Hippeastrum species in areas of restinga in the state of Rio de Janeiro , Brazil : pollen characters

The order Asparagales comprises 14 families, five of which occur in Brazil. Amaryllidaceae is a family of economic relevance and includes numerous ornamental genera. The genus Hippeastrum is widely distributed in Brazil and comprises 34 species, 11 of which occur in areas of restinga (coastal woodland) and Atlantic Forest in the state of Rio de Janeiro. The morphology of Hippeastrum has not been extensively studied in Brazil, where only a few systematic floristic surveys have been carried out with native species. In field studies and reviews of herbarium collections, we identified five Hippeastrum species occurring in areas of restinga in the state of Rio de Janeiro. The five species identified could be distinguished according to the following palynological characteristics: pollen grain size, polarity, pollen units, shape, aperture (number and type), exine sculpture, colpus length and pattern of sexine ornamentation. Of the five species identified, Hippeastrum aulicum Herb. and H. glaucescens (Ker Gawl.) Herb. were not identified in the field. Among the three species that were found in the field, H. striatum had the widest distribution in the study area, whereas the distribution of H. reticulatum was restricted to a single area of restinga, in the Jacone district of the municipality of Saquarema. Through palynological examinations of specimens from herbaria in the state of Rio de Janeiro, we were able to confirm the identity of all five species of Hippeastrum studied. Our data represent a relevant contribution to increasing knowledge of this plant group in the region and will aid in future conservation efforts.


Introduction
The order Asparagales comprises 14 families, five of which occur in Brazil: Amaryllidaceae, Asparagaceae, Hypoxidaceae, Iridaceae, and Orchidaceae (APG III 2009).Amaryllidaceae is widely distributed in temperate and tropical areas worldwide.The family comprises three subfamilies, 73 genera, and 1605 species (APG III 2009).In Brazil, there are 14 recognized genera, comprising 134 native species (Dutilh & Oliveira 2012) in 14 tribes.The genus Hippeastrum belongs to the tribe Hippeastreae (Meerow et al. 2000).Hippeastrum is widely distributed in Brazil, with 34 species, 11 of which occur in areas of restinga (coastal woodland) and Atlantic Forest in the state of Rio de Janeiro (Dutilh & Oliveira 2012).
Amaryllidaceae is a family of economic relevance, including ornamental genera that occur in Brazil, such as Hippeastrum Herb.(Dutilh & Oliveira 2012).The morphology of Hippeastrum has not been extensively studied in Brazil, only a few systematic floristic surveys having been undertaken with native species (Dutilh 1987(Dutilh , 1996(Dutilh , 2005;;Amaral 2007; Alves-Araújo et al. 2009).Information about the morphology and taxonomy of the genus in the state of Rio de Janeiro is still scarce, citations in lists of local floras representing the only records currently available (Araujo 2000;Alves 2001;Argôlo 2001;Braga 2004;Menezes et al. 2005;Bocayuva et al. 2006).
Restingas constitute one of the component habitats of the Atlantic Forest biome, a known biodiversity "hotspot" (Myers et al. 2000).Although restinga habitats were once distributed along most of the coastline of the state of Rio de Janeiro, they have been diminishing at an alarming rate (Rocha et al. 2007) due to the removal of native vegetation for human occupation, the replacement of substrates, the displacement of native vegetation by exotic plant species and the selective extraction of plants with economic value, such as bromeliads, orchids and clusias (Rocha et al. 2007).
In view of the lack of studies focusing on Amaryllidaceae and the genus Hippeastrum in areas of restinga within the state of Rio de Janeiro and the increasing degradation of the original flora, we sought to determine the number of species of this genus existing in those areas.Although many collections of Hippeastrum have been stored in herbaria in the state of Rio de Janeiro during the last century, the preparation of this material often obscures the corona, which is important for species delimitation.We therefore analyzed palynological characteristics relevant to the classification of Hippeastrum species, in order to expand our understanding of this genus in the region and to aid future conservation efforts.

Materials and methods
We conducted regular excursions over a two-year period (from January 2008 to January 2010) in areas of restinga in the state of Rio de Janeiro, observing Hippeastrum species in their natural habitats, determining their geographic distribution, investigating their growth habits, as well as identifying intrapopulation and interpopulation variation.Specimens were identified, photographed and prepared with traditional techniques (Fidalgo & Bononi 1989).The specimens were then deposited in the Herbarium of the Biology Institute of the Federal University of Rio de Janeiro (code, RFA).Duplicates were also provided to the Herbarium of the Botany Department of the National Museum of the Federal University of Rio de Janeiro (code, R).
For palynological studies, we collected flowers or flower buds of Hippeastrum species occurring in the study area from established collections in herbaria in the state of Rio de Janeiro (Appendix 1).The slides prepared during this study were deposited in the Palynology Laboratory of the Botany Department of the National Museum of the Federal University of Rio de Janeiro.
To prepare specimens for analysis under light microscopy (LM), we performed acetolysis with 60% lactic acid (Raynal & Raynal 1971), because the pollen grains showed low resistance to traditional acetolysis.Photomicrographs of pollen grains were obtained with a digital camera (Cybershot DSC-W7; Sony, Tokyo, Japan) coupled to a microscope (Axiostar Plus; Carl Zeiss, Oberkochen, Germany).
For scanning electron microscopy (SEM) analyses, non-acetolyzed pollen grains were spread onto the surface of double-sided carbon tape on aluminum supports.The samples were then transferred to a vacuum chamber, sputter-coated with a thin layer of palladium gold (ca. 2 nm thick) and subsequently observed using a Zeiss scanning electron microscope (SEM) in the Laboratory of Cellular Ultrastructure at the Biophysics Institute of the Federal University of Rio de Janeiro, with a Zeiss scanning electron microscope (DS M960; Carl Zeiss), and in the Laboratory of the Invertebrate Department of the National Museum of the Federal University of Rio de Janeiro, with a JEOL scanning electron microscope (JSM-6390LV; JEOL, Tokyo, Japan).A pollen key was prepared based on the features observed under LM and SEM.
Pollen grain measurements of standard specimens were taken under LM with an ocular micrometer.The largest and smallest equatorial diameters of 25 pollen grains were measured in polar views.Polar and equatorial diameters of ten pollen grains were measured in equatorial views, and ten measurements were made to determine the dimensions of colpi and exine layers.When possible, ten measurements were also made of the lumen of the reticulum (ornamentation).The terminology used in describing pollen shape, size, morphology and ornamentation was based on Punt et al. (2007).
Four specimens of each species were analyzed and compared when possible; one specimen of each species was chosen for statistical treatment and illustration (Appendix 1).To confirm the stability of the reference material data (Mendonça et al. 2010;Carrijo et al. 2011, Lopes et al. 2013), hereafter referred to as comparison material, ten of the same types of measurements were made of pollen grains from another collection.

Results
In areas of restingas in the state of Rio de Janeiro, the genus Hippeastrum is represented by five species (Fig. 1), specimens of which are on deposit in herbaria collections: Hippeastrum aulicum Herb., H. glaucescens (Ker Gawl.)Herb., H. puniceum (Lam.)Kuntze, H. reticulatum Herb., and H. striatum (Lam.)H. E. Moore.Of those five species, only the last three were found in the field during the present study; the first two being represented only by herbarium specimens.

Size
The diameters of pollen grains of the five species evaluated, in polar view and equatorial view, are shown in Tab. 1 As can be seen, the diameters of Hippeastrum aulicum pollen grains were very large (100-110 μm), whereas those of the remaining taxa were classified as large (57.5-77.5 μm).

Exine ornamentation
Most of the Hippeastrum species evaluated showed sexine semitectate, reticulate heterobrochate and sparse perforations, with narrower lumina surrounding the larger ones.Intectate and retipilate exines were seen only in H. puniceum (Fig. 16,17,18,21 and 22).The pila were round, of different diameters and arranged in double rows in an approximately reticulate pattern.The diameters of the "lumina" decreased at the ends of the pollen grains, which were sparsely scabrous (Fig. 22).
The diameter of the lumina decreased at the ends of the apertures in Hippeastrum aulicum (Fig. 7), H reticulatum (Fig. 28) and H. striatum (Fig. 35 and 38), as well as in the median aperture region of H. reticulatum (Fig. 28).Under SEM, the columellae of H. aulicum are clearly visible with straight muri (Fig. 8); in H. glaucescens, the columellae were hardly visible and the muri were sinuous, with scattered granules within the lumen (Fig. 15); in H. reticulatum, the columellae were hardly visible and the muri were straight, with scattered interruptions, whereas the columellae were hardly visible and the lumina showed no granulation (Fig. 29).In H. striatum, the muri were straight and were almost at the level of the lumen, which was densely scabrous (Fig. 36).
Among the reticulate species studied, lumen diameters were largest in Hippeastrum striatum (ca.4.9 μm).Among all of the species studied, lumen diameters were largest in H. puniceum (ca.5.9 μm) and smallest in H. reticulatum (ca.2.4 μm).The largest lumina were found in the central region of the pollen grain in all of the species, lumen diameters decreasing toward the ends (Fig. 7,14,21,28,35,37 and 38).In H. aulicum and H. glaucescens, the sexine was as thick as the nexine and thicker than in the other species (Table 3).The thickest sexine was seen in H. reticulatum (1.6 μm).

Discussion
Our results indicate that, of the five species studied, two (Hippeastrum aulicum and H. glaucescens) are no longer found in their natural environment, probably due to the ornamental value and predatory harvesting of these species.There is considerable taxonomic confusion surrounding the genera Hippeastrum and Amaryllis.However, in an early study, (Herbert 1821) posited that all Amaryllidaceae species occurring in the Americas should be classified as belonging to the genus Hippeastrum, and that all those occurring in Africa should be classified as belonging to the genus Amaryllis.Even today we find erroneous citations of Amaryllis in Brazil, as reported in Zamith & Scarano (2004), who asserted that the genus Amaryllis could be used in order to restore restinga vegetation in the state of Rio de Janeiro.The genus cited by Zamith & Scarano (2004) was most likely Hippeastrum and not Amaryllis.
Among the species evaluated in the present study, Hippeastrum striatum had the widest distribution in restinga vegetation in the state of Rio de Janeiro, whereas the species H. reticulatum was found in only a single area of restinga, in the Jaconé district of the municipality of Saquarema.This finding is worrisome, because it shows that H. reticulatum is vulnerable to local extinction.Due to the difficulty in finding some of these species in the field, we were obliged to use the specimens deposited in herbaria in the state of Rio de Janeiro as sources for palynological studies.This fact showed how the species are vulnerable in this environment.The sizes of the pollen grains of Hippeastrum varied from large to very large, corroborating the results reported by Alves-Araújo et al. (2007); a number of previous studies have reported that, in species belonging to the family Amaryllidaceae, pollen grains are large (Erdtman 1952;Walker & Doyle 1975;Zavada 1983;Roubik & Moreno 1991).
All five of the species analyzed here had heteropolar pollen in monads, a finding that has not been previously described in the literature.As previously reported for Amaryllidaceae (Erdtman 1952;Walker & Doyle 1975;Zavada 1983), we found that all five species showed elliptical pollen grains in polar view.In equatorial view, H. aulicum showed concavo-convex grains, whereas the remaining species showed plano-convex grains.
We found that the apertures of the pollen grains evaluated were monocolpate.The colpi were long and wide, with sharp or rounded ends.This is the most common aperture type among the monocots; within the Asparagales clade, most species have monocolpate pollen grains (Pennet et al. 2005).A recent study confirmed this aperture type in Asparagaceae (Lopes et al. 2013).However, previous studies of Asparagaceae have described bicolpate pollen grains (Dahlgren et al. 1985;Erdtman 1952), with narrow colpi (Roubik & Moreno 1991).Other authors have made observations similar to ours (Alves-Araújo et al. 2007).The fact that Dahlgren et al. (1985) and Erdtman (1952) both examined the genus Amaryllis could explain the apparently conflicting results.
We found that the sexine of H. puniceum was intectate, differing from the semitectate sexine observed in the other four species evaluated.Intectate sexines have also been reported for the genus Amaryllis (Roubik & Moreno 1991), whereas semitectate sexines have been described for the family Amaryllidaceae (Erdtman 1952;Walker & Doyle 1975;Zavada 1983).
Although we found the ornamentation pattern of H. puniceum to be retipilate, that of the other species was reticulate heterobrochate with perforations-the only type of ornamentation previously described for the family (Erdtman 1952;Walker & Doyle 1975;Zavada 1983;Roubik & Moreno 1991;Alves-Araújo et al. 2007).

Figure 1 .
Figure 1.Distribution of Hippeastrum species in areas of restinga (coastal woodland) in the state of Rio de Janeiro, Brazil, based on the specimens examined.

Table 1 .
Diameters of pollen grains of five species of Hippeastrum, in polar and equatorial views.Samples from areas of restinga (coastal woodland) in the state of Rio de Janeiro, Brazil.

Table 2 .
Polar and equatorial diameters of pollen grains of five Hippeastrum species (n = 10 per species), in equatorial view.Samples from areas of restinga (coastal woodland) in the state of Rio de Janeiro, Brazil.

Table 3 .
Means of the measurements of the colpi, exine layers and lumina of pollen grains of five species of Hippeastrum (n = 10 per species).Samples from areas of restinga (coastal woodland) in the state of Rio de Janeiro, Brazil.