (Figs. 2F, 4L, 5A ), but the P/E ratio of V. rupestris is larger than V. caspia (Tab. 2).
Subsection Viola (Figs. 2G-I, 5B-I)
This subsection has three species in Iran, V. alba, V. sintenisii, and V. odorata. Whereas the latter can be easily recognised using morphological characters, i.e. broad stipules and high number of leaf crenulae, the division between two first species is still problematic. The shape in equatorial view of three species is prolate-spheroidal to subprolate (Figs. 2G-I) . Pollen characteristics are also variable between the taxa, particularly, the exine ornamentation shows that V. alba has perforate to subpsilate and perforate to granulate exine (Figs. 5B-C) , V. sintenisii has perforate-granulate (Fig. 5H-I) . Viola odorata has perforate to subpsilate and perforate to granulate exine (Figs. 5D-G) .
Discussion
The exine sculpturing of the pollen grains, their size, shape and aperture numbers can be of taxonomic value in the studied taxa. The pollen size variability is a diagnostic value between some of the taxa within the same section, for example, between V. arvensis and V. kitaibeliana (sect. Melanium), V. modesta and V. occulta (sect. Melanium), V. cinerea and V. stocksii (sect. Sclerosium).
POLLEN STUDIES IN THE GENUS VIOLA (VIOLACEAE) FROM IRAN

Fig. 5. SEM micrographs of pollen grains in Viola rupestris
sintenisii (H-I). (WALKER and DOYLE 1975) .
Fig. 4. SEM micrographs of pollen grains in Viola cinerea
According to the classification of pollen grains based on size (WALKER and DOYLE 1975) , this section has large pollen grains (50-99 mm), while the other sections of Viola distributed in Iran have medium sized pollen grains (25-49 mm).
Pollen grains with six apertures were reported for V. arvensis from Ukraine (GORB 1994). The figures given in ERDTMAN (1952) show that V. arvensis has 4-colpate with a few 5-colpate grains appearing to be based on misidentification with V. tricolor since this type is found in V. tricolor and not V. arvensis. PETTET (1964) noted that the two very variable and often confused species, V. tricolor and V. arvensis, are readily separable on the basis of pollen assemblage.
Earlier studies suggested that section Plagiostigma is not closely related to section Viola, as previously assumed (CLAUSEN 1929 . Our palynological results for Iranian V. stocksii agree with findings for this species in Pakistan (PERVEEN and QAISER 2009) .
Previously these species, i.e.. V. pachyrrhiza and V. spathulata were included in section Plagiostigma subsection Patellares (as section Nomimium grex Adnatae; BECKER 1918). However, this lineage takes a completely different phylogenetic position and will require a section of its own . Yousefi et al. (2012) demonstrated that Viola somchetica (section Plagiostigma) differs from V. spathulata (section Spathulidium) in the number of collenchyma layers at the corners of petiole cross sections (4 vs. 3) and the shape of peduncle cross sections (quadrangular vs. circular).
Morphological and phylogenetic evidence supports two subsections, subsection Rostratae, with aerial floriferous stems and explosive capsules, and subsection Viola, lacking aerial stems and with inexplosive capsules (BECKER 1925, MARCUSSEN and KARLSSON 2010) . Anatomically, V. caspia can be separated from V. reichenbachiana by the presence of a pith region in root cross-sections and by the number of vascular bundles in stem cross sections (YOUSEFI et al. 2012) .
Viola behboudiana (section Sclerosium), V. caspia (section Viola) and V. occulta (section Melanium) showed structural polymorphism. The number of apertures in the two first ones was 3-4, and 4-5 in the last one. All three species are high-polyploids. Viola behboudiana and V. caspia are both octoploids (MARCUSSEN and BORGEN 2011) , and this is probably the case also for V. occulta (n = 10 versus n = 3, 4, 5, 7 in its close tetraploid relatives; YOCKTENG et al. 2003) . In the genus Viola, pollen heteromorphism is a direct result of polyploidy (NADOT et al. 2000) .

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