ISSN 2226-3063 e-ISSN 2227-9555 Modern Phytomorphology 10: 25–38, 2016 Macro- and MIcroMorphologIcal studIes of Clypeola specIes (BrassIcaceae) In Iran Sousan Abbasian & Maryam Keshavarzi * abstract. Clypeola is an annual genus from Brassicaceae with four (C. jonthlaspi, C. aspera, C. lappacea and C. dichotoma) species in Iran. These are plants of different habitats and found as early spring therophytes in semiarid regions of Iran. In this study 63 populations of Clypeola genus have been studied by 49 macro- and micromorphological features. Results were analyzed by use of multivariate statistical methods. Cluster analysis, factor analysis and ordination methods were applied. The result showed that such characters as pedicle, trichome, stamen, petal features and sculpture of fruit surface have valuable diagnostic in separating of these species. In present study it was also found that the use of seed surface character in not effective alone for taxa delimitation except of some C. jonthlaspi subspecies. Among leaf features, trichome ornamentations, their position and branching pattern are effective for species separation. Fruit hairs are of diagnostics importance in species separation too. Key words: Brassicaceae, Clypeola, fruit, micromorphology, seed Plant Science Dept, Faculty of Biological Science, Alzahra University, Tehran, Iran; * neshat112000@yahoo.com, M.keshavarzi@alzahra.ac.ir Introduction Brassicaceae comprise of 338 genera and 3709 species (Franzke et al. 2010). In Brassicaceae, the importance of micromorphological characters such as seed surface and hairs has been emphasized (Al-Shehbaz et al. 2006). Trichomes are divided into three groups based on their form, the branching pattern, types and number of cells. The trichome features are valuable in the classification of Brassicaceae on generic and specific levels. For example, Rollins & Banerjee (1976) studied the leaf trichomes of Lesquerella S. Watson. Ancew & Goranova (2006) studied the seed and leaf trichome morphology of eight taxa from Alysseae tribe. Mummenhoff et al. (1997) pointed to the characters convergence in fruit morphology during their studies on Thlaspi s.l. based on ITS analysis. Based on early researches on seed surface features, characters of anticlinal and periclinal cell walls have been considered significant (Abdel Khalik et al. 2002; Tantawy et al. 2004; El Naggar et al. 2005; Moazzeni et al. 2007; Kasem et al. 2011). In particular, Abdel Khalik et al. (2002) studied morphological features of 45 taxa of Brassicaceae. They pointed on the diagnostic importance of leaf, flower, fruit, seed, embryo and trichome morphology. Bolourian & Pakravan (2011) mentioned that silicle and stamen features are valuable in separation of annual species. Clypeola L., is an annual genus of Alysseae tribe. Distribution of this genus is limited to northern hemisphere. There are 9 species in this genus (Chaytor & Turill 1935). Rechinger (1968) mentioned 5 Clypeola species in Iran: C. aspera (Gruer) Turill, C. lappacea Boiss, C. dichotoma Boiss, C. jonthlaspi L. and C. microcarpa Morise. C. jonthlaspi is idely distributed in Iran and in some papers presence of varieties or subspecies of this taxon is mentioned (Chaytor & Turill 1935; Breistroffer 1936). Due to the different description of C. microcarpa in literatures, it is mainly considered as a subspecies or a variety of C. jonthlaspi. In present study C. microcarpa as a separate species is rejected too. The aim of this research is to evaluate the macro- and micromorphological variation © 2016 The Author(s). Published by Modern Phytomorphology. This is an open access article under the Creative Commons BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) 26 Modern Phytomorphology 10 (2016) in Clypeola in order to find more diagnostic features and to realize a better separation of taxa. Material and methods The herbarium specimens and freshly collected plants from 63 populations belonging to 4 annual taxa (C. jonthlaspi, C. aspera, C. lappacea, C. dichotoma) from different localities in Iran were studied (Tab. 1). Vouchers of the collected plants are deposited in Alzahra University Herbarium (ALUH). Ten individuals were taken from each locality and used for morphological studies based on species distribution patterns. Totally 49 qualitative and quantitative morphological characters, consisting of vegetative and reproductive structures were assessed (Tabs 2 & 3). Characters were selected on the base of Floras and our own field studies. The fruit, seed and leaf surface were studied by use of scanning electron microscopy (SEM). Samples were fixed on aluminum stubs using double-sided adhesive and were coated with a thin layer of gold-palladium. The SEM micrographs were taken by Philips XL30. In text El Naggar (2005) terminology is applied. In phenetic analysis the mean of quantitative characters were used while qualitative characters were coded as binary/multistate characters. Variables were standardized (mean = 0, variance = 1) for multi-variant statistical analyses (Ng et al. 1981). In order to find the species relationships cluster analyses using UPGMA (un-weighted paired group, mean average) and WARD (minimum variance, spherical clusters) as well as ordination based on principal component analysis (PCA) were performed. In order to determine the most variable morphological characters among the species/populations, factor analysis based on principal components analysis (PCA) was performed. Non-variant characters were omitted before factor analysis. results Macromorphological studies form of pedicle show variation in studied species. The pedicle is recurved and this is the diagnostic trait for Alyssum and Clypeola separation. Pedicle is like an umbrella hand in C. jonthlaspi (Fig. 1 E), domical in C. aspera (Fig. 1 F), recurved with a sharp angle in C. lappaceae (Fig. 1 G), and S-shaped in C. dichotoma (Fig. 1 H). fruit trichomes in studied species are columnar or pyramidal. In C. jonthlaspi and C. dichotoma, trichomes are columnar. In C. aspera and C. lappacea, trichomes are pyramidal and branching at the tip and in mature fruit. The branching pattern in different populations of C. lappacea is varied and in C. aspera it is pentameral. The trichome features in C. jonthlaspi showed variation and due to this it has been used to distinguish subspecies and varieties in Floras and papers. In C. lappacea there is a great variety in hair distribution and length. fruit shape is elliptic, orbicular or obovate. The style in C. dichotoma and C. lappacea is long and the tip of fruits are not emarginate (Fig. 1 I, J), though in C. aspera and C. jonthlaspi it is emarginate and style length is often equal to its width (Fig. 1 K, L). In C. jonthlaspi, C. aspera and C. dichotoma there are two distinct parts, fruit disk and wing. the shape of cells in fruit margins differs among three species, they are lobate in C. jonthlaspi (Fig. 2 A), stellate with the presence of stomata in C. aspera (Fig. 2 B), and represented by extensive cell with fine lobes in C. dichotoma (Fig. 2 C). In C. lappacea there is no distinction between central and marginal parts of fruit. The seed is wingless and without mucilage. Seed is elliptic, obovate, oblong and orbicular with different degree of elongation. In all studied species, different form can be seen among populations and even individuals of a population. the calyces are shed after ripening of fruits. In C. aspera, C. jonthlaspi and C. dichotoma, calyx is saccate. In C. lappacea, calyces are nearly straight. Sometimes the margins are purple (C. jonthlaspi). petals in studied species are oblong or triangular (C. jonthlaspi). In C. aspera and C. lappacea, lamina is flattened. In C. dichotoma, at the base of one or two petals, there is a protruding in some flower (Fig. 2 F). The 27 Abbasian S., Keshavarzi M. Morphological studies of Clypeola in Iran tab. 1. Collections data for populations used in macromorphological study. * – stands for accessions used in micromorphological studies by SEM. aluh – vouchers preserved in Herbarium of Alzahra University; farh – in Herbarium of Kharazmi University; fuMh – in Herbarium of Ferdouwsi University; hsBu – in Herbarium of Shahid Beheshti University. Nr Species Voucher Nr Origin Collector 1 C. jonthlaspi 18917-ALUH Tehran: Boumehen Abbasian 2 18918-ALUH Alborz: Baghestan Mosaferi 3 188-ALUH Alborz: Aghesht to Baraghan * Keshavarzi 4 18919-ALUH Gilan: Jirandeh Dadmehr 5 18920-ALUH Qazvin: Abyek Abbasian 6 18921-ALUH Qazvin: 20 km Ange road, Shekarnab village * Abbasian 7 18922-ALUH Fars: Shiraz to Isfahan, 15 km to Saadat Shahr Abbasian 8 18923-ALUH Kermanshah: Taq-e-Bostan Gholami 9 18924-ALUH Tehran: Dar Abad Abbasian, Habibi, Dadmehr 10 18925-ALUH Tehran: Darakeh River near Vanak Keshavarzi, Abbasian, Habibi 11 18926-ALUH Tehran: Bumehen to Tehran road Abbasian 12 18915-ALUH Fars: 35 km Shiraz, 1482 m Abbasian 13 18913-ALUH Yazd: Tabas, Neyzar village, 1010 m Abbasian 14 18911-ALUH Yazd: Tabas, Eshqabad road, Ozbak Kuh Abbasian 15 18999-ALUH Khorasan: Boshrouyeh, Neygenan village Abbasian 16 18918-ALUH Fars: 35 km to Neyriz, 1480 m Abbasian 17 1897-ALUH Fars: Marvdasht road, Naqsh-e-Rostam Abbasian 18 1895-ALUH Yazd: Tabas, Khevr village Abbasian 19 1892-ALUH Yazd: Tabas, Eshqabad road, Kalshane village,1092 m * Abbasian 20 1903-ALUH Kurdistan: Sanandaj road, 15 km Kamyaran Abbasian 21 1904-ALUH Kermanshah: Sanandaj road Abbasian 22 1905-ALUH Kermanshah: 60 km Kermanshah, Bid Sorkh ghaut Abbasian 23 24711-FUMA Khorasan: 70 km to Mashhad, Kalat road, 1600 m * Faqhihi nia & Zanguee 24 16372-FUMA Khorasan: W. Dargaz-Gadganlou, 1200 m Joharchi & Zanguee 25 25371-FUMA Khorasan: Kashmar, SW Kuh Sorkh Faghihi nia & Zanguee 18916-ALUH Fars: 35 km to Shiraz, 1482 m Abbasian 27 26 18914-ALUH Yazd: Tabas, Neyzar village, 1010 m Abbasian 28 18912-ALUH Yazd: Eshqabad road, Ozbak Kuh Abbasian 29 18910-ALUH Khorasan: Boshrouyeh, Neygenan village * Abbasian 30 1898-ALUH Kerman: Kerman, 1770 m Abbasian 31 1896-ALUH Fars: 35 km to Neyriz, 1480 m Abbasian 32 1893-ALUH Yazd: Tabas, Abid village Abbasian 33 1894-ALUH Yazd: 40 km Deyhuk, 1361m Abbasian C. aspera 28 Modern Phytomorphology 10 (2016) tab. 1. Continued. 5044-ALUH Fars: 35 km Marvdasht * Rastipishe 35 34 1891-ALUH Yazd: Tabas, Eshqabad road, Kalshane village, 1092 m Abbasian 36 36394-FUMA Yazd: SE Tabas, NE Deyhuk, 1700 m Zanguee & Rafei 37 18917-ALUH Qazvin: Abyek Abbasian 38 AnonymusHSBU Kerman: Kerman Sonboli 1907-ALUH E Azerbayjan: 48 km NW Marand, salt hills, 1036 m Akhani & Samadi 40 16313-FUMH Khorasan: between Quchan and Dargaz, Allah Akbar ghaut, 1650 m Joharchi & Zanguee 41 24743-FUMH Khorasan: Bshrouyeh road, 1400 m Rafei & Zanguee 42 26738-FUMH Khorasan: SE Birjand, 1650 m Rafei & Zanguee 43 AnonymusFUMH Khorasan: Birjand to Tabas, 1150 m Anonymus 44 10076-FUMH Khorasan: Birjand to Sarchah road, 1400 m Anonymus 45 16609-FUMH Khorasan: SW Bojnurd, 1050 m Joharchi & Zanguee 46 17287-FUMH Khorasan: E Birjand, Gzyk mountains, 1400-1500 m Joharchi & Zanguee 47 13743-FUMH Khorasan: Ferdows, Boshrouye Ayatollahi & Joharchi 48 21809-FUMH Khorasan: Birjand, Hamand, Give road, 1400 m. Faghihinia & Zanguee 49 15109-FUMH Khorasan: Birjand, 8 km after Hamand, 1300-1350 m * Rashed & Zanguee AnonymusHSBU n.a. Anonymus 51 36-HSBU Kermanshah: Songhor, Asadabad, 1650 m Zehzad 52 87410-HSBU Isfahan: between Shahreza and Vanak, 2300 m Khosravi 53 87532-HSBU Chahar Mahal Va Bakhtiari: between Kharadgee and Gandoman, Pare Das mountain, 2150-2300 m * Zehzad 54 AnonymusFARH Kordestan: Sanandaj * Anonymus 55 18814-FARH Iran: Anaran mountain, Ghooch Ali * Anonymus 56 18905-FARH Isfahan: Faridan * Bagheri 57 18904-FARH Kermanshah Hassan pour 58 18906-FARH W. Azerbaijan: Takab * Anonymus 59 11089-FARH Hamadan: Tuyserkan Haj Mohamad Sameeii 60 1901-ALUH Lorestan: Malayer-Borujerd road, 60 km to Borujerd Abbasian 61 1902-ALUH Kermanshah: Bid Sorkh gauth Abbasian 62 74413-HSBU E Azarbaijan: between Tabriz and Marand Zehzad 8624402-HSBU Chahar Mahal Va Bakhtiari: between Gandoman and Ardal, south hills of pond Shaloo, 2350-2450 m Zehzad 39 50 63 C. aspera C. dichotoma C. lappacea Abbasian S., Keshavarzi M. Morphological studies of Clypeola in Iran 29 tab. 2. Qualitative macro- and micromorphological features used in this study. Characters State of character and coding Vegetative form ascending (0), erect and un-branched (1), branched at base (2), ascending with few branches (3), ascending or erect and bifurcate branches (4) Fruit shape ovate (0), elliptic (1), round (2), elliptic-ovate (3) Seed shape oblong (0), elliptic (1), elliptic-ovate (2) Petal shape connate (0), oblong (1), oblong-connate (2) Hair in fruit disc absent (0), present (1) Hair in fruit margin absent (0), present (1) Pedicle shape curved (0), recurved sharply down (1), S-shaped (2), umbrella handle like (3) Leaf shape elliptic and flat (1), oblanceolate-linear (2), oblanceolate and flat-elliptic (3) Hair shape cylindrical (0), conical (1) Anthers shape non-elliptic (0), elliptic (1) Petal base protruding present (0), absent (1) Protruding at the end of filament and its wing present (0), absent (1) Fruit margin smooth (0), crenate (1), serrate (2) Different tissues in fruit margin absent (0), present (1) Fruit hair surface without furrows (0), horizontally furrowed(1) Fruit hair surface tuberculate (1), hispid (2) Hair apex rounded (1), swollen (2), branched (3) Trichome surface ornamentation non-verucate (0), verucate (1) Trichome shape columnar (1), pyramidal (2), infundibular (3) Trichome tapering gradually (1), suddenly (2) Trichome without net-like structure (0), with net-like structure (1) Fruit ornamentation smooth with button shaped particles (0), with cone shaped protruding (1), tuberculate (2), papillat with irregular pattern (3), small tubercules (4), amorphous and branched (5) Fruit surface with deep holes(0), net-like (1), lineolate (2) Seed surface reticulation regular (1), irregular (2) Seed surface domate (0), net like (1), stripped (2), lineolate (3) stamens are winged and wings are terminated to a lobed or acute tip. In some individuals, the stamens with two wings can be seen. Anthers are medifix in C. aspera, C. jonthlaspi and C. dichotoma. In C. lappacea, at the margin of tip of filament and wings, there are sinuate protrudings (Fig. 2 G). Length of filament and wing of stamen are valuable traits in species delimitation. Some populations of C. jonthlaspi showed different combination of flowers (with unusual structures or with both high density of hair and unusual structures) (Fig. 1 C). Micromorphological studies Fruit and seed surface were studied. Fruit and seed sculpture pattern and fruit hairs showed a great variation in C. jonthlaspi and C. lappacea. The surface of fruit in C. jonthlaspi haired on both part of fruits, covered with papilla smaller than hairs, and a net-like sculptured, but in fruits with marginal hairs, surface of fruit have lineolate sculpturing pattern. In C. aspera, surface of fruit covered with cone-shaped protrudings which are branched at tip. The shape of these protrudings and fruit sculpturing 30 Modern Phytomorphology 10 (2016) tab. 3. Quantitative macro- and micromorphological features used in this study. Characters Characters Leaf length Seed length Leaf width Seed width Petal length Fruit length Petal width Fruit width Sepal length Length of style Sepal width Average length of fruit hair Length of pedicle Length of Fruit to seed ratio Length of inflorescence Length of longest filament/length of same anther Length of stamen Fruit/fruit hair length ratio Width of fruit/length of hair Sepal/petal length ratio Length of fruit margin Fruit to style length ratio Length of pedicle/length of fruit Fruit to seed length ratio vary among studied populations (Fig. 3). The surface of C. dichotoma covered with papilate hairs which are smaller than fruit hairs. In most studied populations of C. lappacea, the surface of fruit is covered with branched protrudings, the form of them are different among populations. In Kurdistan population, surface of fruit is stripped and have button-shaped protrudings that scattered irregularly, while vacuolar pattern in Faridan and Paredas populations were observed (Fig. 3). In Azerbaijan population, the fruit is covered with net-like sculptures. In other populations, surface of fruit have stripe or smooth sculpture pattern as in Ilam, Kermanshah, Gandoman, and Faridan (Fig. 3). trichome surface has button-shaped ornamentation in C. jonthaspi and C. dichtoma but barbed-shaped in C. aspera (Fig. 4) and C. lappacea. There was an infudibular-shaped hair base in Faridan accession or the gradually narrowing at the tip of hairs (Fig. 4). seed surface sculpturing is mostly reticulate but three other types were observed too: domate (C. dichotoma), lineolate (in C. jonthlaspi with marginal hair at fruit), or net-like pattern (in C. lappacea) from Azerbaijan (Fig. 4). Other populations of C. lappacea, C. aspera and C. jonthlaspi have reticulate sculpture (Fig. 4). A variation in hair density was observed while studying leaf dorsal epidermis. The hairs have button-shaped sculptures (Fig. 5 A-C). In C. dichotoma sculptures are not prominent and hairs are more slender (Fig. 5 D). In some studied populations of C. lappacea, tip of hairs is curved (Fig. 5 E). It was evident that the lower and upper epidermis of petal is different. In all examined samples, the sculpture of lower epidermis is stripy and these lines are parallel and margin of cells are wavy (Fig. 5 F), but in upper epidermis of four studied species there are differences (Fig. 5 G-J). Interspecific variation based on macromorphological characters The studied species showed significant differences in selected set of characters. The Ward’s phenogram based on morphological features showed that there are two main clusters (Fig. 6). The first cluster consists of C. jonthlaspi, C. aspera and C. dichotoma. In second cluster C. lappacea is grouped. In order to determine the most variable characters among the studied species, factor analysis based on PCA was performed, revealing that the first three factors comprise more than 70% of total observed variation. In the first factor, with more than 50% of the total variation, characters as petal length and width, sepal length and width, filament length and width of all stamens wing, length of pedicle, length of anther, length of style, presence Abbasian S., Keshavarzi M. Morphological studies of Clypeola in Iran 31 fig. 1. a – leaf of Clypeola dichotoma; B – trichome with four main branches at down and three at upper part; c – abnormal (left) and normal (right) flowers of C. jonthlaspi; d – inflorescence of C. dichotoma, e – pedicle of C. jonthlaspi; f – pedicle of C. aspera; g – pedicle of C. lappacea; h – pedicle of C. dichotoma; I – style of C. dichotoma; J – style in C. lappacea; K – style and crenate shape of fruit margin in C. aspera; l – style in C. jonthlaspi. 32 Modern Phytomorphology 10 (2016) fig. 2. Fruit margin in Clypeola jonthlaspi (a), C. aspera (B) and C. dichotoma (c); d – mature fruits; e – emargination in lower part of petal of C. lappacea; f – protruding in basal petal of C. dichotoma; g – protruding in tip of filament and wing of stamen of C. lappacea. of different tissue at fruit margin, presence of protruding in filament and wing margin of stamens, shape of leaf and anther had highest correlation (>0.6). In the second factor, with about 11.53% of the total variation, characters as the height of plant, width of leaf, length of inflorescence, presence of protruding in base of petal and number of leaf veins had highest correlation(>0.6). In the third factor with about 9.44% of the total variation, characters as length of fruit trichomes, ratio of fruit length to seed length, ratio of length of filament to anther length and shape of fruit margin had highest correlation(>0.6). Therefore, these are the most variable morphological characters among the studied characters of studied Clypeola species. Interspecific variation based on micromorphological characters of seed and fruit In order to group the species having micromorphological similarities, cluster analysis using Ward’s method was performed (Fig. 7). It has revealed two main clusters (Nrs 1 & 2). In cluster Nr. 1, C. jonthlaspi, C. dichotoma and one population from C. lappacea are grouped. There are two subsets in this cluster, one of which contains populations from Kalshane and Karaj belonging to C. jonthlaspi. fig. 3. Seed and fruit surface in Clypeola jonthlaspi (a-f), C. aspera (g-I), C. dichotoma (J-K), and C. lappacea from ▶ different populations (l-r). l – Kordestan; M – Faridan; n – Paredas; o – Azerbaijan; p – Isfahan; Q-r – Kermanshah. Abbasian S., Keshavarzi M. Morphological studies of Clypeola in Iran 33 34 Modern Phytomorphology 10 (2016) Abbasian S., Keshavarzi M. Morphological studies of Clypeola in Iran In these populations, trichomes of fruit can be seen only at margin of fruit. In a cluster populations of Karaj, Qazvin and MashhadKalat of C. jonthlaspi are grouped. These taxa have fruit hairs in both parts. Presumably, the 35 reason for grouping population of Azerbaijan from C. lappacea, in this subset, is the presence of net like sculpture on the surface of fruit (Fig. 3), which can be seen in populations of C. jonthlaspi (Fig. 3 A). Cluster Nr. 2 contains fig. 5. Leaf and petal surface. a-e – leaf hairs; a – hair with protruding that are larger in center; B – hair with equal protruding in all parts; c – hairs without protruding in center; d – hair of C. dichotoma; e – hair with curvature in tip in C. lappacea; f – dorsal petal epidermis in C. lappacea; g-J – petal ventral epidermis in C. jonthlaspi (g), C. aspera (h), C. dichotoma (I), and C. lappacea (J). ◀ fig. 4. Fruit and seed surface. a-I – fruit surface. a-c – rounded trichome tip and button shaped sculptures in Clypeola jonthlaspi; d – swollen trichome tip in C. dichotoma; e-f – branched tip and barbed sculptures in C. aspera; g – infundibular-shaped trichomes in C. lappacea; h – narrowed gradually trichomes in C. lappacea; I – sharping tip in C. lappacea. J-o – seed surface sculptures. J – domate in C. dichotoma; K – lineolate in C. jonthlaspi; l – net-like in C. lappacea; M – reticulate in C. aspera; n – reticulate in C. lappacea; o – reticulate in C. jonthlaspi. 36 Modern Phytomorphology 10 (2016) fig. 6. Phenogram (Ward’s method) based on macromorphological characters in four annual Clypeola species. populations of C. lappacea and C. aspera. In f cluster populations of C. aspera are clearly separated. The arrangement of C. lappacea and C. jonthlaspi populations in dendrogram are modified for macro- and micromorphology with pollen morphology results. In order to determine the most variable characters among the studied species, a factor analysis based on PCA was performed, revealing that the first three factors comprise about 61% of total variation. In the first factor with about 31% of the total variation showed the highest correlation (>0,6). Second and third factor with about 16% and 15% of the total variation respectively showed the highest correlation. discussion By considering of different populations of Clypeola species in Iran in present study it was evident that, some morphological characters as pedicle shape, shape of fruit hair, shape of fruit margin, characters of stamen, shape of style and petals are efficient in species separation. A number of authors (Abdel Khalik 2002; Tantawy et al. 2004; El Naggar 2005; Moazzeni et al. 2007; Kasem et al. 2011) demonstrated that seed surface features are valuable in separating species regarding anticlinal and periclinal walls. In this study we demonstrated that the use of seed surface character is not efficient when used alone, but it was also shown that some subspecies of C. jonthlaspi could be separated by these features. The diagnostic importance of fruit characters in present study is in concordant with Kaya et al. (2011). Some micromorphological leaf features as hair position, branch number and diameter are effective in species separation. Rollins & Banejee (1976) mentioned the importance of same features while studying Lesquerella species. It is believed that in dry condition, hairs are denser than humid condition because of adaptive behavior of plants. Results of these study is somewhat in congruent with this idea, as in C. aspera and C. jonthlaspi and C. lappacea (Faridan) from drier location than Ilam and Bakhteeyari belonging to C. lappacea have a higher hair density. C. dichotoma (Birjand population) which grow in dry location, the hair density is low. Trichomes with curvature Abbasian S., Keshavarzi M. Morphological studies of Clypeola in Iran 37 fig. 7. Phenogram (Ward’s method) based on seed and fruit micromorphological characters in four annual Clypeola species. at their tips were observed in populations of Gandoman, Ardal and Ilam of C. lappacea. Fruit micromorphological features especially fruit hairs have diagnostic role in species delimitation but leaf and seed characters are used as complementary. Due to the vast variation which was observed in C. jonthlaspi and C. lappacea populations, the result of micromorphological studies can be helpful to separate these species as it was evident in palynological study of same genus (Keshavarzi et al. 2012). acknowledgments We are very thankful of the support of Mr. Jouharchi (FUMH), Mrs. Tavakoli (FARH) and Dr. Mehrabian (HSBU) to make the possibility to observed more herbarium samples. references abdel Khalik K.e. 2002. Biosystematic studies on Brassicaceae (Cruciferae) in Egypt. Ph.D Thesis, University of Wageningen. al-shehbaz I.a, Beilstein M.a., Kellogg e.a. 2006. Systematic and phylogeny of the Brassicaceae (Cruciferae): An overview. Pl. Syst. Evol. 259: 89–120. ancev M., goranova V. 2006. 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