IRANIAN JOURNAL OF BOTANY 24 (1), 2018 DOI: 10.22092/ijb.2018.110952.1165 PALYNOLOGICAL SURVEY OF THE GENUS HYPERICUM (HYPERICACEAE) IN IRAN AND ITS TAXONOMIC IMPORTANCE M. B. Faghir, M. Razaz, F. Attar, Z. Salehi & M. Vafadar Received 2017. 06. 05; accepted for publication 2018, 05, 23 Faghir, M. B., Razaz, M., Attar, F., Salehi, Z. & Vafadar, M. 2018. 06. 30: Palynological survey of the genus Hypericum (Hypericaceae) in Iran and its taxonomic importance. -Iran. J. Bot. 24 (1): 01-15. Tehran. In this research, pollen grains of ten species and two subspecies of the genus Hypericum in Iran belonging to four sections were studied using light and scanning electron microscopy. The pollen grains are monad, isopolar to subisopolar and heteropolar, prolate, subprolate, spheroidal and prolate- spheroidal in shape, small to medium in size. The outline of pollen grains varies from round to triangular, quadrangular and ovate from polar view and elliptical, tetrahedral, quadrangular, round and ovate from equatorial view; 3 and 4 syncolporate to 3, 4 and 6 zonocolporate. Based on exine sculpturing, pore shape, size and muri thickness, the examined species are divided in two main types including scrobiculate and micro reticulate and 2 subtypes including small pore / thick muri and large pore / thin muri. The pores are arranged from irregular to regular-irregular with regular intervals. The current result revealed taxonomically important palynological data of the genus Hypericum. These traits can be used for infrageneric classification, especially at sectional and species levels. Marzieh Beygom Faghir (corresponence <MarziehBeygomfaghir@gmail.com>), Mahsa Razaz & Zivar Salehi, Department of Biology, Faculty of Science, University of Guilan, Rasht, Iran.- Farideh Attar, Central Herbarium of Tehran University, School of Biology, University College of Science, Tehran, Iran. - Mahnaz Vafadar. Department of Biology, Faculty of Science, University of Zanjan, Zanjan, Iran. Key words: Hypericum; Hypericaceae; taxonomy; pollen morphology; Iran ‫ در ایران و اهمیت تاکسونومیک آن‬Hypericum ‫مطا لعه گردهشناسی سرده‬ ‫ دانشگاه گیالن‬،‫ دانشكده علوم‬،‫ دانشیار گروه زيستشناسي‬:‫مرضیه بیگم فقیر‬ ‫ گرايش سیستماتیک گیاهي دانشگاه گیالن‬،‫ دانشجوي دکتري زيستشناسي‬:‫مهسا رزاز‬ ‫ دانشگاه تهران‬،‫ دانشكده علوم‬،‫ استاد گروه زيستشناسي‬:‫فریده عطار‬ ‫ دانشگاه گیالن‬،‫ دانشكده علوم‬،‫ استاد گروه زيستشناسي‬:‫زیور صالحی‬ ‫ دانشگاه زنجان‬،‫ دانشكده علوم‬،‫ استاديار گروه زيستشناسي‬:‫مهناز وفادار‬ ‫ متعلق به چهار بخشه توسط میكروسكپ نوري و الكتروني مورد بررسي قرار‬،‫ در ايران‬Hypericum ‫در اين مطالعه ده گونه و دو زيرگونه از سرده‬ -‫ کروي و کروي‬،‫ تقريباً استوانهايي‬،‫ داراي اشكال استوانهايي‬،‫ جور قطب تا تقريباً جور قطب و ناجور قطب‬،‫ دانههاي گرده به صورن موناد‬.‫گرفت‬ ‫ تخم مرغي و از نماي استوايي‬،‫ چهار گوش‬،‫ سه گوش‬،‫ طرح کلي دانههاي گرده از نماي قطبي گرد‬.‫ اندازه کوچک تا متوسط ميباشند‬،‫استوانهاي‬ 6 ‫ و‬4 ،3 ‫منفذي پیوسته و‬-‫ شیاري‬4 ‫ الي‬3 ‫نامنظم و نامنظم؛ داراي دريچههاي‬-‫ منظم‬،‫ گرد و تخم مرغي؛ منظم‬،‫ مخروطي‬،‫ چهارگوش‬،‫بیضوي‬ ‫ ضخامت ديوارهها به دو گروه اصلي (زگیلدار‬،‫ شكل و اندازه منفذ‬،‫ براساس تزيینات اگزين‬،‫ گونههاي مورد مطالعه‬.‫منفذي ناپیوسته ميباشند‬-‫شیاري‬ ‫ منظم–نامنظم‬،‫ منافذ داراي فواصل منظم‬.‫ديوارهها نازک) تقسیم ميشوند‬/‫ديوارهها ضخیم و منافذ بزرگ‬/‫و ريز مشبک) و دو زير گروه (منافذ کوچک‬ 2 Palynological survey of the genus Hypericum in Iran IRAN. J. BOT. 24 (1), 2018 ‫ اين صفات ميتوانند براي رده‬.‫ نتايج تحقیق حاضر اهمیت تاکسونومیک صفات ريختشناسي گرده را در سرده آشكار ساخت‬.‫و نامنظم ميباشند‬ .‫ به ويژه در سطوح بخشه و گونه مورد استفاده قرار گیرند‬،‫بندي تحت سردهاي‬ INTRODUCTION Hypericum L. as a species rich (with ca. 500 representatives) and the largest genus in the Hypericaceae family (Nürk 2011), comprising herbs and shrubs, distributed mainly in temperate zones of northern hemisphere (Eurasia and North America). However, some species of Hypericum occur in mountains and higher altitudes of equatorial regions and southern hemisphere (Robson, 1977; Nürk2011; Meseguer and Sanmartin, 2012). Iranian species of this genus grow mainly in north, northwest and center of Iran and form floristic elements of Hyrcanian mountainous areas, Irano-Turanian, Mediterranean and Zagros elements. They generally prefer steep slopes of rocky and calcareous cliffs and margin of mountainous forests (Robson, 1968; Azadi, 1999). Robson (1968) introduced 21 species in the area covered by Flora Iranica. Robson (1977) and Assadi (1984) reported H. fursei N. Robson and H. dogonbadanicum Assadi as two endemics of N and SW of Iran. In Flora of Iran, Azadi (1999) identified 19 species, 4 subspecies arranged in 5 sections (comprising Campylosporus (Spach) R. Keller, Hypericum, Hirtella Stef., Taeniocarpum Jaub. & Spach. and Drosanthe (Spach) Endl.), and two doubtful species including H. heterophyllum Vent. and H. olivieri (Spach) Boiss. In addition to considerable pharmaceutical importance (Ernst, 2003; Maggi, 2004; 2010; Bruni and Sacchetti, 2009; Ozturk & al., 2009; Nürk and Crockett, 2010; Crockett and Robson, 2011), the genus Hypericum has been subject of various investigations due to taxonomical complexities (Robson, 1981, 1985, 1996, 2001, 2006, 2010; Nürk and Blattner, 2010). Primarily, the basic studies on pollen of Hypericaceae were reported by Erdtman, 1952 and Aytug & al 1971. Then, different authors reported various pollen morphological data of the genus Hypericum (Khan, 1969; Thomas, 1970; Clarke, 1975, 1976, 1981; Barros, 1984). This was followed by palynological survey of the selected species of the genus (Martonfi & al., 2002; Ocak & al., 2012; Sentark, 2012; Avato, 2005; Matzka, 2001; Meseguer and Sanmartín, 2012) plus six Iranian species including H. perforatum, H. tetrapterum, H. androsaemum, H. fursei, H. hirsutum (Mahmoudi Otaghvari & al., 2015) and H. dogonbadanicum (Bayat & al., 2015). The main aims of this research are to provide a detailed account of pollen morphological features of 10 species and 2 subspecies of Hypericum belonging to four sections that has not been extensively studied yet. In addition, identification of pollen diagnostic characters for taxonomic treatment at interand intra-specific levels, are among other important objectives of this study. MATERIALS AND METHODS In the current survey, a total of 12 taxa (including 10 species and 2 subspecies) belonging to four sections (Hypericum, Hirtella Stef., Taeniocarpum Jaub. & Spach. and Drosanthe (Spach) Endl.) of Iranian species of Hypericum were undertaken for pollen morphological analysis, by light (LM) and scanning electron microscope (SEM). Pollen grains were collected from both mature flowers of freshly collected specimen (during 2015-2016) and herbarium materials. The voucher specimens are deposited in the herbaria as indicated in table 1. For LM analysis, pollen grains were acetolysed according to Harley (1992), using Olympus light microscope, and photographed by DinoEye camera model AM-423x. At least 30 grains were examined from each sample and several individuals of the same species (table 2). For SEM observation, pollen grains were mounted on the stubs with double-sided cellophane tape and then coated in a sputter coater with 25nm of gold-palladium at an accelerating voltage of 10–15KV, in Guilan University. The samples were photographed by Tescan Vega scanning electron microscope; model VEGA/TESCAN in Razi Metallurgical Research Center (RMRC), Tehran. The pollen terminology in general follows Erdtman (1952), Punt & al. (2007). IRAN. J. BOT. 24 (1), 2018 M. B. Faghir & al. 3 Table 1. Voucher specimens of the studied taxa. Species 1. Sect. Hypericum 1.1 H. triquetrifolium Turra 2. Section: Hirtella Stef. Subsect. Platyadenum N. Robson 2.1 H. scabrum L. Collecting data Kurdestan, Marivan to Khav, 1350 m, Ghahremani & Mozaffarian, 18325 (TARI). Tehran,Haraz road, 5-8 Km to Polur, 2200m, Amin & Bazargan, 75718 (TARI). 2.2 H. asperulum Jaub. & Spach Azarbaiejan, From band Uromieh to Ziveh Margavar region, 1500m, Ghahreman & Aghustin, 13573 (TUH). 2.3 H. hirtellum Boiss. Tehran, Polur, Golkaryeh, 2000m, 13566 (TUH). Subsect. Stenadenum N. Robson 2.4 H. elongatum subsp. micrcalycinum (Boiss. & Heldr.) N. Robson Azarbaiejan, 48 km from Tabriz to Marand, Givan village, Mishudagh Mountain, 1700m, Sabzi & Imani, 6918 (Tabriz Herbarium). 2. 5 H. elongatum subsp. apiculatum N. Robson Azarbaiejan, Arasbaran protected area, 1300m, Ghahremani & Kasebi, 6918 (Tabriz Herbarium). 2. 6 H. davisii N. Robson Azarbaiejan, Kaleybar to Ahar. 20Km to Ahar, Sambran pass, 1735m, Azadi & Nickchehre, 75666 (TARI). 2. 7 H. helianthemoides (Spach) Boiss. Ardabil, Givy, 1400-1700m, Azadi, 75761 (TARI). 2. 8 H. vermiculare Boiss. & Hausskn. ex Boiss. Tehran, Damavand to Firuzkuh, 27 Km to Firuzkuh, 2050-2150m, Azadi, 75722 (TARI). 3. Section: Taeniocarpium 3. 1 H. linarioides Bosse Kurdestan, west of Sanandaj, 1705m, Maroofi, 1259 (Kurdistan Herbarium). 3.2 H. armenum Jaub. & Spach Mazandaran, Yoush, Baladeh, Kamarbon, 3200, Naqinezhad & Gholizadeh, 4366 (Babolsar Herbarium). 4. Section: Drosanthe (Spach) Endl. 4.1 H. hyssopifolium Vill. Azarbaiejan, West of Tabriz, 1700, Mozaffrian, 25859 (TARI). Table 2. Pollen morphological characters of studied species of the genus Hypericum of the current survey and former study of Mahmoudi Otaghvari & al., 2015. Abbreviations: Polar axis length (P), Equatorial axis width (E), ratio of Polar axis / Equatorial axis (P/E), Colpus length (Cl); Apocolpium index (AI): Distance between the apices of two ectocolpi (d) / equatorial diameter (D); Mesocolpium thickness (Meso), Outline from polar view (PO), Outline from equatorial view (EO), Triangular (T), Round (Ro), elliptical (EL),Oval (Ov), Tetrahedral (Th), Quadrangular (Q); Apertures number and types (ANT), Syncolporate (Syn-colr), Zono-colporate (Z-colr); Polarity (Pol), Isopolar (Iso), Sub-isopolar (Sub-iso), Heteropolar (Het), Colpus margin (Cm), Rolled (Rol), Flat (FL); Operculum (O),Pollen size (Ps), Medium (Me), Small (Sm); Pollen shape (Psh), Prolate (Pr), Prolate-spheroidal (PS), Sub- prolate (SubP), Spheroidal (Sph); Irregular (Ir), Regular (Re), Regulare-Irregular (Re-Ir) pollen. Species and subspecies 1. Sect. Hypericum 1.1 H. triquetrifolium 2. Sect.: Hirtella Subsect. Platyadenum 2.1 H. scabrum 2.2 H. asperulum 2.3 H. hirtellum Subsect. Stenadenum 2.4 H. elongatum subsp. microcalycinum 2.5 H. elongatum subsp. apiculatum 2.6 H. apricum 2.7 H. davisii 2.8 H. helianthemoides 2.9 H. vermiculare 3. Sect. Taeniocarpium 3.1 H. linarioides 3.2 H. armenum 4. Sect. Drosanthe 4.1 H. hyssopifolium P (µm) E (µm) P/E (25.1-26.5) 25.8 ±0.51 (11.9-14.5) 13.2 ±0.93 1.95 (26.5-28.6) 27.5 ±0.65 (20.5-22.5) 21.5 ±0.66 1.27 (23.1-23.7)23.4 0.23 (25.1-25.3) 25.2 ±0.10 (19.4-20.1) 19.7 ±0.28 (18.8-19.7) 19.2 ±0.35 (24.8-28.7) 26.7 1.77± (17.5-21.9) 19.7 ±1.89 (24.3-26.4) 25.3 ±0.93 (28.3-26.5) 27.4 ±0.60 (25-25.2) 25.1 ±0.10 (24.4-27.8) 26.1 ±1.31 (27.1-27.5) 27.3 ±0.15 (30.3-30.9) 30.6 ±0.23 (17.3-21.6) 19.4 ±1.63 (23.3-26.9) 25.1 ±1.43 (19.7-23.1) 21.4 ±1.39 (23.1-16.5) 19.8 ±2.6 (17.2-18.9)18 ±0.7 (22.7-25.5) 24.1 1.12 (19.3-19.9) 19.6 ±0.24 (16-16.9)16.4 ±0.38 (15.1-18.9)17 ±1.51 (20.1-22.1)22.1 ±0.94 1.18 1.31 1.35 1.18 1.38 1.39 1.08 1.39 1.86 1.14 1.13 Cl (µm) (24.6-25.1)24.9 ±0.21 d (µm) D (µm) AI (d/D) Meso (µm) _ (18.5-24.5) 21.5 ±2.54 _ (7.2-7.8) 7.5 ±0.17 (20.7-21.3)21 ±0.21 (5.1-5.7) 5.4 ±0.25 (24.3-24.9) 24.6 ±0.23 0.22 (6.6-7.2) 6.9 ±0.23 (18.123.9)21 ±2.45 (16.5-17.1)16.8 ±0.23 (3.9-4.5) 4.2 ±0.23 (1.9-2.5) 2.2 ±0.23 (21-21.6) 21.3 0.25 4.29 (20.7-21.3) 21 ±0.24 9.56 (22.6-23.2)22.9 ±0.23 _ (20.1-23.9) 22 ±1.38 _ (17.1-17.6)17.3 ±0.19 (22.1-20.9)21.5 ±0.51 (21.8-24.1)22.9 ±0.85 (26.2-26.8)26.5 ±0.25 (18.1-18.6)18.3 0.19 (26.8-27.4)27.1 ±0.24 (15.9-16.5)16.2 ±0.23 (19.3-19.9)19.6 ±0.23 (3.3-3.9) 3.6 0.23 (18.6-23) 20.8 ±1.84 (17.8-13.3)15.6 ±1.77 (25.9-26.5)26.2 ±0.23 (23.9-24.5)24.2 ±0.25 (21.9-25.5)23.7 ±1.53 (6.6-7.2) 6.9 ±0.23 (13.3-17)15.1 ±1.58 (4.2-4.8) 4.5 ±0.23 (3-3.6) 3.3 ±0.23 (14-14.6)14.3 ±0.23 (19.1-19.7) 19.4 ±0.25 _ _ _ (3.2-3.8) 3/5 ±0.23 _ _ _ 0.14 0.15 0.45 0.31 0.17 (11.8-12.4) 12.1 ±0.25 (7.6-8.2) 7.9 ±0.25 (8.6-9.2) 8.9 ±0.23 (11.5-12.1) 11.8 ±0.24 (11.1-10.5)10.8 ±0.24 (9.3-9.9)9.6 ±0.23 (6.3-6.9)6.6 ±0.24 (9-9.7)9.4 ±0.27 (9.7-10.3)10 ±0.24 (10.9-11.5)11.2 ±0.24 (14.6-15.2)14.9 ±0.23 Table 2. Continued. Species and subspecies PO EO ANT Ro El 3-Syn-colr T Th R R-Ov 3-Z-colr Ro-Q Ro-Q 4- Syn-colr H. elongatum subsp. microcalycinum T Ov-El 4- Syn-colr H. hirtellum Q Th H. apricum T T Q Th H. helianthemoides T Th H. vermiculare Sect. Taeniocarpium H. linarioides H. armenum Sect. Drosanthe H. hyssopifolium Q 1. Sect. Hypericum H. triquetrifolium 2. Sect.: Hirtella Subsect. Platyadenum H. scabrum H. asperulum Subsect. Stenadenum H. elongatum subsp. apiculatum H. davisii Pol Cm O Ps Psh Iso Rol + Me Pr Het Rol - Me Subp Iso Rol + S Subp Sub-iso* Rol + Me SubP Sub-iso* Rol + Me Pr Het* Rol + Me Subp Re-Ir Ir Sub-iso Rol Me Pr Het Rol + Me Pr 4-Syncolr Ir Het* Rol - Me Pr-sph Th 4-Syncolr Ir Het* Rol - Me Subp Ro El 3- z-colr Iso Rol - Me Pr Ro Ov-Ro 3-z-colr Iso Fl + S Sph T Ro-Ov 3-z-colr Iso Rol + Me Pr-Sphr 6-z-colr 4- z-colr4-syncolr 4-Syn-colr 4-Syn-colr OT Re IR Re Re-Ir Re-Ir Ir Re Re Re 6 Palynological survey of the genus Hypericum in Iran RESULTS The pollen morphological features are presented in tables 2–3 and figures 1-4. Our findings shows that pollen grains are monad, isopolar, subisopolar or heteropolar; prolate (P/E= 1.35-1.95), subprolate (P/E=1.18-1.31), spheroidal (P/E=1.14) and prolatespheoroidal (P/E=1.08-1.13) in shape; small (19.4-23.4 µm) to medium (25.1-30.6 µm) in size (Erdtman 1952). The outline of pollen grains vary from round to ovate, triangular and quadrangular from polar view (fig. 1 AF), elliptical to round (fig. 1 G-I) and tetrahedral from equatorial view (fig. 1 J-L). The outline of pollen are regular in H. triquetrifolium, H. armenum, H. asperulum, H. hyssopifolium and H. linarioides, regular -irregular in H. elongatum subsp. apiculatum, H. elongatum subsp. microcalycinum, H. apricum and irregular in H. helianthemoides, H. vermiculare, H. scabrum, H. hirtellum and H. davisii. The maximum (30.6 µm) and minimum polar axis (19.4 µm) were observed in H. linarioides and H. armenum respectively. The maximum equatorial axis (24.1 µm) was recorded in H. helianthemoides, while minimum equatorial axis (13.2 µm) belongs to H. triquetrifolium. Mesocolpium varied from 6.6 µm in H. helianthemoides to 14.9 µm in H. hyssopifolium. Minimum (16.2 µm) and maximum colpi lengths (27.1µm) were recorded in H. linarioides and H. armenum respectively. Apocolpium Index (AI) (External distance between two colpi/pollen grain diameter and d/D (Punt & al., 2007) differed from 14.3µm in H. armenum to 26.2 µm in H. davisii. Among the studied taxa, apertures number and types changed from 3- syncolporate in H. triquetrifolium; 3zonocolporate in H. asperulum, H. hyssopifolium, H. armenum and H. linarioides; 4- zononcolpaorate in H. hirtellum; 6- zonocolporate in H. scabrum; 4syncolporate in H. hirtellum, H. elongatum subsp. microcalycinum, H. elongatum subsp. apiculatum, H. helianthemoides and H. vermiculare and H. davisii IRAN. J. BOT. 24 (1), 2018 all the examined species had rolled colpus margin, (except H. armenum and H. hyssopifolium, with relatively rolled margin) and psilate colpus membrane (fig. 2. A). In some studied species, operculum was present and obviously protruded e. g. H. hyssopifolium (fig. 2. A) and H. armenum (fig. 4. E and F). In addition, we observed equatorial bridge in H. elongatum subsp. apiculatum (fig. 3. K), H. asperulum and fastigium in H. hirtellum (fig. 4), in different species of the genus. Based on exine sculpturing, pore shape and muri thickness the examined species are divided in two main types and 2 subtypes (presented in table 3, figs. 2-4). Type I: This type includes pollen grains with scrobiculate exine sculpturing and occurred in H. hyssopifolium (fig. 2. C) and H. asperulum (fig. 2. F). In these species pores are small (0.17-0.18 µm) and round in shape. Type II: This type comprises, micro reticulate exine sculpturing and recognized in 13 species (table 2). This type is divided in to two subtypes. Type II sub type A: includes pollen grains with small pore (0.16-0.40 µm) and thick muri (0.30-0. 95µm) and recorded in H. linarioides (fig. 2. I), H. davisii (fig. 2. L), H. scabrum (fig. 3. C), H. triquatrifolium (fig. 3. F) and hirtellum (fig. 4. I). Type II sub type B: this subtype consists of pollen grains with large pore (lumen) (0.24-0.75 µm) and thin muri (0.200.35 µm), it was identified in H. helianthemoides, H. elongatum subsp. microcalycinum (fig. 3. I), H. elongatum subsp. apiculatum (fig. 3. L), H. apricum (fig. 4. C), H. armenum (fig. 4. F), H. vermiculare (fig. 4. L). Our findings showed that the pores are spaced irregularly in H. apricum, H. vermiculare, H. hyssopifolium and H. asperulum, regular-irregular in H. linarioides, H. davisii, H. scabrum, H.triquetrifolium, H. elongatum subsp. apiculatum, H. elongatum subsp. microcalcicum, H. armenum and regular in H. helianthemoides and H. hirtellum. IRAN. J. BOT. 24 (1), 2018 M. B. Faghir & al. 7 Fig. 1. The LM micrographs of pollen grains in :A, H. armenum; B, H. helianthemoidae; C, H. scabrum; D and L, H. elomgatum subsp apiculatum; E, H. hirtellum; F and K, H. elongatum subsp. microcalycinum; G, H. asperulum; H, H. triquetrifolium; I, H. linarioides; J, H. hyssopifolium. 8 Palynological survey of the genus Hypericum in Iran IRAN. J. BOT. 24 (1), 2018 Fig. 2. The SEM micrographs of pollen grains in Hypericum. A-C, H. hysopifolium; D-F, H. asperulum; G-I, H. linarioides; J-L, H. davisii. Red arrow indicates smooth colpus membrane and the blue one shows operculum. IRAN. J. BOT. 24 (1), 2018 M. B. Faghir & al. 9 Fig. 3. The SEM micrographs of pollen grains in Hypericum. A-C, H. scabrum; D-F, H. triquetrifolium; G-I, H. elongatum subsp. microcalycinum; J-L H. elongatum subsp. apiculatum. Arrow indicates equatorial bridge. 10 Palynological survey of the genus Hypericum in Iran IRAN. J. BOT. 24 (1), 2018 Fig. 4. The SEM micrographs of pollen grains in Hypericum. A-C, H. apricum; D-F, H. armenum; G-I, H. hirtellum; J-L, H. vermiculare. Arrow indicates fastigium. Table 3. Grouping based on exine sculpturing, pore shape and muri thickness; Abbreviations: Muri thickness (Mt); Pore (Pd); Pore shape (PS): Round (Ro), Elliptical (EL), Linear (L), Polygonal (Pol); Pore arrangements (PA); Regular (R), Irregular (Ir).* indicates, species from Mahmoudi Otaghvari & al., (2015). Section/ Sub section/Species and subspecies Type I 1. H. hyssopifolium (Sect. Drosanthe) 2. H. asperulum (Sect. Hirtella, Sub sect. Platyadenum) Type II Sub type A 3. H. linarioides (Sect. Taeniocarpium) 4. H. scabrum (Sect. Hirtella, Sub sect. Platyadenum) Mt (µm) (0.20-0.40) 0.3 Pd (µm) (0.14-0.2) 0.17 PS Ro PA Ir (0.35-0.40) 0.35 (0.16-0.2) 0.18 Ro Ir (0.50-0.95) 0.72 (0.50-0.7) 0.60 (0.24-0.27) 0.20 (0.20-0.24) 0.22 Ro-L Ro-L R-Ir R-Ir 5.*H. tetrapterum (Sect, Hypericum) 6. H. triquetrifolium (Sect. Hypericum) >0.6 (0.50-0.68) 0.59 (0.210-0.269) 0.23 (0.16-0.25) 0.20 Ro R-Ir 7. H. davisii (Sect. Hirtella Subsect. Stenadenum) (0.50-0.68) 0.59 (0.29-0.39) 0.34 Ro R-Ir 8. *H. fursei (Sect. Taeniocarpium) 9. H. hirtellum (Sect. Hirtella, Sub sect. Platyadenum) (0.30-0.52) 0.40 (0.211-0.314) 0.26 (0.20-0.40) 0.30 Ro Ir R - (0.184-0.221) 0.20 - Ir <0.6 (0.30-0.4)0.35 (0.20-0.30) 0.30 (0.574-0.673) 0.62 (0.24-0.68) 0.58 (0.50-0.60) 0.55 Ro-L Ro Ir R-Ir Ir 14. H. elongatum subsp. microcalycinum (Sect. Hirtella , Sub sect. Stenadenum) 15. H. elongatum subsp. apiculatum (Sect.: Hirtella, Sub sect. Stenadenum) 16. *H. perforatum (Sect. Hypericum) 17. H. helianthemoides (Sect. Hirtella, Sub sect. Stenadenum) (0.30-0.40) 0.35 (0.49-0.68) 0.58 Ro-L R-Ir 18. H. apricum (Sect. Hirtella, Sub sect. Stenadenum) 10 *H. androsaemum (Sect. Androsaemum) Sub type B 11. *H. hirsutum (Sect. Taeniocarpium) 12. H. armenum (Sect. Taeniocarpium) 13. H. vermiculare (Sect. Hirtella Sub sect. Stenadenum) (0.30-0.40) 0.35 (0.30-0.68) 0.49 PoL-R R-Ir <0.6 (0.25-0.40) 0.32 (0.443-0.562) 0.48 (0.40-0.45) 0.42 Pol-R R (0.25-0.30) 0.25 (0.30-0. 75) 0.52 Ro Ir Sculpturing Scrobiculate Small pore and thick muri Microreticulate Large pore and thin muri 12 Palynological survey of the genus Hypericum in Iran DISCUSSION Palynological analysis of selected species of the genus Hypericum revealed important pollen morphological characters, especially pollen outline, numbers and types of apertures, colpus length; presence and absence of operculum; exine sculpturing type, pore shape, size and arrangements. The current pollen morphological data are in agreement with that of previous authors (Mahmoudi Otaghvari & al, 2015 and Bayat & al., 2015). Based on our findings, the pollen grains were regular (in two species and one subspecies), regular –irregular (in two species and one subspecies) and irregular (in 5 species) in outline. Irregular pollen has been already reported by previous workers (Martonfi & al., 2002; Mesegure and Sanmartin, 2012) in other species e.g. H. fursei, H. desetangsii, H. montanum. Clarke (1981) explained production of 50 to 100% of irregular pollen in different section of the genus (e.g. in Sect. Hirtella Stef). Size of pollen grain varied from small to medium. However, medium pollen type was dominant among the examined species (in 10 representatives). Some researchers believed that the pollen size in the genus Hypericum is affected by hybridization and ploidy level of the species and lack taxonomic importance (Asker & Jerling, 1992; Horandl, 2004; Matzk & al., 2003). In the examined species, apertures number and types changed from 3syncolporate (in 2 species), 3-zonocolporate (in 4 species), 6-zonocolporate (in one species), and 4syncolporate (in 6 species and 2 subspecies). Among the studied species H. hirtellum had both 4-zono and syn-colpotare pollen grains. Previous researches (Ocak & al., 2013; Martonfi & al., 2002) also reported variation in number and types of apertures (from 2 to 4 syncolporate, 3 to 4- zonocolporate, 6, 8 and 12pericolpate) in different species of Hypericum. These traits have been considered as diagnostic evidences and good tool for separating species and subspecies (Walker and Doyle 1975; Moore & al 1991). Rolled colpus margin with smooth external edge are another pollen characteristic feature in Hypericum (Ocak & al., 2013; Martonfi & al., 2002). This has been observed in all the studied taxa (except H. armenum). SEM observation also revealed other important palynological evidences of the genus such as operculum (which is coherent exine structure covering an aperture) (punt & al 2007), equatorial bridge (exine connection between the margins of a colpus in the equatorial region) (Pascoe 2007; punt & al 2007), and fastigium (cavity in colporate grains appearing as separation part of the exine from the domed sexing in the region of endoaparature; (Reitsma 1966; Martonfi & al., 2002). These traits are good tools for separating species of this genus (Hebed & al., 1988; Hebed and Chinepa 1990). Based on exine sculpturing, muri IRAN. J. BOT. 24 (1), 2018 thickness, pore size and shape the examined species are divided in two main types. Scrobiculate exine sculpturing comprises shallow muri forming reticulate pattern surrounding small lumen of less than 1 µm considered as pores (Martonfi & al., 2002; Punt & al). The average distance between pores are greater than their diameters (Vezey & al. 1992) in H. hyssopifolium and H. asperulum. Microreticulate exine sculpturing is the most abundant and recorded in 11 examined species. This type of exine ornamentation was formerly reported in H. bithynicum, H. confertum, H. olympicum, H. orientale and H. adenotrichum (Ocak, & al. 2013), H. tetrapterum, H. perforatum, H. androsaeum (Mahmoudi Otaghvari & al., 2015), and H. dogonbadanicum (Sect. Campylosporus) (Bayat & al., 2015). However, Ocak, & al. (2013) reported other exine sculpturing in different species of the genus e.g. granulated in H. fursei, perforate-microreticulate in H. venustum, H. heterophyllum, H. calycinum, H. avicularifolium, H. montbretii. Our results show that the exine sculpture type classes are not able to delimit species of the same sections, but they can serve as a useful tool for species identification. Palynological study of selected species of the genus Hypericum, render informative data and can be used for classification purpose, especially at sectional, species and subspecies levels. In Iran, the genus Hypericum includes 19 species and 4 subspecies arranged in 5 sections (Robson, 1968 and Azadi, 1999). Among them, Sect. Hirtella Stef, is the largest section (Robson 1986), consists of perennial herbaceous plants having black glands on margin of sepals and petals; clawed petals; stamens in three bundle and elongated gland on the capsule surface. According to our results, these species possess identical pollen morphological characters (polarity, pollen shape and size, type and number of apertures; exine sculpture; pore shape and arrangement). Sect. Hirtella Stef includes two subsections: Subsect. Platyadenum N. Robson and Subsect. Stenadenum N. Robson). Plants with marginal obconical glands, glandular sepal apex, preferring rocky and stony-gypsy steep slopes of NW, W, E and C of Iran, characterize the first subsection. The palynological data of three studied species (H. scabrum, H. hirtellum and H. asperulum) of this subsection, including pollen shape; aperture types, exin sculpture (type I and subtype A) also supports a close relationship among these species. Hypericum scabrum resembles H. hirtellum in having micro reticulate exine sculpture, small pore and thick muri, zono-colporate aparature and irregular folded pollen of medium in size. On the other hand, the exine sculpturing of pollen grains in H. asperulum is scrobiculate, with thin muri and smaller pore diameter,zono-colporate, regular pollen of small in size. The second subsection, possess IRAN. J. BOT. 24 (1), 2018 pollen grains of medium in size; exine sculpturing of type II. sub type B (except for H. davisii) and syncolporate pollen grains with or without operculum. Robson (1968) and Azadi (1999) placed H. Helianthemoides, H. vermiculare , H. apricum , H. davisii and H. elongatum in this subsection, because of their round - elliptical gland at calyx margin and glandular tip of calyx. The two studied subspecies: H. elongatum subsp. microcalcycum and H. elongatum subsp. apiculatum, grow on rocky slopes and forests of N and NW Iran (Robson 1968; Azadi1999), share several pollen morphological characters e.g. pollen of medium size; regular and regular-irregular, subisopolar; micro-reticulate sculpture with large perfora and thin muri (type II sub type B) arranged in regular-irregular intervals. However, they differed by their pollen and perforation shapes. Hypericum vermiculare and H. helianthemoides are placed close to each other because of their capsule and inflorescence shape (Robson 1968). Both species show irregular (folded), heteropolar pollen; micro-reticulate sculpture with large perfora with thin muri (type II sub type B). However, perora shape and arrangement can be helpful to distinguish the two species. Hypericum apricum also resembles to H. vermiculare by microreticulate sculpture, (type II sub type B), perfora and arrangement. Nevertheless, it is characterized by irregularly spaced perfora and regular-irregular pollen. Hypericum davisii is identified by exine sculpturing of type II subtype A, thick muri, small, round regularirregularly spaced perfora. This species is recognized by its round-ovate buds, oblong-elliptical sepals, narrow cylindrical inflorescence (Azadi 1999). Section Taeniocarpium Jaub. & Spach., consists of four species (H. armneum, H. linarioides, H. hirsutum and H. fursei), characterized by their erect or ascending stem; reddish petals, without claw; and linear glands on external surface. The result supports the former palynalogical data (Mahmoudi Otaghvari & al. 2015), and exhibit pollen morphological affinities among different species of the section. This includes medium size (in H. armneum, H. linarioides, and H. hirsutum), zonocolporate, regular (in H. armenum and H. linarioides), regular - irregular (in H. fursei and H. hirsutum), rolled colpus margin (H. linarioides, H. hirsutum and H. fursei), micro-reticulate exine sculpture of type II, subtype A (in H. fursei and H. linarioides) and subtype B (in H. armenum and H. hirsutum) pollen grains. The current result is in controversy with current classification (Robson 1968; Azadi1999), in which H. linarioides, H. fursei and H. armenum (for their glabrous stem, leaves and calyxes); H. fursei and H. armenum (for their dense glands at upper half of the calyx, cylindrical inflorescence) regarded as closely related species. While H. hirsutum M. B. Faghir & al. 13 is separated from them by its hairy stem, leaves and calyx. Hypericum hyssopifolium is the only representative of Sect. Drosanthe (Spach) Endl., characterized by oblong-lanceolate obtuse sepals with marginal sessile glands; obovate petals covered by colored and black marginal glands (Robson, 1968; Azadi ,1999). Based on the present analysis, among the studied species, H. hyssopifolium and H. asperulum (Hirtella Stef, Subsect. Platyadenum) have scrobiculate exine sculpturing. This does not support the relationship between the two species. Sect. Hypericum comprises three herbaceous species (H. tetrapterum and H. perforatum and H. triquetrifolium) which have common seed and petal characters morphological features (Robson, 1968; Azadi, 1999). Among them, H. triquetrifolium and H. tetrapterum (Mahmoudi Otaghvari & al., 2015), resemble to each other by their exine sculpturing pattern (type II sub type A). However, based on Robson, (1968) and Azadi (1999), H. triquetrifolium approaches to H. perforatum for its stem morphology (having two longitudinal line). Our finding showed that, pollen morphological characters of Sect. Hypericum do not support morphological studies and current classification (Robson, 1968; Azadi, 1999). Sect. Campylosporus is identified by tree, shrubs, with dark glands, permanent petals, 5 bundle stamens, 5 more or less fused styles. It includes H. dogonbadanicum growing in W and SW Iran, in Zagros Mountain, at 1000-1500 m a.b.s (Azadi, 1999). Despite to morphological differences, it exhibits similar palynological evidences (Bayat & al. 2015) especially exine sculpturing pattern (type II) with other representatives of the genus especially those having micro reticulate sculpturing. In summary, pollen morphology is very useful in delimitation of Hypericum. These traits are taxonomically informative and can be used for separation of different taxonomic ranks (sections, species and subspecies levels). However, the importance of pollen morphological characters and the species relationship should be discussed based on molecular phylogenetic study of Iranian species of the genus which is in urgent need. ACKNOWLEDGMENT The authors are grateful to the authorities of herbaria of University of Mazandaran, Research Centers of Agricultural and Natural Resources of East Azerbaijan and Kurdistan provinces. We wish to thank Mr. Rezaei for preparing SEM micrographs. 14 Palynological survey of the genus Hypericum in Iran REFERENCES Asker, S. E. & Jerling, L. 1992: Apomixis in Plants, CRC Press. –Boca Raton, FL, USA. Avato, P. 2005: A survey on the Hypericum genus: secondary metabolites and bioactivity. –Stud. Nat. Prod. Chem. 30: 603–634. Aytug, B., Aykut, S., Merev, N. & Edis, G. 1971: Pollen Atlas of plants in and around. Istanbul University Press. –Istanbul. Azadi, R. 1999: Guttiferae. In: Assadi, M. (Eds.), Flora of Iran. 27: 1–62. R.I.F.R. –Tehran. Barros M, Ramos A 1984: Estudio del polen de las especies de Hypericum sect. Hirtella Stef. en la Penı´nsula Ibe´rica y Baleares. V Simposio de Palinologı´a. APLE. Resu´menes, Co´rdoba Bayat, M., Rahiminejad, M. & Ghaem maghami, L. 2015: The anatomical and palynological study of Hypericum dogonbadanicum. –Journal of plant researches. 29 (1): 15-30. Bruni, R. & Sacchetti, G. 2009: Factor affecting polyphenol biosynthesis in wild and field Grown St. John’s Wort (Hypericum perforatum L. Hypericaceae/Guttiferae). – Molecules. 14: 682725. Clarke, G. C. S. 1975: Irregular pollen grains in some Hypericum species. –Grana. 15: 117–125 Clarke, G. C. S. 1976: Guttiferae. Review of Palaeobotany and Palynology. 21(3): 125-142. Clarke, G. C. S. 1981: Pollen morphology. In: Robson N. K. B, Studies in the genus Hypericum L. (Guttiferae). –Bull British Mus Nat Hist (Bot). 8:115–118. Erdtman, G. 1952: Pollen morphology and plant taxonomy. Almqvist and Wiksells. –Stockholm. Ernst, E. 2003: The genus Hypericum. Taylor and Francis. –London, New York. Hebda, R. J. & Chinnappa, C. C. 1990: Studies on pollen morphology of Rosaceae in Canada. –Rev Palaeobot Palynol. 64: 103–108. Hebda, R. J., Chinnappa, C. C. & Smith, B. M. 1988: Pollen morphology of the Rosaceae of western Canada. I1. Dryas, Fragaria, Holodiscus. –Can. J. Bot. 66: 595–612. Horandl, E. 2004: Comparative analysis of genetic divergence among sexual ancestors of apomictic complexes using isozyme data. – Int. J. Plant. Sci. 165: 615–622. Khan, H.A. 1969: Pollen morphology of Indian Hypericaceae. –J. Palynol. 5(97): 99–105. Maggi F, Ferretti G, Pocceschi N, Menghini L, Ricciutelli M. 2004: Morphological, histological and phytochemical investigation of the genus Hypericum of central Italy. –Fitoterapia 75: 702– 711. IRAN. J. BOT. 24 (1), 2018 Maggi, F., Cecchini, C., Cresci, A., Coman, M. M., Tirillini, B., Sagratini, G. & Papa, F. 2010: Chemical composition and antimicrobial activity of the essential oils from several Hypericum taxa (Guttiferae) growing in central Italy. – Chem. Biodiv. 7: 447–466. Mahmoudi otaghvari, A., Alinattaj omrani, K. & Fadaei, F. 2015: The pollen morphology study on some species of Hypericum L. in the northern Iran. – IJBPAS. 4 (11): 957-967. Martonfi, P. Janikova, M. & Zezula, I. 2002: Palynological analysis of seven Hypericum taxa.– Biologia- Bratislava 57(4): 455-460. Matzk, F., Hammer, K. & Schubert, I. 2003: Coevolution of apomixi and genome size within the genus Hypericum. –Sex Plant Reprod. 16: 51-58. Matzk, F., Meister, A., Brutovska, R. & Schubert, I. 2001: Reconstruction of reproductive diversity in Hypericum perforatum L. opens novel strategies to manage apomixis. –Plant. J. 26: 275–282 Meseguer, A. S. & Sanmartín, I. 2012: Paleobiology of the genus Hypericum (Hypericaceae): a survey of the fossil record and its palaeogeographic implications. –Anales del Jardin Botanico de Madrid. 69(1): 97-106. Moor, P. D. & Webb JA, Collinson ME. 1991: Pollen analysis. Oxford: Blackwell. Nürk, N. M. & Blattner, F. R. 2010: Cladistic analysis of morphological characters in Hypericum (Hypericaceae). – Taxon. 59: 1495–1507. Nurk, N. M. & Crockett, S. L. 2011: Morphological and phytochemical diversity among Hypericum species of the Mediterranean basin. –Med. Aromat. Plant Sci. Biotechnol. 5(1):14–28. Nurk, N. M. 2011: Phylogenetic analyses in St. John’s wort (Hypericum), Inferring character evolution and historical biogeography. Dissertation zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften (Dr. rer. nat.), eingereicht im Fachbereich Biologie, Chemie, Pharmazien der Freien Universitat Berlin. Ocak, A. & Potoglu Erkara, I. 2013: Palynological investigations on some Hypericum taxa (Hypericaceae) growing naturally in Turkey. – Plant. Syst. Evol. 299: 379-388. Ozturk, N., Tuncel, M. & Potoglu-Erkara, I. 2009: Phenolic compounds and antioxidant activities of some Hypericum species: A comparative study with H. perforatum. –Pharmaceutical Biology. 47(2): 120–127. Punt, W., Hoen, P. O., Blackmore, S., Nilsson, S. & Thomas, A. L. 2007: Glossary of pollen terminology. –Rev. Palaeobot. Palynol. 143:1–81. IRAN. J. BOT. 24 (1), 2018 Reitsma, T. J. 1966: Pollen morphology of some European Rosaceae. –Acta Bot Neerl 15: 290–307. Robson, N. K. B. 1968: Hypericum L. In: Rechinger, K. H. (Ed), Flora Iranica. 2–20. –Graz. Robson, N. K. B. 1977: Studies in the genus Hypericum L. (Guttiferae): 1. Infrageneric classification. -Bull. Brit. Mus. (Nat. Hist.) Bot. 5: 291–355. Robson, N. K. B. 1981: Studies in the genus Hypericum L. (Guttiferae): 2. Characters of the genus. –Bull. Brit. Mus. (Nat. Hist.) Bot. 8: 55–226. Robson, N. K. B. 1985: Studies in the genus Hypericum L. (Guttiferae): 3. Sections 1. Campylosporus to 6a. Umbraculoides. –Bull. Brit. Mus. (Nat. Hist.) Bot. 12: 1–325. Robson, N. K. B. 1996: Studies in the genus Hypericum L. (Guttiferae), 6. Sections 20 (Myriandra) to 28 (Elodes) - Bull. Nat. Hist. Mus. Lond. Bot. 26: 75– 217. Robson, N. K. B. 2006: Studies in the genus Hypericum L. (Clusiaceae), Sections 9. Hypericum sensu lato (part 3): subsection 1. Hypericum series 2. M. B. Faghir & al. 15 Senanensia, subsection 2. Erecta and section 9b. Graveolentia. – Syst Biodivers 4: 19–98. Robson, N. K. B. 2010: Studies in the genus Hypericum L. (Hypericaceae) 5 (2). Sections 17. Hirtella to 19. Coridium. – Phytotaxa 4: 127–258. Senturk, H., Kabay, S., Ozden, H., Bayramoglu, G., Ustuner M. C., Ozturk, N., Guven, G., Kutlu, A., Bilgi, G., Ustuner, D. & Gunes¸ V. 2012: The protective effect of Hypericum origanifolium in experimental renal ischemia/reperfusion injury in rats. –African J. Pharm. Pharmacol. Thomas, J. L. 1970: Haploid, diploid pollen in Hypericum patulum. J. –Arnold. Arboretum. 51: 247–250 Vezey, E. L., Shah, V. P and Skvarla, J. J. 1992: A numerical approach to pollen sculpture terminology. –Pl. Syst. Evol. 181: 245-254. Walker, J.W. & Doyle, J. A. 1975: The basis of Angiosperm phylogeny: palynology. –Ann Bot Gard; 62:666-723