Morphoanatomical study of some species from Liliaceae Juss. Family in Ghurfa-Adhaim district

Four species from Liliaceae family were studied: Bellevalia glauca (Lindl.) Kunth. , Bellevalia kurdistanica Feinbrum , Gagea reticulata Salisb. and Ornithogalum brachystachys C.Koch, morphologically and anatomically. Plants were collected from Gurfa-Adhim district between March and May, 2016. Bulb, scape, stem leaves, inflorescence and flowers were examined and variations in size, shape and dimensions were recorded. The results revealed close morphological relationship between Bellevalia galuca and B. kurdistanica in characters: bulbs, stem leaves and stamens, absence of cambium and xylem fibers in outer part of vascular bundle; meanwhile, the two Bellevalia species are varied from Gagea reticulata and Ornithoigalium brachystachys in morphological and anatomical features.


Introduction
Liliaceae is a monocot family plants, with 250 genera and more than 3,500 species distribute in southern hemisphere (Africa, South America, Australia, and India) and in East Asia [1].More than 460 species belong to 36 genera present in Turkey [2]; 21 genera represented in Iraq [3], with high variation in morphological characters regardless genomic correlation between species [4].The family characterized by bulb or rhizome root, tepals (not distinguish calyx from corolla) colour which grouped in two cycle, superior ovary and fruits capsule [5].Different Liliaceae species are cultivated and using as food such as onion Allium sativum L. and garlic Allium cepa L., other beautiful ornamental species are cultivated all over the world, but many species are wild plants.Several Liliaceae plants used as traditional medicinal uses such as bulbs of Fritillaria roylei Hook.used in tuberculosis [6], and Hosta ventricosa used as mastitis, otitis media, folliculitis and snake bites [7].The genus Gagea Salisb.comprise 200 species distributed in Mediterranean region [8], 9 species in Iraq are found: G. confuse A., G. anisanthos C.Loch., G. dubia A.Terr., G. linearifolia A. Terr., G. commutate C.Koch., G. reticulata (Pall.)J. Aet J., G. chloranthus (M.B.) J. A. etJ.H. Shultes, G. uliginosa Siche et Pascher and G. gageoides (Zucc.)Vved.[3].Taxonomically, the genus Gagea L consisted of two subgenera: Gagea (Eugagea Pascher) with spherical seed, and subgenera Hornungia (Bern.)distinguished by triangular seed shape [9].Many morphological characters were used to identify plant species of lily family, like outer and inner tepals of Lloydea serotine (L.) Salsb.ex.Reich.[10]; bulb size, bracts and pedicel of Gagea Saliasb.[11]; gynoecium character is anatropous bitegmic of Gagea villosa (M.Bieb.)Duby.[2];pollen morphology characters includs size and exine sculpture of Erythronium caucasicum, E. gegantum and E. sibiricum [12]; pollen shape monosulcate heteropolar subprolate of Fritillaria mughlae M.Tesen & Aytac [13].Anatomical characters also used in Liliaceae species that phloem elements below the endodermis layer are arranged in single layer alternating with pericycle cells of Lilium candidum L. [14]; root and leaf sections were used in identification of Hyacinthella glabrescens (K.Koch) Schur.[15].Embryonic feature is trilocular ovary with anatropous ovule in each and cellular characters of plastids of Gagea bohimica (Zauschn.)Schult.&Schult.F.[16].Some rare species of Liliaceae such as Tulipa akamasica Christodoulou, Hand & Charalambous exists in small area in Cyprus which morphologically characters are illustrated as globose-elongate bulb 17-25 × 12-18 mm in dimension and red bright distinct tepals of solitary flowers [17]; chromosome number is hexaploid of sterile plant and the reproductive (flowering) depends on bulbils of Gagea lutea (L.) Ker Gawl and Gagea spathacea (Hayne) Salisb [18]; systematic revision of Gagea villosa [19]; phylogenetic study based on plastid DNA of 7 Gagea species in Germany revealed close related between species rather than diversification [20].The genus Bellevalia Lapeyr. is a member of subfamilies Scilloideae (Engler, 1887) which is closely related to the genus Muscari Mill.within the family Liliaceae [21], 6 species of Bellevalia were used to investigate their relationship based on karyotypic and was found both diploid and tetraploid variation between the studied species [22] and [23].Recently, Bellevalia considered from Hyacinthaceae/ Aspargaceae family [24].The genus Bellevalia of about 65 species, 10 species found in Iraq [3].The genus Ornithogalum L. consists of 150 species, only 8 species in Iraq [3].Several studies dealt with Ornithogalum species and described morphological features such as seed micromorphology of Ornithogalum species [25]; ploidy levels of chromosome number of Ornithogalum umbellatum [26]; 31 morphological analysis of Ornithogalum using dichotomous key [27]; as well as study of cytotoxic effect of Ornithogalum cuspidatum Bertol.on Prostate Cancer [28], and antioxidant action of phenolic and flavonoid compounds of bulb and vegetative part extracted from Ornithogalum sintenisii L. [29].However, wild species from Liliaceae family in DGA (Ghurfa-Adhaim district) in Iraq are not examined; the aims of this study are to understand the variations, diversity, species limitation and relationships based on morphological and anatomical attributes of species included in the this study.

Materials and methods
Plant samples were collected from Ghurfa-Adhaim district, particularly from West of Amirli town, and in Adhaim dam near Tigris River at altitude of 110-160 m with limestone soil, no dense vegetation and absence of tree species.The collected plants were dried by using newspaper.Measurements were recorded after two weeks.The changes in the colour of leaves and bulbs were noticed after collecting, morphological characters were observed using dissecting binocular microscope type Stereo TS-70 made in Germany.For anatomical study, cross sections of leaves and stem were made [30] by razor blade, the sections were placed in Ethanol 90% for 5 minutes, then washed in D.W., they stained with safranin stain was used, then mounted with 50% Aqueous Glycerine, coverslip were placed, the measurements by micrometres were taken using Altay binocular microscope made in Italy, and photos were taken by digital camera type Sony.Stomatal index and stomatal frequency were measured as follow: Stomatal frequency = Number of stomata in one microscope field × 40

Results
The taxonomic descriptions of the studied taxa as follows (Table 1,      Bellevalia kurdistanica: this species demonstrate similarities in anatomical characters with the B. glauca, but only with thinner collenchyma tissue below the hypodermis layer cells which 3-layers cells arranged.The leaf anatomy shows similarities of that lower surface (abaxial surface) with anomocytic type of stomata complex in both adaxial and abaxial surfaces, stomatal index 24 and stomata frequency 3.9%, with two of mesophyll layer.The vascular bundle in a single row with uniseriate of upper epidermis, and the cell wall with less cuticle thickness.
Gagea reticulata: the anatomical characters of this species shows similarities with the B. glauca, only with thinner collenchyma tissue below the hypodermis layer cells which 4-layers cells arranged.Stem contain vascular bundles distributed in the ground tissue, in the inner parts the xylem parenchyma and tracheids, in the outer parts the phloem tissue consists of phloem sclerenchyma, phloem parenchyma and companion cells.The cross section of leaf demonstrate that both abaxial and adaxial surfaces with anomocytic type of stomata complex, stomatal index 34 and stomata frequency 8.8%, with two of mesophyll layer.The vascular bundle in a single row, and with uniseriate of upper epidermis contain less thick cuticle.
Ornithogalum brachystachys: the anatomical characters of this species shows similarities with the B. glauca, only with thinner collenchyma tissue below the hypodermis layer cells which 4-layers cells arranged.The cross section of stem reveals that vascular bundles distributed in the ground tissue randomly, the xylem parenchyma and tracheids in inner part of the bundle, the phloem tissue consists of phloem sclerenchyma, phloem parenchyma and companion cells in the outer part of the bundle.The cross section of leaf demonstrate that both abaxial and adaxial surfaces with anomocytic type of stomata complex, stomatal index 31 and stomata frequency 7.6%, with two of mesophyll layer, and the vascular bundle in a single row, and with uniseriate upper epidermis contain thick cuticle.

Table 1 )
: the short stamens and anther in B. glauca (3 mm length, 1 mm width), in O. brachystachys 6-6.2 mm length and 2 mm anther length; also variations in stamens colour revealed from black purplish in B. glauca to white in O. brachystachys.Pistil is small 3 × 1.5 mm, but bigger in O brachystachys 5 × 2-2.6 mm.The anatomical characters reveals variation between the studied taxa (Figs.3; 4 & 5), however the most important anatomical character in most Liliaceae family species in monocotyledon plant is the presence of stomata in both upper and lower surfaces, with more stomata dense in lower surfaces [36].Thick cuticle layer in B. glauca and O. brachystachys, but in B. kurdistanica and G. reticulata with thin cuticle, this character considered significance anatomical distinguish feature among the species [37] & [38].The hypodermis is distinct multilayer in B. glauca, G. reticulata and O. brachystachys, only B. kurdistanica is varied with only 3 layers of hypodermist.Thick layer of collenchyma tissue present in B. glauca, meanwhile thinner collenchyma is present in both G. reticulata and O. brachystachys, the significance character is recorded for the first time in this study in B. kurdistanica with sclerenchyma tissue replaced collenchyma tissue, this could be high variation feature due to ecological effect and isolation [39].