Petal and Sepal Epidermal Micromorphology of Six Lathyrus Taxa ( Fabaceae ) and their Systematic Value

In this study, the epidermal types and their distribution on dorsal and lateral petals, the trichome types and their density on sepals of Lathyrus chloranthus , L. digitatus , L. laxiflorus subsp. laxiflorus , L. roseus subsp. roseus , L. sativus, and L. tuberosus , belonging to sections Lathyrus, Lathyrostylis, Pratensis, and Orobon of the genus Lathyrus in Turkey were investigated using light microscopy (LM) and scanning electron microscopy (SEM), and the systematic significance of these characters was evaluated. These taxa, except L. sativus, are studied for the first time under aspects of the petal and sepal micromorphology. Three major epidermal types were recognized on the petal: tabular rugose striate cells (TRS), areolate cells with more or less striations (AS), and papillose conical striate cells (PCS). TRS and AS were further subdivided into three subtypes. TRS was found on the dorsal and lateral petals of L. chloranthus , L. sativus, and L. tuberosus : the dorsal petals of L. roseus subsp. roseus and the lateral petals of L. laxiflorus subsp. laxiflorus , while AS was present on the dorsal and lateral petals of L. digitatus and the lateral petals of L. roseus subsp. roseus . PCS was found only on the dorsal petals of L. digitatus . Three main types of trichomes on the sepal were observed: peltate glandular, capitate glandular, and nonglandular trichomes. The capitate glandular and nonglandular trichomes were further subdivided into three subtypes. The peltate glandular trichomes were present only in L. chloranthus , but absent in the others. The capitate glandular trichomes were found in L. chloranthus , L. laxiflorus subsp. laxiflorus , L. roseus subsp. roseus, and L. tuberosus . The nonglandular trichomes were always present in L. chloranthus and L. laxiflorus subsp. laxiflorus . The present results show that the petal and sepal micromorphology can be used in delimitation of the taxa based on petal and sepal micromorphology.


Introduction
Lathyrus L. (Fabaceae) is the largest genus in the economically important tribe Fabeae of the subfamily Papilionoideae, with approximately 170 species worldwide (ILDIS, 2010;Lewis et al., 2005). The eastern Mediterranean region is the main center of diversity for the genus, which is less diversified in North and South America (Kupicha, 1983). Many species of the genus are found throughout the temperate northern hemisphere (Asmussen and Liston, 1998;Kupicha, 1983). Moreover, a few species are distributed in the tropical zones and the highlands of East Africa (Davis, 1970;Kenicer et al., 2005;Senn, 1938). Lathyrus species are economically important as food and fodder crops, ornamentals, soil nitrifiers, dune stabilizers, significant agricultural weeds, and model organisms for genetic and ecological research (Chittenden, 1951;Kenicer et al., 2005).
The epidermal surface of petals is significant in pollination because it affects the way that pollinators perceive and interact with the flower (Ojeda et al., 2009). Many legume species have a specialized flower morphology that promotes pollinator specificity (Ojeda et al., 2009). In Compositae (Baagoe, 1977(Baagoe, , 1980Hansen, 1991) and Fabaceae East Technical University (METU), Ankara. The voucher specimens are listed in Tab. 1.
Petals and sepals of fully opened flowers were investigated using a Leica DM1000 LM and a QUANTA 400F SEM. After a number of specimens had been compared under LM, three to six petals and sepals were examined for each species under SEM. Two kinds of petals (a dorsal petal, called a 'standard' and two lateral petals or 'wings') were examined. The epidermal types of the petals were classified based on cell size, shape (the primary sculpture), and on the fine relief of the cell wall, using the terminology of Barthlott (1977) and Kay et al. (1981). To investigate trichomes on the sepals, sections were made by hand using commercial razor blades. The trichome types and densities are described. The general trichome terminology follows Metcalfe and Chalk (1957) and Kahraman et al. (2010).
Type 1. Tabular rugose striate cells (TRS): This type was further subdivided into three subtypes based on several properties, such as cell size, shape and density of striations.
Type 2. Areolate cells with more or less striations (AS): This type was further subdivided into three subtypes based (Stirton, 1981) the properties of the petal epidermal types have been used for taxonomic and phylogenetic analyses.
Studies on the petal and sepal epidermal micromorphology of leguminous species are rather limited (Christensen and Hansen, 1998;Hammett et al., 1994;Ojeda et al., 2009;Stirton, 1981). Stirton (1981) examined the lateral petals of the papilionoids. Hammett et al. (1994) reported the epidermal type of the dorsal and lateral petals in 2 Lathyrus species, and Ojeda et al. (2009) reported the epidermal types on the dorsal, lateral, and ventral petals of 7 species of the same genus. Except for L. sativus, the petal, and sepal micromorphology of L. chloranthus, L. digitatus, L. laxiflorus subsp. laxiflorus, L. roseus subsp. roseus, and L. tuberosus have not been analyzed previously. Therefore, this study aims to investigate the epidermal types and their distribution on dorsal, and lateral petals and the trichome types and their density on sepals of these six taxa from four sections, namely Lathyrus, Lathyrostylis, Pratensis, and Orobon distributed in Turkey, and to evaluate the systematic value of these characters.

Material and methods
Specimens of the six Lathyrus taxa studied were collected from their natural habitats in Central Anatolia, Turkey. The collected specimens were deposited in the herbarium of Department of Biological Sciences, Middle on several properties, such as cell size, shape and waviness of the cell margin. Subtype AS i : Elongated cells with straight anticlinal walls. Observed in the dorsal petals of L. digitatus (Fig.  3). Subtype AS ii : Isodiametric cells with straight or weakly curved anticlinal walls. Observed in the lateral petals of L. roseus subsp. roseus (Fig. 11-12). Subtype AS iii : Isodiametric cells with curved anticlinal walls. Observed in the lateral petals of L. digitatus (Fig. 4).  (Fig. 5-6).
The peltate glandular trichomes had a basal epidermal cell, one neck cell and multicellular secretory head. They were found in the grooves of the calyces of L. chloranthus (Fig. 17, 18), but absent on those of the others.
The capitate glandular trichomes had a basal epidermal cell, unicellular stalk, a neck cell and a cutinized secretory head. They were subdivided into two subtypes on the basis of their structure: (i) composed of a unicellular globose secretory head. This subtype was found only in L. chloranthus (Fig. 17-19). (ii) composed of a multicellular oval sectetory head. This subtype was mainly situated on the sepal teeth of L. roseus subsp. roseus and L. tuberosus and the sides of the sepal ribs of L. laxiflorus subsp. laxiflorus (Fig. 22-25, 27, 28).
The nonglandular trichomes were subdivided into two subtypes on the basis of their structure: (i) composed of short acicular epidermal cells. This subtype was always present in L. chloranthus and L. laxiflorus subsp. laxiflorus, but very rare or absent in the others; (ii) composed of long filamentous epidermal cells. They were situated on the teeth and ribs of the calyces of L. chloranthus and L. laxiflorus subsp. laxiflorus (Fig. 17-20, 22).

Conclusions
Little is known of the petal and sepal micromorphology of the genus Lathyrus. Therefore, the present study provides a more extensive knowledge of petal and sepal surface properties of the genus. Lathyrus taxa can be separated on the basis of the petal and sepal micromorphology. Different petals within a flower of Lathyrus can also be separated micromorphologically.
lateral, and ventral petals occurred, but found only smaller differences among Lathyrus species. TRS was found on the three petal types of some species, such as L. japonicus, L. latifolius, L. sativus, and L. sylvestris. In L. venatus, the dorsal and lateral petals had papillose knobby rugose cells (PKR), whereas the ventral petals had TRS. In L. vernus, the dorsal and ventral petals had TRS while the lateral petals had PKR. The present study showed that there was micromorphological variation between the dorsal and lateral petals of the taxa. TRS was the dominant epidermal type in the dorsal and lateral petals of the taxa. PCS and AS epidermal types were observed in L. digitatus, L. laxiflorus subsp. laxiflorus, and L. roseus subsp. roseus, but PKR was not recognized in any taxa. The dorsal and lateral petals of L. chloranthus, L. digitatus, L. sativus, and L. tuberosus had the same major micromorphology. However, this was not the case in L. laxiflorus subsp. laxiflorus with PCS on the dorsal petal and TRS on the lateral petal; and L. roseus subsp. roseus with TRS on the dorsal petal and AS on the lateral petal.
L. chloranthus, L. sativus, and L. tuberosus had the same main epidermal type (TRS) on their dorsal and lateral petals, however, minor differences in cell size, shape (isodiametric or elongated), and density of striations allow clear petal micromorphological identification of these taxa. TRS was subdivided into three subtypes: TRS i , TRS ii and TRS iii . TRS i was found on the dorsal and lateral petals of L. sativus and the dorsal petals of L. tuberosus, but TRS ii was found on the dorsal and lateral petals of L. chloranthus and the lateral petals of L. tuberosus.
Among the taxa L. digitatus and L. roseus subsp. roseus had AS cell type, which was differentiated by cell size, shape (isodiametric or elongated), and waviness of the cell margin. AS i was subdivided into three subtypes: AS i , AS ii and AS iii . AS i was observed on the dorsal petal of L. digitatus, AS ii on the lateral petal of L. roseus subsp. roseus and AS iii on the lateral petal of L. digitatus.
Molecular genetic studies show that each petal in a papilionoid legume flower has a distinct molecular identity that may be marked by epidermal type (Cronk, 2006;Weng et al., 2011). Many studies have focused on the molecular genetics of PCS cell type. MIXTA, an MYB transcription factor, is responsible for the differentiation of PCS in Anthirrinum majus and in some species of Solanaceae (Glover and Martin, 2002;Noda et al., 1994). However, there is not yet any evidence that MIXTA plays a role in PCS differentiation in legumes (Ojeda et al., 2009). In Lotus japonicus, the genes, CYCLOIDEA 2 (LjCyc2) and KEELED WING 1 (KEW1 or LjCyc3) are associated with dorsal and lateral petal identity, respectively (Weng et al., 2011). Now, there is no information about the precise pathway by which petal identity genes promote the differentiation of the epidermal types described in this study.
Legumes show a variety of uni-and multicellular trichomes, both secretory and nonsecretory, on the vegetative (Franceschi and Giaquinta, 1983;Retallack and