Pollen Morphology of Oxalis Species from Buenos Aires Province (argentina) Introduction

Pollen morphology and the degree of pollen variability within nine species and two varieties of Oxalis species from Buenos Aires Province (Argentina) were studied using light microscopy (LM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Oxalis pollen grain is generally 3-colpate and the shape is prolate spheroidal, oblate spheroidal or spheroi-dal. The exine is microreticulate. The brochi are circular to polygonal; brochi sizes decrease near the colpi. The colpus membrane observed in unacetolysed material, is crustate or granular. The colpus length varies within a species; the largest grains are produced in stamens with long filaments and the smallest ones in stamens with short filaments. Four pollen types are distinguished by means of LM, SEM and TEM. They are recognized mostly on the basis of their colpus membrane structure. In conclusion, the morphological similarity among Oxalis species is also reflected in their pollen morphology.


Introduction
The genus Oxalis L. comprises about 800 cosmopolitan species.
Previous studies of pollen morphology in this genus are meagre and they were conducted using only light microscopy (Erdtman, 1952;Huynh, 1969 a,b) or SEM (Cerceau, 1995).Most of these reports analysed the formation of aberrant pollen.According to the apertures number and position, seven categories of grain aberrations and two succesiform series were identified by Dreyer and Van Wyk (1998) in Southern African Oxalis.Ghosh and Verma (1985) recognized three types of pollen in O. corymbosa DC. and O. dehradunebsis Raizada, and two types in O.corniculata Linn.Pollen morphology of species of this genus from Pakistan and Taiwan (Yueyange Lake) was studied by Perveen and Qaiser (2003) and Wang and Chen (2001), respectively.
The aim of the present study was to provide detailed pollen morphological and structural descriptions and to analyse the degree of pollen variability within Oxalis species from Buenos Aires Province (Argentina) using LM, SEM and TEM.

Materials and Methods
This study is based on materials of nine species and two varieties of Oxalis collected from Buenos Aires Province and Buenos Aires city, Argentina.For scanning electron microscopy (SEM), anthers fixed in FAA (formalin, alcohol, acetic acid) were transferred to 100º acetone and then, air dried.For exine sections, anthers were embedded in paraffin wax.Sections of 3 μm thick were transferred to xilene for ten min and air dried.The sputtering treatment was made with gold-palladium for 3 min.Scanning micrographs were taken with a Philips XL 30 microscope.
For transmission electron microscopy (TEM) studies, the material was pre-fixed in 2,5% glutaraldehyde in phosphate buffer (pH 7,2) for 2 h and post-fixed in OsO 4 at 2ºC in the same buffer for 3 h.Then, it was dehydrated in ethanol series and embedded in Spurr's resin.Fine sections were made on a Sorvall ultramicrotome, stained with uranyl acetate and lead citrate (O'Brien and Mc Cully, 1981).The sections were observed and photographed in a JEOL -JEM 1200 EX II TEM at 85.0 Kv.
The palynological terminology used in this work is in accordance with Punt et al. (1994).Unacetolysed pollen grains from mature but not dehiscent anthers of 10 flowers were mounted in glycerine and the pollen diameter was measured using a light microscope (LM) with a calibrated ocular micrometer.

General morphology and structure
Oxalis pollen grain is generally 3-colpate and the shape is prolate spheroidal, oblate spheroidal or spheroidal.Amb rounded or triangular.The exine is microreticulate .The brochi are circular to polygonal.The brochi sizes decrease near the colpi (Figs.,4,6,8,10,12,14,16,18,20,22).The colpus membrane is granular.The colpus length varies within a species.The largest grains are produced in stamens with long filament and the smallest ones in stamens with short filament (Table 1).
Four pollen types are distinguished by means of LM, SEM and TEM.They are recognised mostly on the basis of colpus membrane ornamentation : Colpus membrane granular.Small granules arranged in an irregular file on the colpus membrane margin.Granules more o less assembled in clusters irregularly scattered on the colpus membrane.
Colpus membrane with granules irregularly and densely scattered.
Colpus membrane with granules arranged in a file on the colpus margin and assembled in central small clusters (Figs. 16,18,20).
Colpus membrane with granules arranged in a row on the colpus margin and assembled in central conspicuous clusters (Fig. 22).

Discussion
In the present study, four pollen types have been distinguished mainly on the basis of the colpus membrane ornamentation.
According to Huynh (1969a), pollen grains from 424 species of Oxalis can be ranged into 13 pollen-types.The species studied in this paper could be referred to the group A of the Huynh's classification according to the general morphology of the pollen grain.This author established types within each group; however, he didn't distinguish between the pollen of the long and short stamen filaments, and the sizes are too variable.For this reason, the pollen studied can not be referred to none of the Huynh's types.The pollen grain wall section is another feature with diagnostic value in the genus Oxalis.In the type I, the exine is thin and the foot layer is from extremely thin to absent .The endexine is continuous.In types II, III and IV, the exine is thick and the endexine is discontinuous or absent .However, the foot layer is thicker in the type II  than in types .Type IV is differentiated from type III by the presence of straight columellae that are arranged equally distant (Fig. 33).
The occurrence of aberrant pollen in the genus Oxalis has been reported previously by Dreyer and Van Wyk (1998).These authors showed that the aberrations always affect the number and arrangement of the colpi, but never alter the exine structure of the grain.
Dreyer and Van Wyk (op.cit) identified 7 types or categories of pollen in the genus Oxalis, based on aperture number and arrangement.Some individual grains of O. corniculata var.atropurpurea could be referred to the category C (6-pantocolpate) (Fig. 7).In O. corniculata var.atropurpurea two types of pollen are observed; one type is 3-colpate and the other one is 6-pantocolpate.In concordance with these observations, Ghosh and Verma (1985) found two types of pollen in this species.
The majority of O. debilis pollen grains are aberrant, with numerous variable forms, and cannot be referred to any category identified by Dreyer and Van Wyk (1998) (Figs. 17-18).This fact might be related to the absence of seed production in this species (Jehlík, 1995).
In a previous study on orbicule morphology in Oxalis species from Buenos Aires Province (Rosenfeldt and Galati, in press), four types were distinguished.The taxa included in each of these orbicule types are in concordance with the ones present in pollen types identified in this work.
The taxa included in the Type I belong to the Section Articulatae, the ones of the Types II and IV are in the Section Corniculatae and the Type III agrees with the Section Ionoxalis (Lourteig, 1983(Lourteig, , 2000)).
Our results indicate that the close morphological similarity among Oxalis species is also reflected in their pollen and orbicules.