DOI: http://dx.doi.org/10.22092/ijb.2013.3002 ANATOMICAL AND POLLEN CHARACTERS IN ACANTHOPHYLLUM C. A. MEY. (CARYOPHYLLACEAE) FROM NORTHEAST OF IRAN M. Mahmoudi Shamsabad, H. Ejtehadi, J. Vaezi & F. Memariani Received 28.07.2012. Accepted for publication 08.11.2012. Mahmoudi Shamsabad, M., Ejtehadi, H., Vaezi, J., Memariani, F. 2013 06 30: Anatomical and Pollen Characters in Acanthophyllum C. A. Mey. (Caryophyllaceae) from northeast of Iran. –Iran. J. Bot. 19 (1): 107-118. Tehran. Palynological and anatomical features of 11 species from NE Iran belonging to Acanthophyllum sect. Oligosperma were carried out and evaluated by numerical methods to determine the taxonomic value of endomorphic characters. The cross section of stems, peduncles and leaves were prepared and stained with Safranin and Fast-green and pollen morphology has been examined by light and scanning electron microscope. The principal component analysis and Cluster Analysis results showed that the type of stomata, shape and size of epidermal cells, trichomes, the number of epidermal cells and stomata, the shape of stem cross section, arrangment of xylem elements in peduncle, arrangment of mesophyll in leaf and the number of sclerenchymatous layers are significant to separating species. The pollen grains were spheroid, pantoporate, ornamentation was scabrate-punctate and exine structure was spinulose-punctate. Masoumeh Mahmoudi Shamsabad (correspondence, <ma_ma648@stu-mail.um.ac.ir>), Department of Biology, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad, Iran. –Hamid Ejtehadi, Department of Biology, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad, Iran -Jamil Vaezi, Department of Biology, Faculty of Sciences, Shahid Chamran University of Ahvaz, Ahvaz, Iran. –Farshid Memariani, Research Centre for Plant Sciences, Ferdowsi University of Mashhad, Mashhad, Iran. Key words. Anatomy, Pollen Morphology, Acanthophyllum, Caryophyllaceae, Numerical Analysis, Iran. ‫( در ﺷﻤﺎل ﺷﺮق اﻳﺮان‬Acanthophyllum C. A. Mey) ‫ﺑﺮرﺳﻲ ﺻﻔﺎت ﺗﺸﺮﻳﺤﻲ و ﮔﺮده ﺷﻨﺎﺳﻲ ﺟﻨﺲ ﭼﻮﺑﻚ‬ .‫ داﻧﺸﺠﻮي ﻛﺎرﺷﻨﺎﺳﻲ ارﺷﺪ داﻧﺸﮕﺎه ﻓﺮدوﺳﻲ ﻣﺸﻬﺪ‬،‫ﻣﻌﺼﻮﻣﻪ ﻣﺤﻤﻮدي ﺷﻤﺲآﺑﺎد‬ .‫ اﺳﺘﺎد ﮔﺮوه زﻳﺴﺖﺷﻨﺎﺳﻲ داﻧﺸﮕﺎه ﻓﺮدوﺳﻲ ﻣﺸﻬﺪ‬،‫ﺣﻤﻴﺪ اﺟﺘﻬﺎدي‬ .‫ اﺳﺘﺎدﻳﺎر ﮔﺮوه زﻳﺴﺖﺷﻨﺎﺳﻲ داﻧﺸﮕﺎه ﺷﻬﻴﺪ ﭼﻤﺮان اﻫﻮاز‬،‫ﺟﻤﻴﻞ واﻋﻈﻲ‬ .‫ ﻋﻀﻮ ﻫﻴﺄت ﻋﻠﻤﻲ ﭘﮋوﻫﺸﻜﺪه ﻋﻠﻮم ﮔﻴﺎﻫﻲ داﻧﺸﮕﺎه ﻓﺮدوﺳﻲ ﻣﺸﻬﺪ‬،‫ﻓﺮﺷﻴﺪ ﻣﻌﻤﺎرﻳﺎﻧﻲ‬ ‫ در ﺷﻤﺎل ﺷﺮﻗﻲ اﻳﺮان ﺑﺎ اﺳﺘﻔﺎده از ﺗﺎﻛﺴﻮﻧﻮﻣﻲ‬Sect. Oligosperma ‫ از ﺑﺨﺶ‬Acanthophyllum ‫ ﮔﻮﻧﻪ‬11 ‫ﺻﻔﺎت ﺗﺸﺮﻳﺤﻲ و ﮔﺮدهﺷﻨﺎﺳﻲ‬ ‫ دﻣﮕﻞ و ﺑﺮگ ﺗﻬﻴﻪ و ﺳﭙﺲ ﺑﺎ ﺳﺎﻓﺮاﻧﻴﻦ و ﻓﺴﺖﮔﺮﻳﻦ رﻧﮓ آﻣﻴﺰي ﺷﺪ و‬،‫ ﺑﺮشﻫﺎﻳﻲ از ﺳﻄﺢ ﻣﻘﻄﻊ ﺳﺎﻗﻪ‬.‫ﻋﺪدي ﻣﻮرد ﺑﺮرﺳﻲ ﻗﺮار ﮔﺮﻓﺖ‬ ‫ ﺷﻜﻞ و اﻧﺪازه‬،‫ ﻧﺘﺎﻳﺞ ﻧﺸﺎن داد ﺻﻔﺎت ﻧﻮع روزﻧﻪ‬.‫ﻣﻮرﻓﻮﻟﻮژي داﻧﻪﻫﺎي ﮔﺮده ﺑﺎ اﺳﺘﻔﺎده از ﻣﻴﻜﺮوﺳﻜﭗ ﻧﻮري و اﻟﻜﺘﺮوﻧﻲ ﺑﺮرﺳﻲ ﺷﺪﻧﺪ‬ ‫ آراﻳﺶ آوﻧﺪﻫﺎي ﭼﻮب در‬،‫ ﺷﻜﻞ ﺳﻄﺢ ﻣﻘﻄﻊ ﺳﺎﻗﻪ‬،‫ ﺗﻌﺪاد ﺳﻠﻮلﻫﺎي اﭘﻴﺪرﻣﻲ و روزﻧﻪ در واﺣﺪ ﺳﻄﺢ‬،‫ ﻧﻮع ﻛﺮك‬،‫ﺳﻠﻮلﻫﺎي اﭘﻴﺪرﻣﻲ‬ ‫ داﻧﻪﻫﺎي ﮔﺮده در اﻳﻦ ﺟﻨﺲ‬.‫ آراﻳﺶ ﻣﺰوﻓﻴﻞ در ﺑﺮگ و ﺗﻌﺪاد ﻻﻳﻪﻫﺎي اﺳﻜﻠﺮاﻧﺸﻴﻤﻲ در ﺗﻔﻜﻴﻚ ﮔﻮﻧﻪﻫﺎي اﻳﻦ ﺑﺨﺶ ﻣﻔﻴﺪ ﺑﻮدﻧﺪ‬،‫دﻣﮕﻞآذﻳﻦ‬ .‫ زﺑﺮﻧﻘﻄﻪاي و ﺳﺎﺧﺘﺎر اﮔﺰﻳﻦ ﺧﺎردار ﻧﻘﻄﻪاي اﺳﺖ‬،‫ ﭼﻨﺪ ﻣﻨﻔﺬي‬،‫ﻛﺮوي‬ INTRODUCTION Acanthophyllum C. A. Mey. belongs to the family Caryophyllaceae, subfamily Caryophylloideae. Acanthophyllum with about 60 species is distributed in the Irano-Turanian region (Takhtajan 1986, SchimanCzeika 1988) and adapted to deserts, mountains and temperate areas (Ghaffari 2002). The northeast of Iran and adjacent regions in Afghanistan and Turkmenistan are the main centers of diversification for the genus (Ghaffari 2004). Acanthophyllum is represented in Iran with 33 species, 23 out of which are endemics. However, in recent investigation on the Iranian Acanthophyllum species, Basiri & al. (2011) have introduced seven synonyms and five reductions to the IRAN. J. BOT. 19 (1), 2013 Mahmoudi Shamsabad & al. 108 Table 1. Voucher specimens of Acanthophyllum species used in the study. species localities A. adenophorum Freyn Khorassan, NE Bojnourd, between Ali Muhammad and Robat, 1500 m, 4/7/1993, 23578, Faghihnia and Zangooei A. borsczowii Bunge ex Boiss. Khorassan, S Sabzevar, Hares Abad desert park, 800 m, 1/6/1991, 20553, Joharchi and Zangooei A. korshinskyi Schischk. Khorassan, NE Qayen, Tikab, 1200 m, 28/5/1995, 25602, Rafei and Zangooei A. speciosum Rech. f. & Schiman- Czeika A. laxiusculum SchimanCzeika A. heratense Schiman- Czeika A. lilacinum Schischk A. squarrosum Boiss. A. diezianum Hand-Mzt. A. brevibracteatum Lipskyi A. pachystegium Rech. f. Khorasan, NE Kalat Naderi, 1200 m, 24/5/1994, 24041, Faghihnia and Zangooei Khorassan, E Bajestan, between Hojat Abad and Helali, 1250 m, 9/5/1998, 30525, Rafei and Zangooei Khorassan, N Bojnurd, 8 km north of Jow-Darreh, 1600 m, 11/6/1996, 27190, Rafei and Zangooei Khorassan, NW Gonabad, 1300 m, 14/5/1997, 26895, Rafei and Zangooei Khorassan, NE Bojnurd, between Naveh & Qatlish, ca. 3 km on Izman bifurcation road, 1250 m, 17/6/2009, 43145, Memariani and Zangooei Khorassan, NW Qayen, 6 km east of Karghand village, 1700 m, 19/5/1998, 30764, Joharchi and Rafei Khorassan, N Kashmar, 10 km south of Ataieh, 1700 m, 25/5/1999, 32615, Hojjat and Zangooei Khorassan, NW Bojnurd, Jargalan area, 3 km from Baqleq towards Guy-Nik, 1430 m, 11/6/2008, 40772, Memariani and Zangooei rank of variety for the genus; accepting 21 species in Iran. Based on the Flora Iranica (Schiman-Czeika 1988), the genus has been divided into seven sections. The sect. Oligosperma, with 23 species worldwide, stand as the largest section of the genus of which 16 occur in Iran (Schiman-Czeika 1988). The members of this section are identified by dense flowers, spherical terminal heads, calyx (4) 6-12 mm long, calyx-teeth 1-2 mm long and 4-ovuled ovary (Schiman-Czeika 1988, Shishkin, 1936). Anatomical characters are less influenced by environmental condition than morphological characters and are more uniform from one population to another (Bokhari 1987). Generally, variation of morphological characters within family Caryophyllaceae makes taxa complicated to be delineated and identified (Fior & al. 2006). In Acanthophyllum species like other Caryophyllaceae genera there are extreme variety in population that causes difficalties to distinguish. In order to resolve problems, with respect to effect of environmental codition on morphology characters, the need of survey on endomorphic characters is nessesary and helps to identify species (Sahreen & al. 2008, Kilic 2009). There have been no anatomical and palynological studies on this genus so far. The previous studies that carried out on anatomy of Caryophyllaceae had been done by Metcalfe and Chalk (1983). Schwingruber (2007) described and analysed the xylem and phloem of 88 species from Caryophyllaceae. Kilic (2009) investigated anatomical and pollen characters in the genus Silene from Turkey. The earlier palynology studies on Caryophyllaceae had been done by Perveen and Qaiser (2006); they studied 74 species of the family Caryophyllaceae from Pakistan. The aim of this paper is to do anatomical and palynological studies for 11 species of the Acanthophyllum sect. Oligosperma in northeastern Iran including A. borsczowii Litw., A. speciosum Rech. f. & Schiman-Czeika, A. korshinskyi Schischk., A. pachystegium Rech. f., A. adenophorum Freyn., A. lilacinum Schischk., A. brevibracteatum Lipsky., A. diezianum Hand.-Mzt., A. laxiusculum SchimanCzeika, A. squarrosum Boiss., and A. heratense Schiman-Czeika and to evaluate their taxonomical application MATERIALS AND METHODS The anatomical and palynological investigations are based on the herbarium specimens obtained from FUMH (Ferdowsi University of Mashhad Herbarium). The list of these species is given in Table 1. The pollen grains extracted from the anther and dehydrated by acetic acid, then acetolyzed according to Erdtman (1960). Pollen grains were mounted in glycerine-jelly to make permanent slides and observed with Olympus BH-2 microscope under oil immersion. For scanning electron microscope studies, the pollen grains coated 109 Anatomical and pollen characters in Acanthophyllum with gold and examination was carried out on LEO1450VP microscope. Pollen terminology followed here is based on Punt & al. (1994). Measurements were based on 20 pollen grains per specimen. For anatomical study, leaves and stem were taken from the middle parts of specimens and epidermal surface were taken from the lower parts of leaves. The number of stomata and epidermal cell was considerd in 1mm². For soften, all materials were embeded in glycerine: ethanol: distilled water for three months, then fixed in FAA for 72 h. All sections cut by hand and staind with Safranin and Fast-green and mounted in entellan to make permanent slides. Pictures were taken by Olympus BH-2 microscope that connected to a Dino lite cammera. Qualitative and quantitative characters were based on 10 observations Anatomical and palynological characters. A total of 63 including 53 quantitative and 10 qualitative characters were examined on each specimen (Table 2). Qualitative characters were scored as binary or multistate characters. In this investigation missing data replacement were made with the means of variables (Legendre and Legendre 1998). Numerical methods. For collection, the characters that found to be functional in separating an apriori group several runs of PCA were carried out. Only those quantative characters allow to be contributed that variability of the first three axes of the PCA (r>0. 5) and had the least correlation (r <0.5) were used to differentiate specimens from each other. The results of these investigations are based on Principal Component Analysis (PCA) and Cluster Analysis (CA) that carried out based on UPGMA method and Euclidian distance as a disimilarity cofficience and was performed using NTSYS software Version 2 (Rohlf 1998). For selecting diagnostic characters, PCA was performed using CONACO software Version 4.5 (Ter Braak 1988). RESULTS Leaf epidermal anatomy Stomata. A. speciosum, A. korshinskyi, A. pachystegium, A. adenophorum, A. lilacinum, A.brevibracteatum, A. diezianum, A. laxiusculum and A. heratense have diacytic, anomocytic and anisocytic types of stomata while A. borsczwii has only anomocytic and A. squarrosum has diacytic type of stomata (Fig. 1). Trichomes. Acanthophyllum pachystegium, A. adenophorum and A. lilacinum have glandular and eglandular hairs, A. brevibracteatum, A. diezianum, A. laxiusculum, A. heratense and A. squarrosum have multicellular and unicellular eglandular hairs while A. korshinskyi has short glandular hairs. A. speciosum has 2-4 celled glandular hairs and in A. borsczowii unicellular eglandular hairs are observed. IRAN. J. BOT. 19 (1), 2013 Shape of epidermal cells. The shape of epidermal cells is irregular, rectangular and polygonal with crenate or entire subsidary cells. Acanthophyllum pachystegium, A. adenophorum, A. lilacinum, A. brevibracteatum, A. speciosum and A. korshinskyi have irregular epidermal cells with crenate walls and A. borsczowii has polygonal cells with entire walls while A. heratense, A. laxiusculum, A. squarrosum and A. diezianum have rectangular cells with entire walls. Size of epidermal cells. The lenght of epidermal cells in Acanthophyllum species are diffrent from .053 µm (smallest) in A. heratense to 0.12 µm (largest) in A. laxiusculum (Table 3) Palynological analysis Pollen shape, type, size, ornamentation and structure. Pollen grains are radially symmetrical and spheroidal; the type of pollen grain is pantoporate. The grains have median size (23-31 µm), this character is diffrent from 24.5µm polar axes and 23.8 µm equatorial axis in A. borsczowii (smallest) to 30.7 µm polar axes and 30.3µm equatorial axis in A. adenophorum (largest) (Table 4). Pollen ornamentation is scabrate-punctate and exin structure is spinulose-punctate (Figs. 2 and 3) Anatomical characters Cuticular layer 2-4 µm thick. Epidermis consist of a single layer of orbicular or rectangular cells located close to several parenchymatous layers with druse crystals. Several sclerenchymatous layers including thick and thin walled cells that surrounded vascular bundle. The pith is hollow or filled with large thin walled cells in some species contain druse crystals. The important indicative anatomical characters that we found for separation of taxa are as follows: Stem cross section shape and size. In A. borsczowii the stem in cross section is rectangular-elliptical and have the greatest size (1.8 mm length and 1.135 mm width), in other species stem cross section is more or less elliptical-circular and A. pachystegium has the smallest stem cross section (0.979 mm length and 0.793 mm width) (Fig. 4). The number of sclerenchymatous and parenchymatous layers in stem. Sclerenchymatous thin walled cells layers are well developed in A. borsczowii (8-9 layers) and occupied about 70% of stem radious whereas sclerenchymatous thick walled cell layers are expanded in A. korshinskyi and A. adenophorum, this character defined in the fewest amount in A. lilacinum, A. pachystegium, A. borsczowii and A. brevibracteatum (0-2 layers). Parenchymatous cell layers defind in the greatest amount in A. brevibracteatum and A. speciosum and occupied 30-40% of stem radious (about 6 layers). IRAN. J. BOT. 19 (1), 2013 Mahmoudi Shamsabad & al. 110 Table 2. Anatomical and palynological characters used in this study, followed by their abbreviations. The qualitative characters denoted by asterisks, character states and considered scores are given in square brackets. Pollen Leaf epidermis Indumentum Stem Peduncle Leaf Equatorial axis Polar axis Length of pores P/E rate Exine thickness Average distance between pores Number of micropores (mm²) *type of epidermal cells [ entire:1, crenate:2] Length of epidermal cells Width of epidermal cells Number of epidermal cells Length/width of epidermal cells Length of stomata Width of stomata Length/Width of stomata Guard cell length Guard cell width Length/Width of guard cell Number of stomata *Unicellular simple hair [absent:0, present:1] *Multicellular simple hair [absent:0, present:1] *Short glandular hair [absent:0, present:1] *Long glandular hair [absent:0, present:1] *Crystal in parenchymatous layer [absent:0, present:1] *Shape of stem cross section [Rounded = 0, elliptic = 1] Length of stem cross section Width of stem cross section Number of sclerenchymatus layers cell Number of parenchymatous layer Size of phloem Size of xylem Width of vascular bundle Size of xylem/phloem ratio Size of pith Length of vascular bundle Size of greatest xylem element Size of cuticle Length of peduncle cross section Width of peduncle cross section Length/width of peduncle cross section Size of cuticle Size of epidermal layer Size of parenchymatous layer Number of sclerenchymatus layer Number of parenchymatous layer Size of phloem Size of xylem Size of pith Width of vascular bundle Length of vascular bundle Length/width of vascular bundle Size of greatest xylem element *Arrangment of xylem elements [ solitary:1, radial chain pore:2, cluster:3] Size of greatest xylem element Number of sclerenchymatus layers cell Size of lower epidermal cell Size lower parenchymatous layer Lenght of leaf cross section Width of leaf cross section Length/width of leaf cross section *Crystal in leaf [ scarcely:0, densely:1] *Arrangment of mesophyll [ isolateral 1, dorsiventral 2] Length of lamina EQAP POAP LEPP PERP EXTP ADBP NMIP TEPE LECE WECE NUCE LWIE LSTE WSTE LWSE GCLE GCWE LWGE NSTE UNSH MUSH SGLH LGLH CRPS SHAS LCRS WCRS NSCS NPAS SPHS SXYS WVAS SXPS SPIS LVAS SGXS SICS LCRI WCRI LWCI SCUI SEPI SPAI NSCI NPAI SPHI SXYI SPII WVAI LVAI LWVI SXYI AXYI SGXI NSCL SLEL SLPL LCRL WCRL LWCL CRYL ARML LELL IRAN. J. BOT. 19 (1), 2013 111 Anatomical and pollen characters in Acanthophyllum Table 3. Leaf epidermal features of Acanthophyllum species in adaxial surface. The measurements are in mm. Taxon Epidermal cells lenght A. adenophorum 0.1 0.051 Number of Stomata epidermal cell in width 1mm² 14 0.02 A. 0.106 brevibracteatum 0.051 7 0.029 0.043 2 0.041 0.012 A. diezianum 0.082 0.066 11 0.03 0.033 3 0.028 0.013 A. borsczowii A. heratense 0.054 0.053 0.071 0.037 11 16 0.026 0.027 0.027 0.031 3 5 0.027 0.029 0.013 0.012 A. korshinskyi 0.11 0.058 12 0.032 0.034 3 0.035 0.01 A. laxiusculum 0.12 0.051 11 0.028 0.035 2 0.031 0.029 A. lilacinum 0.093 0.039 13 0.029 0.036 2 0.032 0.01 0.062 12 0.028 0.035 3 0.035 0.013 A. pachystegium 0.07 Epidermal cells width Stomata lenght 0.03 Number of stomata in 1mm² 4 Guard cell lenght 0.025 Guard cell width 0.014 A. speciosum 0.095 0.032 16 0.026 0.035 5 0.028 0.012 A. squarrosum 0.082 0.055 15 0.024 0.026 5 0.029 0.011 Table 4. General pollen characters of the examined Acanthophyllum species. Taxon Polar axis Equatorial axis P/E Pores diameters 30.7 30.3 1.013201 4.5 A. adenophorum 28.9 28.2 1.024823 4.5 A.brevibracteatum 24.7 24.6 1.004065 3.2 A.diezianum 24.5 23.875 1.026178 3.7 A. borsczowii 25.4 24.9 1.02008 3.7 A.heratense 27.6 26.8 1.029851 3.6 A.korshinsky 26.1 25.2 1.035714 4.5 A.laxiusculum 29.2 28.9 0.989726 5.2 A.lilacinum 29.4 29.4 1 6 A.pachystegium 30.6 30.25 1.001637 5 A.speciosum 26.75 26.4 1.013258 4.1 A.squarrosum Pith size and distribution of crystals. Size of pith in A. adenophorum and A. borsczowii is the greates amount (about .28 mm) and druse crystals are seen densly in the pith of A. laxiusculum. The arrangment of xylem elements in peduncle and size of them. This character is different among the Acanthophyllum species and divided this section into three groups as i) A. borsczowii with solitary elements Exine thickness 1.4 1.5 1.4 1.6 1.9 1.5 1.6 1.9 2 2 1.47 Stomata type diacytic, anomocytic, anisocytic diacytic, anomocytic, anisocytic diacytic, anomocytic, anisocytic anomocytic diacytic, anomocytic, anisocytic diacytic, anomocytic, anisocytic diacytic, anomocytic, anisocytic diacytic, anomocytic, anisocytic diacytic, anomocytic, anisocytic diacytic, anomocytic, anisocytic diacytic Pores distance 10.2 8.5 8 7 8.3 6.4 8.7 10.8 8.6 11.5 7.92 ii) A. diezianum, A. lilacinum, A. heratense, and A. squarrosum have radially chain pore arrangement iii) cluster or radially chain pore arrangement with cluster elements are observed in A. laxiusculum, A. pachystegium, A. brevibracteatum, A. korshinskyi, and A. adenophorum (Fig. 5). The size of xylem elements is maximum in A. borsczowii, A. adenophorum, A. lilacinum, and A. korshinskyi (> 0.02 mm). This IRAN. J. BOT. 19 (1), 2013 Mahmoudi Shamsabad & al. 112 A B C D Fig. 1. Epidermal surface of some Acanthophyllum species. A: A. borsczowii, B: A. squarrosum, C: A. laxiusculum, D: A. lilacinum. Fig. 2. The SEM micrographs of pollen grains in Acanthophyllum adenophorum. 113 Anatomical and pollen characters in Acanthophyllum IRAN. J. BOT. 19 (1), 2013 Fig. 3. The LM micrographs of pollen grains in Acanthophyllum adenophorum. A B Fig. 4. The cross section of stem in Acanthophyllum borsczowii (A) and A. korshinskyi (B). E= Epidermis, P= Parenchymatous layers, Cr= Crystal, S= Sclerenchymatous layers, Ph= phloem, X= xylem, Pi= pith IRAN. J. BOT. 19 (1), 2013 Mahmoudi Shamsabad & al. 114 A B C Fig. 5. The peduncle cross section of some Acanthophyllum species. A: A. borsczowii, B: A. squarrosum, C: A. adenophorum. A B Fig. 6. The leaf cross section in Acanthophyllum diezianum (A) and A. korshinskyi (B). 115 Anatomical and pollen characters in Acanthophyllum IRAN. J. BOT. 19 (1), 2013 Fig. 7. Phenogram resulting from the UPGMA of Acanthophyllum sect. Oligosperma in Khorassan provinces. OTU’S represented by: A: A. adenophorum, P: A. pachystegium, K: A. korshinskyi, Li: A. lilacinum, Sp: A. speciosum, D: A. diezianum, Sq: A. squarrosum, H: A. heratense, La: A. laxiusculum, Br: A. brevibracteatum, B: A. borsczowii. character is minimum in A. laxiusculum, A. heratense, A. squarrosum, and A. pachystegium (<0.02 mm). The number of sclerenchymatous layers in peduncle. Sclerenchymatous layers are not observed in A. borcszowii peduncle while A. pachystegium, A. brevibracteatum, A. diezianum, A. squarrosum and A. adenophorum, have greatest amount of sclerenchymatous layers (4-7 layers). The arrangment of mesophyll. This character is different among Acanthophyllum species and divided examined species into two groups as A. diezianum, A. laxiusculum, A. squarrosum and A. heratense had dorsi-ventral arrangment while A. borsczowii, A. korshinskyi, A. pachystegium, A. speciosum, A. adenophorum and A. brevibracteatum had isolateral arrangment (Fig. 6). The number of sclerenchymatous layers in leaves. Sclerenchymatous thick walled cells layers are not observed in A. borsczowii while these layers difind in the greatest amount in A. korshinskyi, A. adenophorum, A. brevibracteatum and A. pachystegium. Lamina size and druse crystal distribution. Lamina size in A. borsczowii is longer than 1 mm and druse crystal are seen densly in its lamina while in all other examined species lamina size is smaller than 0.5 mm. Cluster analysis of micromorphological characters The UPGMA of the OTU’S used in this study is shown in Fig 7. The arrangement of species in cluster was written in the left of cluster. The phenogram is divided into two clusters. Acanthophyllum borsczowii is separated from the rest of species in a single species cluster. In addition, the other cluster is divided into two branches. In the upper branch located A. adenophorum, A. pachystegium, A. korshinskyi, A. lilacinum and A. speciosum, in the lower branch A. laxiusculum, A. heratense and A. squarrosum located close together. Principal component analysis of micromorphological characters. Only characters that have high eigenvalue on the first three principal component (r>0.5) and had the least correlation coefficient (r< 0.5) were selected to separate OTU’s (Table 5). The first three components explain 94 % of the total character variation 64%, 23% and 7 % for the respective axes. In a plot of the first and second PCs (Fig. 8); the number of sclerenchyma thick walled layers in leaf (NSCL) character are isolated A. korshinskyi and A. pachystegium from the other taxa. Lenght of lamina (LELL), the size of greatest xylem elements in stem IRAN. J. BOT. 19 (1), 2013 Mahmoudi Shamsabad & al. 116 PC2 PC3 PC1 Fig. 8. Principal Component Analysis- Scatter diagram of specimens and characters from 11 species of Acanthophyllum sect. Oligosperma in Khorassan provinces. 1: A. adenophorum, 2: A. brevibracteatum, 3: A. borsczowii, 4: A. diezianum, 5: A. heratense, 6: A. pachystegium, 7: A. laxiusculum, 8: A. lilacinum, 9: A. korshinskyi, 10: A. speciosum, 11: A. squarrosum. (SXYS) and the size of the greatest xylem elements in peduncle (SXYI) are the characters that separated A. borsczowii. Acanthophyllum heratense and A. squarrosum are isolated from the rest by length/width of epidermal cells (LWIE); the number of epidermal cells (NUCE) separates A. speciosum. Long glandular hairs (LGLH) character differentiates A. lilacinum. Multicellular simple hairs (MUSH) and arrangement of xylem elements (AXYI) are the characters that excluded A. adenophorum. A. laxiusculum is distinguished by size of phloem in peduncle (SPHI) and the number of sclerenchymatous layers in peduncle (NSCI) character exluded A. diezianum from the other taxa. IRAN. J. BOT. 19 (1), 2013 117 Anatomical and pollen characters in Acanthophyllum Table 5. Eigen vectors of the characters used on the first three axes in the PCA. N NAME AX1 AX2 AX3 AX4 1 MUSH 0.5833 -0.037 0.0831 0.0052 3 LGLH 0.5148 -0.2294 0.005 0.7133 4 AXYI 0.5457 -0.2162 0.3366 0.7491 8 NUCE 0.3274 -0.9442 0.0042 -0.0359 9 LWIE 0.9909 0.1293 -0.0336 -0.0156 10 SXYS -0.0084 0.5253 0.3243 -0.224 12 SGXS 0.5606 0.3005 0.0164 -0.0071 13 SCPI -0.4968 0.5469 0.174 -0.2294 14 SPAI -0.2518 0.5479 0.0886 0.2556 15 NSCI 0.5708 0.7156 -0.1937 -0.1455 16 SPHI 0.0107 0.5748 -0.4096 0.3501 17 SXYI 0.5156 0.4294 -0.2917 -0.2049 22 NSCL 0.1791 0.0709 0.9737 -0.1205 25 LELL 0.1392 0.5612 0.1306 -0.3428 DISCUSSION The present study matches with the results of SchimanCzeika (1988) and Shishkin (1936) who reported different type of trichomes in Acanthophyllum species. Schiman-Czeika (1988), in Flora Iranica, described A. borsczowii as a glabrous plant but our result is in agreement with Bidi (2007) who reported unicellular simple hairs on A. borsczowii. The basic type of stomata is diacytic in Caryophyllaceae family according to Metcalfe and Chalk (1983) while in A. borsczowii the anomocytic type of stomata was observed. Some other stomata types such as anisocytic and anomocytic were observed in several species (Table 3). This study is also in agreement with Jafari & al. (2008) who reported crenate and entire subsidiary cell walls in some species of Silene. Similarly, anisocytic and anomocytic types of stomata were reported in some Silene species by Sahreen et al. (2010). In Acanthophyllum species pollen grains are fairly uniform with radially symmetrical and pantoporate grains, although, our results showed differences in pollen size, distance between two pori and number of pores but these variation were not remarkable and are not important characters for taxonomy of Acanthophyllum species. Stem anatomy was somewhat simillar in the examined taxa, but stem cross section shape and size, the number of sclerenchymatous layers are taxonomically significant to identify species. In this study calcium oxalate crystals are observed both in endodermis and pith that are dissent with Metcalfe and Chalk (1950). They reported that crystals in Caryophyllaceae are placed only in endodermis. However, it is well known that it is an environmentally influenced anatomical character and we cannot use it as a strong taxonomic character for grouping species (Kilic 2009). The number of sclerenchymatous layers difined in the greatest amount in A. borsczowii, it should be mentioned that the stem in this species contrary to the other species of the section except for A. elatius Bunge ex Boiss. is vertical. The arrangment of xylem in peduncle and the arrangment of mesophyll in leaf were qualitative characters that taxonomically significant to separating Acanthophyllum species. The second character was removed in the PCA analysis due to the high correlation coefficient on the PCA analysis. Examined species have been divided to three groups by the arrangment of xylem elements character in peduncle; these species are well separated by morphological characters too. The results of the cluster analysis (Fig. 7) indicated that A. laxiusculum, A. squarrosum and A. heratense are linked together that are in agreement with Basiri & al. (2011) taxonomic results. They concluded that the species A. heratense and A. laxiusculum are as synonyms. With respect to the proximity of A. heratense habitat to A. laxiusculum and A. squarrosum, molecular study and a ditailed morphological study on IRAN. J. BOT. 19 (1), 2013 these three species in the field are suggested. The results showed endomorphic characters in Acanthophyllum species are only useful to separate the species that are morphologically apart, as the species A. adenophorum, A. pachystegium and A. lilacinum that are morphologically similar having more or less irregular epidermal cells with wavy walls, glandular and eglandular hairs, cluster or radial chain pore with cluster elements, leaf with isolateral mesophyl and also generally greater sclerenchymatous layers while A. laxiusculum, A. heratense and A. squarrosum have rectangular epidermal cells with entire walls, eglandular hairs, radial chain pore or radial chain pore with cluster elements and dorsi-ventral arrangement in leaf mesophyll. Acanthophyllum borsczowii with vertical stem, ovate-lanceolate leaves, anomocytic stomata, polygonal epidermal cells, solitary xylem element arrangement and rectangular stem cross section was different from all examined species in this genus. 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