Pollen and seed morphology of Zygophylum fabago and Peganum harmala ( Zygophyllaceae ) from Bulgaria

Resumen. Se llevó a cabo un estudio de la morfología del polen y las semillas de poblaciones búlgaras nativas de Peganum harmala L. y Zygophyllum.fabago L. (Zygophyllaceae) con microscopico electrónico de barrido (MEB). Se estableció que los granos de polen en Z. fabago son 3-zonocolporados. La ornamentación de la exina es reticulada. Los granos de polen en P. harmala son óvalo alargados, colporados, exina estriado-rugulada. Las semillas de P. harmala son tetraédricas de forma romboidal, con bordes puntiagudos, arco-curva lateralmente. La escultura de la semilla corresponde al tipo Tubularcóncavo, que se caracteriza con células poligonales y tejado reticulado. Las semillas de Z. fabago son de forma oval-romboidales. La escultura de la semilla corresponde al tipo cóncavo-convexo que se caracteriza con células poligonales, variables en tamaño y con pliegues de cera cuticular.


INTRODUCTION
Zygophyllaceae R. Br. is a large and heterogeneous family that comprises 27 genera and 285 species subdivided into five subfamilies widespread in arid and semi-arid areas in the world (Sheahan & Chase 1996, 2000).
In Bulgaria this family is represented with three taxa: Tribulus terrestris, Peganum harmala and Zygophyllum fabago (Petrova, 1979).The three species are important medicinal plants used as compounds of pharmaceuticals products or as a popular remedy (Kuzmanov et al., 2007;Moloudizargari et al., 2013).
In a palynological aspect, Zygophyllaceae is an euripalynous family (Punt et al., 2007).Therefore, the study of pollen morphology in that family is an important step in the establishment of relationships and dependencies between the comprising taxa.The pollen morphological analysis has a proved taxonomical significance and is successfully used as an additional criterion for delimitation of the taxa (Terziysky & Atanasov, 1977).
From the Bulgarian representatives of the family Zygophyllaceae, the pollen morphology was studied only on T. terrestris (Semerdjieva et al., 2011).
The importance of structural pattern analysis of the seed coat observed under the SEM as a reliable approach for identifying the species and assessing taxonomic relationships has been recognized well (Bartholtt, 1981;Koul et al., 2009;Gammarra et al., 2007).Until recently, the morphology of the seed coat sculpture under the SEM on the representatives of Zygophyllaceae family was studied in Fagonia schweinfurthia, Peganum harmala and Zygophyllum simplex L. from Saudi Arabia (Solman et al., 2010) and T. terrestris from Bulgaria (Semerdjieva et al., 2011).
The present study of the sculpture of pollen and seed of P. harmala and Z. fabago is a continuation of the undertaken investigation of pollen and seed morphology of the Zygophyllaceaee family in Bulgaria (Semerdjieva et al., 2011).The present study aimed to reveal the peculiarities of spermoderm and sporoderm in P. harmala and Z. fabago, and provide important information that will complete the characteristic of the taxa included in the family Zygophyllaceae in Bulgaria.

MATERIALS AND METHODS
The material was collected from native Bulgarian populations of Zygophyllum fabago and Peganum harmala in the town Balchik, near to the Black sea.The specimens were preserved in Herbarium of Agricultural University, Plovdiv (SOA).
The scanning electron microscope (SEM) used in this investigation was a Jeol 5510, which belongs to the Faculty of Chemistry of Sofia University.The samples were covered with gold in a vacuum-evaporator for 60 seconds in an ionizing argon environment.The clamp holder for providing contact was made with silver paste.The method suggested by Terziyski (1981) was followed, namely: the objects were directly observed, without any preliminary physical or chemical treatments, the seeds were observed in air-dry conditions and the pollen grains were preserved in 95% ethanol.For the seed classification, the morphological classes of Thompson (1993) were used.The structure of spermoderm was determined accordingly to the terminology and classification given by Barthlott and Ehler (1977).For the pollen morphology, the terminology by Punt et al. (2007) was used.Additionally, a light microscope (LM) investigation on pollen grains was made in order to elucidate the peculiarities of the main parameters of the pollen surface and size.Measurements were made with an eyepiece micrometer (16x) and a microscope "Amplival", and the pictures were taken with a light digital camera Motic DMBA-210.The data were processed mathematically according to the descriptive statistics method (program Statistica for Windows - Statsofting, 2007).For each indication, 50 measurements were made.

RESULTS
Pollen morphology.The study of pollen morphology carried out revealed the characteristics of sporoderm (exine) in the two studied species (Table 1).The comparative analysis of values of main parameters of pollen surface showed that they differed between two studied species.Regardless of the established differences, concerning the P/E axis type, the pollen grains of P. harmala and Z. fabago fell into the same type -longiaxe (P/E>1.8)(the P/E was 2.15 in P. harmala and 2.30 in Z. fabago).
According to Erdman (1952) the pollen in two species can be defined as small (10-25 µm).
The pollen in Z. fabago was oblong-oval and slightly flattened in the side (incorrect tetrahedral to rhomboid) in shape (Fig. 2a., Fig 5c,d).The apertures were pori, which borders had a thickened walls and a smooth membrane (Fig. 2b.).The exine ornamentation was reticulate with clearly to slightly expressed wavy barriers with simple smooth texture.The brochus had a conically enlarged tip and ranging in size lumen (heterobrochate).The murus measures were from 0.05 µm to 0.19 µm approximately.The brochi were irregular in shape and variable in size (from 0.53 µm to 1.47 µm) (Fig. 2c.).

Seed coat.
Using SEM allowed us a detailed study of the micro-morphology of the spermoderm in P. harmala and Z. fabago.The analyses of the results showed that: according to the classification of Thompson (1993), the seeds of P. harmala were irregular tetrahedral to rhomboid in shape, with a peaked edge (Fig. 3a,b,c).The seeds coat was multilayered, flat to slightly concave, the seeds were laterally arc-curved.On the upper side of the seed surface, the reticulum formed a triangular area of raised cell layer that continued towards the base and defined an area with an irregular polygonal shape.The seed surface was flat to slightly concave, with reticulate tectum.The cells of spermoderm were isodiametric.The anticlinal walls were smooth, with rounded edge and cuticular striations.On the upper surface of the seed, they were higher than the other seed surfaces and formed striations (Fig. 3d).The periclinal walls were wavy folded, with many striations on them.
The shape of the seeds in Z. fabago can be defined as ovalrhomboid according to the classification of Thompson (1993) (Fig. 4a.).The spermoderm cells were polygonal, ranging in size, with epicuticular wax folds (Fig. 4b,c.).On its surface, irregular granulate areas formed.The anticlinal walls were straight to arc-curved, with striation and rounded edge.The external periclinal walls were smooth to arc-curved, with striations and irregularly disposed of small papillae (Fig. 4b,c.).
The disposition of the pores on the whole surface of pollen grains in Z. fabago has given us enough reasons to determine the pollen in this species as pantoporate.Pantoporate pollen was described also in T. terrestris (Yunus & Nair, 1988;Perveen & Qaiser, 2006;Ben Nasri-Ayachi & Nabli, 2009;Semerdjieva et al., 2011) that belongs to the T. terrestris-type of the pollen classification of Perveen & Qaiser (2006).These findings agree with the proposal of El-Atrush et al. ( 2015) that the Peganum species should be placed in the Peganiaceae family preceding the Zygophyllaceae regarding evolutionary trends, and Tribulus species should be placed in the Tribulaceae family following the Zygophyllaceae.
The pollen of P. harmala was defined as colporate (the pori are singles), as it was already described in "Results" On the basis of exine ornamentation, Perveen & Qaiser (2006) distinguished four distinct pollen types in the family Zygophyllaceae: Nitraria retusa-type, Peganum harmala-type, Tribulus terrestris -type and Zygophyllum simplex-type.These authors determined the pollen of Z. fabago as Zygophyllum simplex-types, and pollen of P. harmala as P. harmala-type.Perveen & Qaiser (2006) show that the two types differ only in the ornamentation.However, in the present study, it was established that the two studied species (belonging to the two described types) differed also in the pollen class: tri-colporate for P. harmala and pantoporate for Z. fabago.The importance of pollen morphology in the identification and classification of plants (Doyle & Walker, 1975;Martens & Felz, 1980;Ray, 1983;Blackmore, 1984) has been widely recognized.We established differences in the characteristics of the pollen surface in the two studied species, which corresponded to two different pollen-types.This justifies that the two cited taxa can be assigned to different taxonomic groups.The same suggestion was made by Sheahan & Chase (1996, 2000) and El-Atrush et al. (2015), who recommended to separate Peganum species from Zygophyllaceae.
Seed coat.The shape of seeds is more or less genetically determined, but in some cases, even within the same type, there can exist differences (Werker, 1997).In the present study, we showed characteristics of the shape of seeds of Z. fabago and P. harmala which make them species-specific.These results agree with those established by Zhang et al. (2013): the characteristic of the seed surface in Zygophyllaceae species      is different among the distinct species but is relatively stable within the species.
As noted by Axelius (1992) and Watanabe (1999), the seed surface ornamentation is an important diagnostic feature of seeds that can be used in taxonomic decisions in many families (Esau, 1953;Takhtajan, 1991).An important feature for the spermoderm specificity is the shape of cells of its surface layer, their dimensions, type of anticlinal and periclinal walls, and the presence of wax secretions (Deborah, 2005).The secondary sculpture of the periclinal walls can also be used for diagnosis of the seeds (Hufford, 1995;Koul et al., 2000).The present SEM study of micromorphology of spermoderm of Z. fabago and P. harmala showed that at the submicroscopic level, the seed surface of the two studied species differed considerably.It may be used as a criterion for distinguishing them.In Z. fabago, the surface can be defined as Concave to Convex-type, and in P. harmala as Tabular to lightly Concave-type, according to the classification of Bartholtt & Ehler (1977).The same type of sculpture was determined in T. terrestris seed (Semerdjieva et al., 2011).
This agrees with the consideration of Zhang et al. (2013): the species of Peganiaceae (in which are placed Peganum species from Dahlgren, 1989) could be separated from Zygophyllaceae, on the base of their seed morphological characteristics.

CONCLUSIONS
In the present and previous studies carried out, it was revealed the pollen morphology of the representatives of Zygophyllaceaee family from Bulgarian flora.Despite the isolated    position of T. terrestris from Z. fabago and P. harmala regarding pollen grains sculpture, T. terrestris shows some similarities with the other two species, especially with Z. fabago.Thus, this latter species, typical of the Zygophyllum simplex pollen type, could be considered as a transitional one toward the Tribulus terrestris-type and Peganum harmala-type.
The results of the present study on pollen and seed morphology of the two Zygophyllaceae species support the proposed by others authors separation of Peganum species from Zygophyllaceae in a different family: Peganiaceae.
A comparative analysis of the seed surface studied in Z. fabago and P. harmala, and the previously studied T. terres-tris, shows that the characteristics of the spermoderm of Z. fabago significantly differ from those in P. harmala and T. terrestris.The seed surface of the latter two species showed a similar character (Tabular to slightly Concave-type).Therefore, the submicroscopic characteristics of the seed coat can be used to solve biosystematic problems in the family Zygophyllaceae.