Orbicules Diversity in Oxalis Species from the Province of Buenos Aires (argentina) Introduction

Eleven Oxalis L. species from the province of Buenos Aires (Argentina) were investigated with scanning and transmission electron microscopes. We identified four different types and two subtypes of orbicules. We conclude that the close morphological similarity between these species is also reflected in their orbicules, and we suggest that the orbicules morphology may be a useful character in systematic studies.


Introduction
observed granular bodies along the inner tangential walls of the tapetal cells in Oxalis rosea, and she pointed out the similarities of these bodies to pollen exine in staining properties.However, until now, ultrastructural and ontegenetic studies of these bodies have been completed for only three species of this genus (Carniel, 1967;Rosenfeldt and Galati, 2005).Kosmath (1927) studied the presence of orbicules in 69 taxa from 35 families.Later, Bhandari and Kishori (1971) considered that the orbicules are composed of sporopollenin because the pollen exine and Ubisch bodies have a specific staining pattern with spirit-soluble aniline blue.
Heslop-Harrison (1968) named these bodies "orbicules", but Bhandari (1984) preferes the term Ubisch bodies because it has been long used in the literature and is well understood both by LM and EM embryologists.
The orbicules can be defined as corpuscules of variable size (0.14 μm-20 μm) that show the same electron density, autofluorescence, resistance to acetolysis, and reaction to the dyes as pollen exine.
The parallelism between the pollen exine and the orbicule wall is explained by Hess (1985Hess ( , 1986) ) as rooted in the homology of tapetum and sporogenous tissue.Because ornamentation of the pollen exine offers useful characters for systematics, orbicules might also have taxonomic value.
The aim of this study was 1) to provide detailed orbicules morphological and ultrastructural descriptions in 11 species of Oxalis with the use of SEM and TEM and 2) to explore the possibilities of employing these characters in taxonomic considerations.

Materials and methods
This study was based on materials from 11 species and two varieties of Oxalis collected from the province Buenos Aires and the city of Buenos Aires, Argentina.For Scanning Electron Microscopy (SEM) studies, the anthers were fixed in FAA (formalin, alcohol, acetic acid), were transferred to 100% acetone and were air dried.The sputtering treatment was made with goldpalladium for 3 min.Scanning micrographs were taken with a Philips XL 30 microscope.
For Transmission Electron Microscopy (TEM) studies, the anthers were pre-fixed in 2.5% glutaraldehyde in phosphate buffer (pH 7.2) for 2 h and then post-fixed in OsO 4 at 2ºC in the same buffer for 3 h.Following dehydration in ethanol series, the material was embedded in Spurr's resin.Fine sections were made on a Sorvall ultramicrotome and then stained with uranyl acetate and lead citrate (O'Brien and Mc Cully, 1981).The sections were observed and photographed with a JEOL -JEM 1200 EX II TEM at 85.0 Kv.

A) Morphology
The orbicules are randomly dispersed on the inner surface of the anther locule.They are plate-like and more or less rounded in most of the species studied (Figs.1A,   C, E, G; 2A, C, E and 3F).
The surface of the orbicules is generally smooth (Figs.1A, C, E and 2A, C).In a few cases, the surface of orbicules has minute verrucoid processes (Figs.3C,  D).Perforations are found in some species (Figs.

1F, G and 2C, E).
There is also considerable variation in the size of orbicules (1-13 μm), even within a single species.
In some cases, the orbicules fuse their corpus, which results in aggregates of two or more orbicules that are observed as rod-like to elongated units with a lightly wavy margin (Figs.3A, C).

B) Ultrastructure
The orbicules of the species studied have one to several central cores and a corpus or wall that is similar to the ektexine in electron-density (Figs.1B, D, H;  2B, D, F and 3B, E, G).The central core has the appearance of a vesicle, transparent to the electrons in medium section (Figs.1B, D, H; 2B, D, F and 3B, E,  G).Its diameter varying between 0.28 μm and 0.8 μm.In some species, the central core is in contact with the inner loculus surface (Figs.1B and 2B, D, F) and the perforations end in the surface of the central core (arrows) (Figs.1H and 2D).
In some species, the surface of the orbicule that is in contact with the microsporangium wall presents irregular invaginations (arrow heads) (Figs.2D, F; 3B,  E, G).
A very electron-dense peripheral layer, which delimits the homogeneous electron-dense orbicule wall, is present in all the species studied (Figs.1B, D; 2B, D and 3B, E, G).This layer is less evident when the orbicules are not fully mature (Figs.1H; 2D, F).

Orbicules types (Table):
Type I: The orbicules are plate-like, more or less rounded, rarely irregular in form, and with a smooth surface.Taxa included: O articulata Savigny: O.lasiopetala Zuccarini, O. floribunda Lehmann (Figs. 1A-E).Type III: The orbicules are plate-like and irregular in shape.The orbicules are fused, resulting in rod-like aggregates.The central cores are small (0.28-0.33 μm) (Fig. 3E, arrow head) and, thus, cannot be observed with a light microscope.
Subtype III A: The surface of the orbicules is smooth to slightly verrucose and the margin is straight.Taxa included: O. hispidula Zuccarini, O. perdicaria Bertero (Figs. 3C-G).
Subtype III B: The surface of the orbicules is smooth and the margin is wavy.Occasional perforations are present.Taxa included: O.debilis Humboldt, Bonpland et Kunth (Figs. 3A, B).

Type IV:
The orbicules are plate-like and have a irregular contour, a smooth surface, and numerous perforations.Taxa included: O. niederleinii Knuth and O. refracta Saint Hilaire (Figs. 1F-H).

Discussion
The orbicules of the species of Oxalis L. studied are morphologically diverse in size and shape.Varia-tion in the size of the orbicules has previously been observed in several species of Euphorbia (El-Ghazaly, 1989;El-Ghazaly and Chaudhary, 1993) and in species of Cinchonoideae (Rubiaceae) (Huysmans et al., 1997).According to Rowley andWalles (1985, 1987) and El -Ghazaly and Nilsson (1991), this variation in the size of the orbicules is due to the cycle of hyperactivity in tapetal cells during their development.
No intraspecific variation was observed, and all the specimens of the same species had the same orbicule morphology.
The general morphology of the Oxalis orbicules is plate-like with a smooth surface.The orbicules have one to several central cores, but multiple cores are not the result of the aggregate of several orbicules.In some species, several plate-like orbicules are aggregated resulting in orbicules that are rod-like or elongated units with a lightly wavy margin.All these characteristics define a new type of orbicules that can be added to Galati's classification (2003).
We observed an electron-dense peripheral sheet that covered the whole orbicular surface.According to Clément and Audran (1993a, b, c), this sheet can be found in each stage of orbicule development.However, in the Oxalis species that we studied, the sheet was not yet developed when the orbicule was not fully mature.Oxalis orbicules can be grouped into four types and two subtypes according to their external and internal morphology.These types correspond with floral and vegetative characters used to separate species into different sections (Lourteig, 1983(Lourteig, , 2000;;Cabrera, 1965).The systematic value of the orbicule types was tested by comparing it to the most recent classification of the genus Oxalis (Lourteig, 2000).In a previous study about the pollen morphology in the same species of Oxalis (Rosenfeldt and Galati, 2007), four pollen types were described as follows: Type I: granules assembled in clusters irregularly scattered on the colpus membrane.Taxa included: O. articulata, O. lasiopetala, and O. floribunda.Type II: colpus membrane with irregularly scattered granules.Strong correlations between pollen exine and orbicule surface ornamentation within the same species have been recorded for other genera (Huysmans et al., 1997;Vinckier et al., 2000;Vinckier and Smets, 2002a, b).However, in the Oxalis species we studied, the ornamentation of the pollen exine and that of the orbicule wall do not show striking analogies.Nevertheless, the Oxalis species belonging to each pollen type coincide with the species belonging to each orbicule type.
We conclude that the close morphological and palynological similarity between species is also reflected in their orbicules, and therefore the orbicule morphology can be used in the taxonomy of the genus Oxalis.

Type II :
The orbicules are plate-like, with smooth surface.They may have no or one perforation.When a single perforation is present, the orbicules are doughnut shaped.The orbicules fuse resulting in aggregates with two or three perforations.Taxa included: O. paludosa Saint Hilaire, O. conorrhiza Jacquin, O .corniculataL. var atropurpurea Planchon and O. corniculata L. var corniculata (Figs.2A-F).
Type I orbicules: O. lasiopetala, O. floribunda and O. articulata belong to the section Articulatae Knuth and are characterized by a cymose inflorescence with numerous flowers, a pink corolla, and an articulate rhizome.Type II orbicules: O. conorrhiza, O. corniculata and O. paludosa belong to the section Corniculatae D.C. and have cymose inflorescence with one to three flower and a yellow corolla.Type III orbicules: O. hispidula, O. perdicaria and O. debilis belong to the section Ionoxalis (Small) Knuth.All these species have bulbs.The first two species are grouped in orbicules subtype A and have a cymose inflorescence with one to three flowers.In contrast, O. debilis has a cymose inflorescence with numerous flowers and a bulb with bulbils.Type IV orbicules: O. niederleinii and O. refracta belong to the section Corniculatae D.C, have a cymose inflorescence with numerous flowers, a yellow corolla, and very branched stem with notable glandular hairs.
Taxa included: O. paludosa, O. conorhiza and O. corniculata.Type III: granules assamble in small clusters more or less aligned on the colpus membrane center.Taxa included: O. hispidula, O. perdicaria and O.debilis.Type IV: granules assembled in comspicuous clusters extensively aligned on the colpus membrane center.Taxa included: O. niederleinii.