Pollen morphology of selected species of Passiflora L. (Passifloraceae) from the Atlantic Forest

Pollen grains from twelve species of the Passifloraceae family from the Brazilian Atlantic Forest were analyzed: Passiflora subg. Astrophea (1sp.), P. subg. Decaloba (1sp.) and P. subg. Passiflora (10spp.). The current study aims to acquire additional information and feature the pollen morphology of the herein studied species to help setting a more precise taxa delimitation. Acetolyzed pollen grains were measured, described and illustrated using light and scanning electron microscopy. The pollen grains were medium or large sized, oblate spheroidal, suboblate, prolate spheroidal and subprolate; 4-colpate (P. kermesina), 6-12-pantocolporate (P. mediterranea), 6-colpate (P. mucronata), 6-colporate (P. pentagona), 12-colporate (P. misera) or 6-syncolpate (in most species). The presence of reticulate sexine, pseudopercula, pontopercula and/or opercula was observed. The endoaperture was just found in P. pentagona and P. misera. It was concluded that pollen morphology is an important source of taxonomic features useful for distinguishing species and characterizing the three subgenera. The current study provides additional information that, along with other previously published studies, will enable a better understanding of phylogenetic relations among these strains.

Brazil hosts approximately 150 Passifloraceae species, 87 of them are endemic (Bernacci et al. 2014).The State of Rio de Janeiro is considered to be one of the greatest fauna and flora endemism centers in the country (Bergallo et al. 2000;Rocha et al. 2003).According to Bernacci et al. (2014), the state is represented by 41 species.
Most of the herein analyzed species (Passiflora alata, P. amethystina, P. edulis, P. farneyi, P. foetida, P. kermesina, P. mediterranea, P. misera, P. mucronata, P. pentagona, P. racemosa and P. setacea) can be found in sandbanks in the State of Rio de Janeiro.These species show wide distribution and are found in almost all Brazilian regions, except for Passiflora kermesina, P. pentagona and P. mucronata, which are restricted to the Southeast region of the country and P. farneyi, which is just found in the State of Rio de Janeiro (Bernacci et al. 2014).
The Atlantic Forest is an important "hotspot", i.e., one of the richest and most threatened biological regions in the world; its endemic rate is particularly high (Mittermeier et al. 2000).As already highlighted by Candido et al. (2013) as well as by preceding authors (Myers et al. 2000;Rocha et al. 2007), "although restinga habitats were once distributed along most all the coastline in the State of Rio de Janeiro, they have been diminishing at alarming rates due to native vegetation removal for human occupation purposes".
The current study aims to feature pollen morphology and identify informative characters that may help increasing knowledge on the genus and subgenus of this plant group.It also aims to study the phylogeny of the group and to gather helpful information to further conservation efforts.

Material and methods
The current study investigated pollen grains of 12 Passiflora species, subgenus Astrophea (1 sp.), P. subg.Decaloba (1 sp.) and P. subg.Passiflora (10 spp.).The herein used botanical material consisted of flowers in anthesis and /or flower buds picked from specimens deposited in different herbaria in the State of Rio de Janeiro; their acronyms were related according to the Index Herbariorum (Thiers, continuously updated).
Material used in the study: Standard specimens marked with an asterisk (*) next to the collector's name were chosen for each studied species.Three specimens were chosen for comparison to corroborate the results.The following criteria were adopted to choose the standard material: being preferably collected in the State of Rio de Janeiro and being identified by an expert on the family.The used slides were deposited in the Palynology Collection of Álvaro Xavier Moreira Palynology Laboratory, in the Botany Department at National Museum of Federal University of Rio de Janeiro.
The pollen samples to be analyzed under light microscopy were prepared according to the acetolytic method by Erdtman (1952), which was modified by Melhem et al. (2003); and measured after seven preparation days (Salgado-Labouriau 1973).
Twenty-five (25) polar diameter (PD) and equatorial diameter (ED) measurements of pollen grains in equatorial view, 10 equatorial diameter measurements and measurements of the side of the apocolpus -both in polar view (EDPV) -were performed.The grains were distributed among three slides.The statistical analysis was done by calculating the arithmetic mean (x), sample standard deviation (s), mean deviation (s x -), the coefficient of variability (CV%), the confidence interval of 95% and the variation rate.Ten (10) pollen grains were measured to set other features such as apertures and exine layers.Ten (10) similar pollen grain measurements were obtained from additional material deposited in another collection (from this point on referred to as comparison material).Such measures were taken to check the stability of data collected from the reference material (Abreu et al. 2012;2014;Moreira et al. 2013).
The terminology herein used meets that by Barth & Melhem (1988), Punt et al. (2007).It takes into account the size, shape and ornamental pattern of sexine.The definitions by Presting (1965) and Dettke & Santos (2009) were applied to apertures description.
Non-acetolyzed pollen grains were assembled on stubs with double-sided carbon tape and sputter-coated (for 3 min) with a thin palladium-gold layer.The photomicrographs were taken under scanning electron microscopy using the JSM-5310 microscope (JEOL, Ltd., Tokyo, Japan), in the Electron Microscopy Center of Invertebrates Department at National Museum of Federal University of Rio de Janeiro.
The palynological description was arranged according to the following pollen features (Figs. 1 e 2): size; polarity; dispersal unit; size, shape, and type of apertures; opercula types; exine sculpture.The results were presented in a single description and summarized in Tables 1 to 5.

Polarity, dispersal unit, size and shape
Isopolar pollen grains were found in most of the species and the apolar grains were found in P. kermesina and P. mediterranea (P.subg.Passiflora) (Tabs.2-4, Fig. 2); they were considered to be large sized in most of the species.Medium sized pollen grains were just found in P. pentagona.Just five species were analyzed in equatorial view.Their polar and equatorial diameters were measured and shapes established.Thus, the suboblate shape was found in P. edulis and P. mucronata; oblate spheroidal in P. alata, prolate spheroidal in P. misera and subprolate in P. pentagona.Passiflora pentagona presented the smallest polar (48.6 m) and equatorial diameters (42.6 m); whereas P. edulis showed the largest polar (63.0 m) and equatorial diameters (67.5 m), in equatorial view (Tab.2).P. pentagona showed the lowest equatorial diameter, in polar view (44.2 m) and P. amethystina showed the largest one (87.4m); P. farneyi had the lowest value related to apocolpium side (14.3 m) and P. amethystina showed the largest one (25.0m) (Tab.4).P. kermesina species had the lowest values in D1 and D2 (Tab.3).
The scanning electron microscopy showed that muri are supported by conspicuous columellae (except in P. misera and P. pentagona).The upper part of the muri was psilate, in all studied species.

Discussion
The 12 species subordinated to the three palynologically studied subgenera could be initially separated into two major groups according to apertural type (colpate and colporate).The group with colpate aperture can be syncolpate and it coveres most of the species: apolar colpate (P.kermesina and P. mediterranea) or 6-colpate (P.mucronata); all species with colpate aperture belong to the Passiflora subgenus.The other group consists of two species, P. misera and P. pentagona, which belong to the Decaloba and Astrophea subgenera, respectively.P. mis-era pollen grains are 12-colporate with 6 endoapertures, whereas P. pentagona pollen grains are 6-colporate with 3 endoapertures.
The exine in most of the species presented large and very ornamented lumina.P. setacea was different, because it had a bigger amount of densely distributed bacula in the lumina in comparison to other species.Passiflora mucronata stood out for the presence of perforations; a feature not found in other species from the Passiflora subgenus.The ornamentation in P. misera and P. pentagona species is similar because these species show reticula with smaller and little ornamented lumina and perforated muri.The herein elaborated pollen key allowed separating the species among subgenera, although some attributes were found in more than one subgenus.Passiflora subg.Passiflora showed apolar and isopolar pollen grains, besides syncolpate (one species) or non-syncolpate apertures.Passiflora subg.Astrophea and Passiflora subg.Decaloba were close in the key.However, they were separated from Passiflora subg.Passiflora, due to these species colpori-type aperture.Other important features that allowed separating the species regard details of lumen ornamentation, muri sinuosity, polar area and apertures size as well as exine thickness.
Previous studies showed great controversy in describing the types and number of apertures in genus Passiflora (see Tab. 6).Such difficulty concerned terminology (colpus, colporus, operculum types) and the level of observation (light microscope x scanning electron microscope).Some of these differences could be especially seen in older articles.
The 6-colporate aperture type found in P. pentagona by Mezzonato-Pires ( 2013) was the same one found by the current study.
P. farneyi is an endemic species in the State of Rio de Janeiro (Bernacci et al. 2014), and it is not easily found in the field.Its first pollen grain analysis was done in the current study.
As it was already emphasized by other authors, it is worth analyzing pollen morphology under transmission electron microscope as well as studying its ontogeny to confirm whether or not and when aperture fusion takes place in pollen grains from this family.
As it was evidenced in previous studies, pollen morphology in the Passifloraceae family is very important for the taxonomy of the group.The family is eurypalynous and in-depth knowledge of its features will enable assessing the constituencies and the organization of its infrageneric categories, since such attributes have been completely recorded.The current study is another source of information that, along with other previously published studies, will enable better understanding the already known phylogenetic strains.

Table 5 .
Measures (in m) of the apertures, murus width and lumen length of Passiflora species pollen grains (n=10).