Int. J. Biosci. 2014

International Journal of Biosciences | IJB |

ISSN: 2220-6655 (Print) 2222-5234 (Online) http://www.innspub.net Vol. 5, No. 7, p. 50-57, 2014

RESEARCH PAPER OPEN ACCESS

Evaluation of generic concept in trin. and Catabrosella tzvel. (Tzvel.): a new subspecies in family in Iran

Maryam Abbasi1*, Mostafa Asadi2

1Department of Biology, College of Basic Sciences, Central Tehran Branch, Islamic Azad University, Tehran, Iran 2Research Institute of Forests and Rangelands, Tehran, Iran

Key words: Iran, , morphology, micromorphology, anatomy.

http://dx.doi.org/10.12692/ijb/5.7.50-57 Article published on October 06, 2014

Abstract

Flora Iranica provides a description of the genera and identification keys for species, however; as a result of variability of morphological traits and intermediate characters there is some ambiguities in positions of taxa in Poaceae family. Therefore, we studied some taxonomic changes in belong to Poaceae family. After sampling, morphological investigations, anatomical studies of leaves, micromorphological studies of abaxial and adaxial surface of leaves, lemma and gluma were performed. The results were conducted to some numerical analysis aspects On the basis of PCA (Principal component analysis). Additionally, inter-species taxonomic distances and inter-genus borders were determined. According to the results, in Flora Iranica, four species of Colpodium Trin. genus are named which are reduced to two species in this study. In other words, taxonomic positions of C. violaceum (Boiss.) Griseb. and C. versicolor (Stev.) Schmalh are preserved whereas C. parviflorum Boiss. and C. humile (M.B.) Griseb. were transferred to Catabrosella Tzvel.(Tzvel.) genus and introduced as Catabrosella parviflora (Boiss. and Buhs.) Alex. ex Mill., Comb. Nov species, and C. humilis subsp. humilis. Furthermore, C. humilis subsp. Calvertii (Boiss.) Tzvel., is reported for the first time in Iran. The populations of Catabrosella Tzvel. (Tzvel.) in high regions, with moist and humous beds forms mountain meadows. In Flora Iranica (Bor, 1970) there is no report about species belong to this genus. * Corresponding Author: Maryam Abbasi  [email protected]

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Introduction boll and picked and oval lemma. In addition, these Poaceae (grasses) is one of the most species-rich species are morphologically different from flowering families and includes many C.parviflorum and C.humile in terms having basal economically important crops. Parallel evolution of boll, two or three flowers in each spikelet and long such features as the annual habit, C4 photosynthesis and oval lemma. Micromorphology and anatomical and several highly characteristic reproductive studies confirmed similarity between C. violaceum structures has facilitated a series of major radiations and C. versicolor and difference between C. within Poaceae, culminating in the existing global violaceum and C. versicolor and two other species. distribution of about 10000 species and 700 genera. Colpodinum Trin. was introduced for the first time in (Rudall, 2005; Clayton and Renvoize, 1986). 1820 by Trin that are growing in mountainous regions, rocky hillsides, flat plains and mountainous Iran is one of the most important regions for being pastures. In addition, these species can be found unique in terms of diversity and speciation process of beside of the rivers and clay-sandy river beds as well due to different climates. Plants belong to as semi marshy areas. Catabrosella Tzvel. (Tzvel.) Poaceae family are spread in Iran and due to the wide was introduced for the first time in 1965 by Tzvelev diversity and structural complexities make many who reported four species for this genus in Grasses of taxonomic problems (Assare, 2004). Successful the Soviet Union book (Tzvel., 1976). The aim of this distribution of Poaceae family is indebted to some study was to collect some information from specific characteristics such as strong resistance to morphology, anatomy and micromorphology features grazing due to having particular meristems, stigma to evaluate taxa. structure, pollinated by the wind (anemophilous), having hollow stems called culms plugged at intervals Materials and methods and finally leaf shape, which increase plant Plant materials and experimental conditions compatibility with arid and cold regions such as For anatomical studies, basal leaves were selected and deserts and steppes (Judd et al., 1999). Flora Iranica middle part of blade was investigated. Plant samples helps to provide taxonomic borders with presenting were fixed in alcohol and glycerin (1:1) solution for genus descriptions and identification keys for species. two months. Sections were prepared manually using However, as morphological traits are variable and razor blade and were stained using methyl green and there are some intermediate traits, there are many Bismarck brown. Appropriate samples were ambiguities in real positions of taxa; consequently photographed by Olympus VANOX AHBS3 light keys are not efficient most of the time. On the one microscope. The selection of Characters was based on hand, plant systematic is based on morphology Ellis ( 1976) and Metcalf (1960). (Baum et al., 2004), on the other hand, Poaceae family because of high diversity and structural Tissue samples for scanning electron microscopy complexity makes many taxonomic problems for (SEM) were taken from the basal leaves and lemma of taxonomists who are studying just on morphological middle spikelets. Micrographs with different traits (Strivastava, 1978). Therefore, we decided to magnifying were prepared from abaxial and adaxial taxonomic evaluation of some genera in Poaceae surfaces. According to previous studies and consider filmily. In Flora Iranica, Colpodium Trin. with four to this possibility that indumentums on trichomes species i.e. C. Parviflorum Boiss. and C. humile and epidermis have some differences, sampling was (M.B.) Griseb, C. violaceum (Boiss.) Griseb. and C. performed from epidermis belong to middle spikelets versicolor (Stev.) Schmalh is introduced for Iran ( Abbasi, 2010; Liu, 2010; Acedo, 2001). After (Bor, 1970). At first, morphological studies indicated washing and drying, samples were placed on stapes that, C. violaceum and C. versicolor are similar to and put into Sputter coater to cover them with gold. each other having single-flower spikelets, lack of basal Micrographs were prepared with different

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Int. J. Biosci. 2014 magnifying. Most of micro-graphs were taken using sheath, outer layer of sheath is bigger than inner cell scanning electron microscope Vega-Tescan. layer and smaller than chlorenchyma cells, inner layer cell with thick wall, outer phloem, metaxylem and Numerical analysis: in this section, morphological, protoxylem in center of vascular bundle, concurrent anatomical and micromorphological qualitative and blade, flat on upper and lower surface, fibrous quantitative traits were studied and coded. In sclerenchyma mass on the edge of leaves as pointed anatomical investigations three populations were cap, one row vascular bundle placed in the middle of selected from each species. In the next step, in order blade, lack of collenchyma and segregating cells in to multivariate statistic analyses, qualitative traits mesophyll and having fibrous sclerenchyma stripe were coded from 2 or more states and for quantitative under lower and upper epidermis along with vascular traits, average values were used finally data were bundles, bubble cells with thickness less than ¼ of standardized (Stace, 1989 and Osman, 2011). blade, placed in keel and blade conjunction on lower Classification was done using principle components surface, stomata placed on upper and lower surface analysis (PCA). and lower than epidermis cells, bubble shape epidermis attachments placed in upper surface of Results lower epidermis, lack of trichome on the surface of Anatomical studies upper and lower epidermis. Whereas some characters During anatomical studies, individual variable traits were different between two species used for and diagnostic characters were investigated. diagnostic characters (Table 2). The number of Individual variable traits, which have different anatomical characters that represents similarity quantitative and qualitative traits due to ecological between species of Catabrosella Tzvel. (Tzvel.) are conditions, have no value in segregating species and included: concurrent blade, flat on upper and lower include related traits to blade and keel such as angle surface, fibrous sclerenchyma mass on the edge of between two mid, blade thickness and length, shape leaves as pointed cap, lack of collenchyma and and number of vascular bundle, being complete or segregating cells in mesophyll and having fibrous partial vascular bundles sheath, cell layer number in sclerenchyma stripe under lower and upper epidermis mesophyll part, vascular bundles number and cuticle along with vascular bundles, stomata placed on upper thickness in blades and keel thickness, midrib length and lower surface and lower than epidermis cells, keel and width, distance between midrib and upper and with type one vascular bundle in central, , lack of lower surface of keel, chlorenchyma fibrous stripe Lysogen sinuses and air sinuses, lack of sclerenchyma thickness in upper and lower surface and protoxylem between vascular bundles, Midrib with a bilayer number in keels (Table 1). Traits, which are not vascular sheath, outer layer of sheath is bigger than affected by different ecological conditions and are inner cell layer and smaller than chlorenchyma cells, fixed in different populations, are called diagnostic lack of trichome on the surface of upper and lower characters. These kinds of traits are valuable in epidermis (Figure 2 and 3). Regarding anatomical segregating different species (Table 2 and 3). It is features, species can be separated from each other in notable that these traits in C. violaceum and C. terms of characteristic, presented in Table 3. veriscolor belong to Colpodium Trin. genus are similar to each other ( Figure 1). The number of these Identification keys for Catabrosella Tzvel. based on traits represents similarity between these two species anatomical traits of blade: based on anatomical traits are included; V shape keel 1: Circular keel with circular upper surface………. C. with protuberant upper surface (flat to globular) and parviflora (Boiss. and Buhse) Alexeevex Mill type one vascular bundle in central, fibrous 2: V shape keel with protuberant upper surface or sclerenchyma on lower surface, lack of Lysogen circular……………… C. humilis (Bieb.) Tzvel. sinuses and air sinuses. Midrib with a bilayer vascular 3: upper surface of keel with a fibrous sclerenchyma

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Int. J. Biosci. 2014 stripe, type one vascular bundle in blade having sclerenchyma column narrowing towards vascular Micromorphological observations bundle…………………..……… C. humilis subsp. humilis The leaf blades are usually divided into longitudinal 4: lack of fibrous stripe on upper surface of keel, type zone with the costal zone lying opposite the veins and one vascular bundle in blade with fibrous the intercostal zone present between the veins sclerenchyma stripe placed under upper and lower (Metcalf, 1960; Zhang et al., 2014). epidermis...... C. humilis subsp. Calverti (Boiss.) Tzvel.

Table 1. individual variable traits in Colpodium Trin. Angel between two half of blades 7-12 degree Blade length 0.21-0.52 mm Blade thickness 0.004-0.007 mm Vascular bundle number 1 Vascular bundle shape Oval and globular Keel thickness 0.01 mm Keel abaxial surface sclerenchyma fibrous mass thickness 0.004 mm MIdrib length 0.002-0.003 mm MIdrib width 0.001-0.002 mm Distance between midrib and abaxial surface 0.004-0.009 mm Distance between midrib and adaxial surface 0.006-0.008 mm Outer sheath cell number 1 Mesophyll layer number 1

Table 2. Diagnostic characters in Colpodium Trin based on anatomical studies of blade.

Traits Leaf Angel between Blade Vascular bundle Upper surface Inner Bubble cell shape Chlorenchyma section two half of thickness number in half of keel phloem status Species blades blade C.violaceum(Boiss.) V shape 45 degree 0.129 mm 9-10 Fibrous mass + Triangular Equal Griseb. with low thickness C.versicolor (Stev.) U shape Parallel 0.175 mm 8 Without fiber - Similar and Unequal Schmalh bigger than epidermis cells

In Colpodium Trin. type of indumentums of leaf edge distribution and epidermis cell type are similar in is considered as a diagnostic characters (Figure 4 four species are not considered as segregating traits, and5). Leaf border in C. versicolor have prickle but in whereas some traits such as papilla on dorsal C. violaceum it’s smooth and without any trichome. epidermis surface of blade, cell type of vascular area In addition, epidermis of abaxial surface of lemma and hair density on dorsal surface of lemma are consists of long and rectangular cells in which cell considered as valuable traits in identification of walls are sinuate wall. Junction of these cells, oval or Catabrosella (Tzvel.) Tzvel. genus (Figure 6 and globular verruca indumentums are clearly visible. Table 5). Hairs are considered as identification traits in this genus. In C. violaceum there is no hair while in C. Identification keys based on micromorphological versicolor scattered tubular hairs with bulbous traits in Catabrosella (Tzvel.) Tzvel.: tubular hair are obvious (Table 4). 1: Globular and clavate papilla on dorsal epidermis of vascular and inter vascular area...... C. In Catabrosella (Tzvel.) Tzvel. epidermis studies on Parviflora abaxial surface of leaf and palea indicated that related Dorsal epidermis surface of blade in both areas traits to stomata, long and short cells, stomata without

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Int. J. Biosci. 2014 papilla…………………………………………………..……..…….2 Dorsal epidermis surface of blade in inter vascular 2: Dorsal epidermis surface of blade in inter vascular area consists long, tubular and protuberant area consists long and hexagonal cells which are not cells………………………………………………………………… C. protuberant………………………………………… C. humilis humilis subsp. Humilis subsp. Calvertii

Table 3. Diagnostic characters in Catabrosella (Tzvel.) Tzvel. based on anatomical studies of blade. Angel between two Blade length Blade Vascular Keel shape Upper surface Bundle Bubble cells Species half of blades length bundle of main vein sheath cell number in number half blade C . parviflora - 1.13-1.88 mm 1.13-1.88 8-10 Circular Circular 14 In furrow mm C. humilis 90 degree 1.98 mm 1.98 mm 8-9 V shape Circular 17 - subsp. Calvertii C . humilis 90 degree 2.04-2.38 mm 2.04-2.38 8-9 V shape Flat to 11 - subsp.humilis mm protuberant C . parviflora Fibrous Cut by Circular rib Flat to Less than 1.4 4-5 Triangular Fibrous mass sclerenchyma chlorenchyma circular rib blade column narrowing thickness towards midrib C. humilis Fibrous Cut by flat flat Less than 1.4 7-8 Similar to Without fibrous subsp. sclerenchyma chlorenchyma blade epidermis sclerenchyma Calvertii stripe thickness cells C . humilis Fibrous Complete flat Flat More than 1.4 4-6 Triangular Fibrous subsp.humilis sclerenchyma blade sclerenchyma column narrowing thickness column towards midrib narrowing towards midrib

Table 4. Diagnostic characters based on micromorphological traits in Colpodium Trin. Species \traits Costal zone cells Blade in margin Dorsal surface of lemma C. violaceum (Boiss.) Griseb. Without papilla Flat Without hair C. versicolor (stev.) Schmalh. Clavate papilla Prickle Tubular and long hairs with surface rib base

Table 5. Diagnostic characters in Catabrosella(Tzvel.) Tzvel based on micromorphological triats. Species Blade border Costal zone cell type Intercostals zone cell typr C. Parviflora Globular clavate papilla Globular clavate papilla Long cells with globular papilla C. humilis subsp. Calvertii Dome-shaped papilla Long cells with short cells Long and hexagonal cells and between them stomata C. humilis subsp. Humilis Clavate papilla Short tubular cells and Tubular and long cell with stomata sinuate walls

Discussion performed (Figure 7). The obtained results were in Change taxonomic rank in some species agreement with Tzvel in 1976 by which C. In Flora Iranica, Colpodium Trin. with four species parviflorum and C. humile species separated from i.e. C. Parviflorum Boiss. & Bohse., C. humile (M.B.) Colpodium Trin genus. These species were joined to Griseb, C. violaceum (Boiss.) Griseb. and C. Catabrosella Tzvel genus, in addition, Colpodium versicolor (Stev.) Schmalh is introduced for Iran. humile was considered as subspecies of Catabrosella Therefore we decided to collect some information humilis (Bieb.) Tzvel. This is the first time that from morphology, anatomy and micromorphology anatomical and micromorphology studies are carried studies to evaluation taxa. The data were converted to out in addition to morphology studies. Tzvelev’s numerical data and principle component analysis was studies (1976) were based on morphologic aspects.

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According to anatomic, morphologic and micromorphologic studies as well as numerical data it appears that what in Tzvelev’s studiesis was considered as taxon, C. humilis subsp. Parviflora (Boiss.) Tzvel. is an independent species Catabrosella parviflora (Boiss. and Buhse) Alexeev ex Mill.

Fig. 3. Cross section of leaf in Catabrosella humilis subsp. Calverti. 1 and 2: keel and midrib structure. 3 and 4: type one vascular bundle structure in blade.

Fig. 1. Cross section of leaf in Colpodium versicolor - and Colpodium violaceum. Figure 1, 3 and 5 Colpodium violaceum. 1: leaf shape, 3: keel and midrib structure, 5: bade structure. Figure 2, 4 and 6 C. versicolor. 2: leaf shape, 4: midrib structure, 6: blade and keel structure.

Fig. 4. Scanning electron micrograghs of Colpodium violaceum (Boiss.) Griseb. Figure A: abaxial surface of leaf 50 µm , Figure B: adaxial surface of leaf 50 µm, Figure C: dorsal surface of lemma 20 µm, figure D: dorsal surface of palea 50 µm. V.O: dome- shaped indumentums. LC: long and rectangular cells with sinuate walls. T.C: tubular cells with globular papilla in one end.

Introducing new subspecies for Iran Fig. 2. Cross section of Leaf in Catabrosella Catabrosella humilis subsp.Calvertii (Boiss.) Tzvel. parviflora, C. humilis subsp. humilis. Figure 1, 3 and As a new subspecies is introduced for Iran. Its type is 5: C. Parviflora. 1: leaf shape, 3: midrib and keel from Turkey and has wide distribution. C. humilis structure, 5: blade structure. Figure 2, 4 and 6: C. subsp. Calvertii has the highest similar traits with C. humilis subsp. humilis. 2: leaf shape, 4: midrib and humilis subsp. humilis. Among key morphological keel structure and 6: blade structure. traits for segregating taxa, both of them had dense

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Int. J. Biosci. 2014 hair on dorsal surface of lemma and keels of palea. surface 50 µm, Figure B: abaxial epidermis surface of Study on anatomical and micromorphological traits lemma 20 µm. Catabrosellahumilis subsp. Calvertii: indicated that irrespective of similarity between these Figure C: adaxial epidermis surface of leaf 50 µm, species it is possible to find more segregating traits, Figure D: dorsal epidermis surface of lemma 20 µm. which are shown in table 3 and 5. For example, upper Catabrosella humilis subsp. humilis : Figure E: surface shape of main vein, fibrous sclerenchyma on adaxial epidermis surface of laef 50 µm, Figure F: upper surface of keel, collenchyma in midrib. In dorsal epidermis surface of lemma 20 µm. addition, in micro-morphological studies differences were included; papilla shape on the leaf border and cell type in costal zone and intercostals zone.

Fig. 7. Principle components analysis ( PCA) of 5 taxons.1: Colpodium violaceum (Boiss.) Griseb. 2: colpodium versicolor (Stev.) Schmalh. 3: Catabrosellahumilis subsp. Calvertii (Boiss.) Tzvel. 4: Fig. 5. Scanning electron micrograghs of Colpodium Catabrosella humilis subsp. Humilis. 5: Catabrosella versicolor (Boiss.) Griseb. Figure E: abaxial surface of parviflora (Boiss. and Buhse) Alex. ex Mill., Comb. leaf 20 µm, Figure F: abaxial surface of leaf 30 µm, Nov. Figure G: dorsal surface of lemma 20 µm, Figure H: dorsal surface of palea 50 µm, L.H: tubular long hair, Regarding to anatomical and micromorphological V.O: dome shaped papilla, L.C: long and rectangular features C. humilis subsp. Calvertii can be separated cells with sinuate walls, T.C: tubular cells with from C. humilis subsp. humilis.( Figure 2, 3 and 6). In globular papilla in one end. Turkey Flora (Mill, 1985) this taxon is considered as subspecies unit of Catabrosella parviflora (Boiss.) Tzvel. while our results demonstrate that if anatomical, morphological and micromorphological traits are used in determining real position of taxa, above mentioned taxon should be considered as subspecies unit of Catabrosella parviflora (Boiss.) Tzvel.

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