Comparative pollen morphological analysis in the subgenera Passiflora and Decaloba

ABSTRACT

The genus Passiflora is the most diversified of the Passifloraceae, and its palynology presents wide morphological variability. The objective of the study was to evaluate the pollen morphology of 18 Passiflora species in order to identify informative pollinic characteristics to contribute to the taxonomic classification of the genus. The morphology of the pollen grains and the exine structure were investigated using light microscopy and scanning electron microscopy. Differences in the pollen morphology were found in the studied species, mainly in terms of shape, pollen aperture and ornamentation pattern of the exine. Most of the species belonging to the subgenus Passiflora presented ornamented 6-syncolpate pollen grains with an oblate-spheroidal shape and an exine with large lumens. In the subgenus Decaloba the pollen grains were 6-colporate, 12-colpate and 12-colporate, with subprolate to prolate-spheroidal shape, as well as an exine with smaller lumen size and few ornamented. The pollen morphology of the species of the subgenera Passiflora and Decaloba has shown palynological characteristics that have specific diagnostic value, thus allowing a better understanding of the taxonomy of the genus Passiflora.

Key words:
Passifloraceae; pollen grains; scanning electron microscopy; exine; morphological characterization

INTRODUCTION

The genus Passiflora L. is the most representative of the Passifloraceae, with approximately 576 species (Ocampo and D’Eeckenbrugge 2017) from the tropical and temperate regions of South America (Krosnick et al. 2013). Brazil has approximately 150 species, and most of them are endemic in the country (Bernacci et al. 2015). This genus was initially divided into 23 subgenera according to Killip (1938) and subsequently reduced to only five: Astrophea (DC.) Mast., Deidamioides (Harms) Killip, Decaloba (DC.) Rchb., Tetrapathea (DC.) Reichb. and Passiflora L. (Feuillet and MacDougal 2003, Krosnick et al. 2009). However, the two largest subgenera are Passiflora (~ 250 species) and Decaloba (~ 230 species) (Porter-Utley 2014).

Due to the great morphological diversity, pollen grains have been used for taxonomic purposes, assisting in the morphological characterization and identification of some species to generate phylogenetic information that more accurately expresses the origin and evolution of the organisms (Judd et al. 2007). The pollen characteristics are fundamental for taxonomic classification because the pollen grains present the shape, size, color and ornamentation of the exine defined for each species, genus and family (Mert 2010, Fazal et al. 2013, Silvério and Mariath 2014, Mezzonato-Pires et al. 2015a, Silva et al. 2016).

Passifloraceae is very interesting from the palynological point of view because the pollen grains present great phenotypic variability for several characteristics, some that have still not been explored according to systematic and phylogenetic aspects (Dettke and Santos 2009). Mohl (1834) and Fritzsche (1837) were the first researchers to use the pollen morphology of Passiflora species for taxonomic purposes, highlighting the shape and ornamentation as good taxonomic characteristics. Based on these studies, some other research have focused on palynology in the differentiation of Passiflora and/or species (Amela Garcia et al. 2002, Milward-de-Azevedo et al. 2010, 2014a, Gabarayeva et al. 2013, Mezzonato-Pires et al. 2015a,b). However, few studies simultaneously analyzed different subgenera of the Passifloraceae (Mezzonato-Pires et al. 2015b). Therefore, the objective of this work was to evaluate the pollen morphology of eighteen Passiflora species in order to identify informative pollinic characteristics to contribute to the taxonomic classification of the genus.

MATERIALS AND METHODS

PLANT MATERIAL

Eighteen Passiflora species, represented for two subgenera according the classification of Feuillet and MacDougal (2003) were analyzed (Table I; Figure 1): Passiflora alata Curtis. (BGP-004, HURB-20749), P. gibertii N. E. Br. (BGP-008, HURB-20753), P. edulis Sims f. flavicarpa Degener (BGP-038, HURB-20754), P. edmundoi Sacco (BGP-046, HURB-20745), P. tenuifila Killip (BGP-105, HURB-20750), P. morifolia Mast. (BGP-107, HURB-20740), P. galbana Mast. (BGP-109, HURB-20743), P. muchronata Sessé & Moc. (BGP-114, HURB-20848), P. rubra L. (BGP-125, HURB-20752), P. suberosa L. (BGP-152, HURB-20742), P. foetida L. (BGP-153, HURB-20741), P. malacophylla Mast. (BGP-170, HURB-20747), P. racemosa Brot. (BGP-172, HURB-20744), P. setacea L. (BGP-237, HURB-20755), P. cincinnata Mast. (BGP-312, HURB-20746), P. maliformis L. (BGP-379, HURB-20756), P. subrotunda L. (BGP-394, HURB-20751) and P. coccinea Aubl. (BGP-408, HURB-20748). These species belonging to the Active Germplasm Bank of Passion fruit (AGP) from Embrapa Mandioca e Fruticultura located in Cruz das Almas, Bahia Brazil (12º 48’38’’S and 39º06’26’’O; 220 m). Specimen of each taxon were deposited in the HURB herbarium (Universidade Federal do Recôncavo da Bahia).

TABLE I
List of 18 Passiflora species of the genus Passiflora L. used in this study.

Access	Species	Subgenus	Supersection/section or series	Pollinator	2n	Compat.* Systematic
BGP-004	P. alata Curtis	Passiflora L.	Laurifolia (Cervi) Feuillet & MacDougal/ Quadrangulares Feuillet & MacDougal	Bees	18	SI
BGP-008	P. gibertii N. E. Br.	Passiflora L.	Stipulata Feuillet & MacDougal/ Granadillastrum Triana & Planch.	Bees	18	SI
BGP-038	P. edulis Sims f. flavicarpa Deg.	Passiflora L.	Passiflora/Passiflora	Bees	18	SI
BGP-046	P. edmundoi Sacco	Passiflora L.	Stipulata Feuillet & MacDougal /Kermesinae Killip ex Cervi	Hummingbirds	18	SI
BGP-105	P. tenuifila Killip	Passiflora L.	Passiflora/Passiflora	Bees	18	SC
BGP-107	P. morifolia Mast.	Decaloba D.C Rchb.	Bryonioides (Harms) MacDougal & Feuillet/ Cieca (Medic.) MacDougal & Feuillet	Wasps	12	SC
BGP-109	P. galbana Mast.	Passiflora L.	Passiflora/Simplicifoliae Harms) Killip	Bats	18	SI
BGP-114	P. muchronata Sessé & Moc.	Passiflora L.	Passiflora/Simplicifoliae Harms) Killip	Bats	18	SI
BGP-125	P. rubra L.	Decaloba D.C Rchb.	Decaloba (DC.) MacDougal & Feuillet/ Xerogona (Raf.) Killip	Wasps	12	SC
BGP-152	P. suberosa L.	Decaloba D.C Rchb.	Decaloba (DC.) MacDougal & Feuillet/ Cieca (Medic.) MacDougal & Feuillet	Wasps	24	SC
BGP-153	P. foetida L.	Passiflora L.	Stipulata Feuillet & MacDougal/Dysosmia DC.	Bees	18, 20, 22	SC
BGP-170	P. malacophylla Mast.	Passiflora L.	Passiflora/Serratifoliae	Bees	18	SI
BGP-172	P. racemosa Brot.	Passiflora L.	Stipulata Feuillet & MacDougal/ Calopathanthus Harms	Bees	18	SI
BGP-237	P. setacea L.	Passiflora L.	Passiflora/Setaceae Killip ex Cervi	Bats	18	SI
BGP-312	P. cincinnata Mast.	Passiflora L.	Passiflora/Passiflora	Bees	18	SI
BGP-379	P. maliformis L.	Passiflora L.	Laurifolia (Cervi) Feuillet & MacDougal/Laurifoliae Killip ex Cervi	Bees	18	SI
BGP-394	P. subrotunda L.	Passiflora L.	Passiflora/Simplicifoliae	Bees	18	SI
BGP-408	P. coccinea Aubl	Passiflora L.	Coccinea Feuillet & MacDougal	Hummingbirds	18	SI

• Taxonomic classification, their pollinators, diploid numbers, compatibility systematic, according to Feuillet and MacDougal (2003), Melo et al. (2001), Melo and Guerra (2003), Soares et al. (2015). *SI = self-incompatible, SC = self-compatible.

Figure 1
General view of flowers of 18 Passiflora species used in this study: a) P. alata, b) P. gibertii, c) P. edulis f. flavicarpa, d) P. edmundoi, e) P. tenuifila, f) P. morifolia, g) P. galbana, h) P. muchronata, i) P. rubra, j) P. suberosa, k) P. foetida, l) P. malacophylla, m) P. racemosa, n) P. setacea, o) P. cincinnata, p) P. maliformis, q) P. subrotunda and r) P. coccinea.

In this study, we followed the two taxonomic treatments of genus Passiflora proposed by Killip 1938 (with emends by Escobar 1988, 1989, 1994; and MacDougal 1994) by Feuillet and MacDougal (2003) as a point of comparison and discussion of our data. Most of these species were preserved in greenhouses, except for P. setacea, P. maliformis and P. alata, which were preserved under field conditions. Each Passiflora species was represented by one accession identified by the BGP code.

MORPHOPOLLINIC CHARACTERIZATION

For the morphopollinic characterization, the pollen grains were fixed in modified Karnovsky’s solution (Karnovsky 1965) [glutaraldehyde (2%), paraformaldehyde (2%), CaCl₂ (0.001 M), sodium cacodylate buffer (0.05 M), pH 7.2] for 48 hours and then dehydrated in increasing concentrations of ethanol (35-100%) for 20 minutes each. The samples were dried out in hexamethyldisilazane, and the dried samples were then mounted on metallic supports (stubs), metallized with gold for 180 s using a sputter coater (MED 010, Balzers Union, Balzers, Liechtenstein). The images were obtained using the LEO 435 VP variable pressure scanning electron microscope (Carl Zeiss, Jena, Germany), at 20 kv.

The pollen grains were submitted to weak lactic acetolysis (ACLAC 40), according to Raynal and Raynal (1979), and the assessments were obtained using the ImageJ 1.46r program (Rasband 1997-2016). Quantitative characters, polar (PD) and equatorial (ED) diameters of 25 pollen grains per species were measured (µm) at random in equatorial view. We also computed the shape (PD/ED ratio), number and aperture type, and exine ornamentation of the pollen grains. The pollen grains were characterized according to the nomenclature described by Punt et al. (2007) and Hesse et al. (2009), and for the general description of the pollen openings, we adopted the definition of Presting (1965).

The pollen grains were photographed digitally using the Olympus microscope (DM1000; Leica Microsystems, Wetzlar, Germany) coupled with a Sony (Sony, Tokyo, Japan) video camera and a microcomputer using Image Pro-plus Software version 3.0 for Windows (Media Cybernetics, Inc., Bethesda, MD, USA).

DATA ANALYSIS

Qualitative (aperture and shape) and quantitative data were submitted to multivariate analysis to determine genetic distance using the Gower distance (1971). The validation of the clusters was determined by the cophenetic correlation (cc) (Sokal and Rohlf 1962). The R (R Development Core Team 2015) program was used to obtain the genetic distance matrix and cophenetic correlation. A dendrogram was generated using the MEGA 5.0 program (Tamura et al. 2011) based on the distance matrix and the clustering method UPGMA (unweighted pair group method with arithmetic means).

RESULTS

Pollen grains of Passiflora were characterized by their morphological characteristics (shape, number and aperture type, and exine ornamentation) (Table II and Figs. 2, 3, 4). The multivariate analysis of the 18 Passiflora species allowed for the formation of three groups (Fig. 2) using the mean genetic dissimilarity (Ddg = 0.36) as a cutoff. The cophenetic correlations, which express the relationship between the original distances and graphically distances was 0.88 (P <0.0001, 10,000 permutations). Some authors have affirmed that cophenetic correlation above 0.8 shows the adequacy of the clustering method to represent the genetic diversity among the assessed individuals (Rohlf and Fisher 1968).

TABLE II
Morphopollinic characterization of 18 Passiflora species belonging to the subgenera Passiflora and Decaloba.

Accessions	Species	Polar Diameter (PD)		Equatorial Diameter (ED)		PD/ED	Shape	Size	Aperture	Group*
		VF	x ± sx	VF	x ± sx
BGP-004	P. alata	85.1 - 63.1	71.0 ± 8.0	83.5 - 59.7	73.3 ± 9.2	0.97	oblate-spheroidal	Large	6-syncolpate	G1
BGP-008	P. gibertii	80.5 - 64.0	71.7 ± 6.3	83.4 - 61.8	72.1 ± 7.6	0.99	oblate-spheroidal	Large	6-syncolpate	G1
BGP-038	P. edulis	76.4 - 63.4	68.6 ± 3.7	77.5 - 63.7	71.3 ± 5.0	0.98	oblate-spheroidal	Large	6-syncolpate	G1
BGP-046	P. edmundoi	78.6 - 77.6	78.2 ± 0.2	83.6 - 82.4	83.1 ± 0.3	0.94	oblate-spheroidal	Large	6- syncolpate	G1
BGP-105	P. tenuifila	82.1 - 79.5	80.8 ± 1.8	82.0 - 78.9	80.4 ± 2.1	0.94	oblate-spheroidal	Large	6- syncolpate	G1
BGP-107	P. morifolia	72.5 - 58.0	62.9 ± 3.9	58.4 - 52.7	54.6 ± 1.3	1.15	subprolate	Large	6-colporate	G3
BGP-109	P. galbana	88.0 - 87.1	87.6 ± 0.4	89.8 -89.5	89.7 ± 0.3	0.98	oblate-spheroidal	Large	6-syncolpate	G1
BGP-114	P. muchronata	88.9 - 87.4	88.1 ± 0.3	90.7 - 89.4	90.1 ± 0.3	0.97	oblate-spheroidal	Large	6-syncolpate	G1
BGP-125	P. rubra	62.5 - 47.2	52.9 ± 5.1	63.2 - 46.0	52.4 ± 5.4	1.01	prolato-spheroidal	Large	12-colporate	G3
BGP-152	P. suberosa	69.1 - 58.2	64.1 ± 2.9	66.8 - 51.0	54.9 ± 3.2	1.16	subprolate	Large	12-colpate	G3
BGP-153	P. foetida	73.5 - 64.2	67.3 ± 2.6	76.2 - 66.7	69.7 ± 2.9	0.96	oblate-spheroidal	Large	6-syncolpate	G1
BGP-170	P. mallacophyla	81.7 - 78.4	79.4 ± 0.8	82.0 - 78.2	80.5 ± 0.8	0.99	oblate-spheroidal	Large	6-syncolpate	G1
BGP-172	P. racemosa	83.4 - 79.1	81.6 ± 0.9	84.2 - 80.2	82.8 ± 0.9	0.99	oblate-spheroidal	Large	6-syncolpate	G1
BGP-237	P. setacea	80.3 - 55.8	69.0 ± 5.8	86.2 - 68.1	75.0 ± 5.9	0.92	oblate-spheroidal	Large	6-syncolpate	G1
BGP-312	P. cincinnata	81.6 - 79.8	80.6 ± 0.5	87.8 - 86.4	87.1 ± 0.3	0.92	oblate-spheroidal	Large	6-syncolpate	G1
BGP-379	P. maliformis	72.0 - 58.1	64.9 ± 3.4	72.30 - 57.4	65.7 ± 4.5	0.99	oblate-spheroidal	Large	6-syncolpate	G1
BGP-394	P. subrotunda	78.5 - 58.1	68.0 ± 5.2	80.2 - 63.20	71.1 ± 5.3	0.96	oblate-spheroidal	Large	6-syncolpate	G1
BGP-408	P. coccinea	61.9 - 50.2	55.3 ± 3.4	76.8 - 64.2	69.6 ± 4.6	0.79	suboblate	Large	6-colporate	G2

• VF = range of the characteristic, x = overall mean, s_x standard deviation of the mean; *groups formed by the clustering analysis.

Figure 2
Dendrogram of the 18 Passiflora species, obtained by UPGMA (unweighted pair group method using arithmetic averages) based on the Gower distance, using the qualitative and quantitative morphopollinic characteristics.

Figure 3
Pollen grains of Passiflora using a light microscope (LM) and a scanning electron microscope (SEM). a-c) P. maliformis (BGP-379); d-f) P. alata (BGP-004); g-i) P. gibertii (BGP-008); j-l) P. subrotunda (BGP-394); m-o) P. foetida (BGP-153); p-r) P. edulis (BGP-038); a, d, g, j, m, p) LM overview; b, e, h, k, n, q) SEM overview; c, f, i, l, o, r) details of the exine and colpi using an SEM. ap: apocolpus; ba: bacula; co: calcium oxalate crystals; mu: muri; pk: pollenkitt; ps: pseudoperculum; pt: pontoperculum; arrow: apertures; *fusion of the apertures within the apocolpus. Bars: a, b, d, e, g, h, j, k, m, n, p, q = 20 μm; c, f, i, l, o, r = 10 μm.

Figure 4
Pollen grains of Passiflora using a light microscope (LM) and a scanning electron microscope (SEM); a-c) P. setacea (BGP-237); d-f) P. cincinnata (BGP-312); g-i) P. racemosa (BGP-172); j-l) P. mallacophyla (BGP-170); m-o) P. tenuifila (BGP-105) p-r); P. edmundoi (BGP-046); a, d, g, j, m, p) LM overview; b, e, h, k, n, q) SEM overview; c, f, i, l, o, r) details of the exine and colpi using an SEM. Bars: a, b, d, e, g, h, j, k, m, n, p, q = 20 μm; c, f, i, l, o, r = 10 μm.

The majority of Passiflora species were allocated in Group 1 (G1): P. alata, P. gibertii, P. edulis, P. edmundoi, P. muchronata, P. racemosa, P. setacea, P. galbana, P. maliformis, P. subrotunda, P. cincinnata, P. tenuifila and P. foetida (Table II and Fig. 2). All species of Group 1 belong to the subgenus Passiflora and form a homogeneous palynological complex, mainly in terms of the shape, size and type of the pollen grain openings (Table II and Figs. 2, 3, 4, 5a-i).

The palynological analysis performed using light microscopy on the accessions of Group 1 (G1) showed the presence of large pollen grains ranging from 50.2 μm to 88.9 μm (major axis - polar diameter), oblate spheroidal shape and 6-syncolpate aperture (six colpos fused in pairs in the apocolpus region). The pollen grains of this group were classified as isopolar, of circular scope, with a thick heteroreticulate exine, columellate and simple muri, and with sinuous and apparent columelles (Table II and Figs. 3a-o, 4a-o, 5a-f). The most notable pollen characteristic that distinguishes the species of this group is the presence of bacula inserted into the lumen, with some variation in the number and height of the bacula. Some species have large lumens with many bacula inserted in their interior (Figs. 3c, 3f, 3i, 4c, 4f, 4i, 4l, 5c, 5f, 5i, 5l, 5r, 5o), and others presented few bacula (Fig. 3l, 3o, 3r, 4o). Although belonging to the subgenus Passiflora (supersection Coccinea), P. coccinea presented an exclusive shape suboblate type and was therefore allocated in Group 2 (G2), which has this characteristic as the main differential (Table II and Fig. 5g-i).

Figure 5
Pollen grains of Passiflora using a light microscope (LM) and a scanning electron microscope (SEM); a-c) P. muchronata (BGP-114); d-f) P. galbana (BGP-109) g-i); P. coccinea (BGP-408); j-l) P. suberosa (BGP-152); m-o) P. morifolia (BGP-107); p-r) P. rubra (BGP-125); a, d, g, j, m, p) LM overview; b, e, h, k, n, q) SEM overview; c, f, i, l, o, r) details of the exine and colpi using an SEM. co: calcium oxalate crystals. Bars: a, b, d, e, g, h, j, k, m, n, p, q = 20 μm; c, f, i, l, o, r = 10 μm.

The species (P. morifolia, P. suberosa, and P. rubra) belonging to the subgenus Decaloba formed Group 3 (G3) (Table II, Fig. 2). The pollen grains of this subgenus ranged from 47.2 to 72.5 μm, being considered large, with distinct pollen aperture (6-colporate - P. morifolia, 6-colpate - P. suberosa and 12-colporate - P. rubra) (Fig. 5j-r). Regarding the classification of pollen grains based on the PD/ED ratio, these were categorized as subprolate (P. suberosa and P. morifolia) and spheroidal prolate (P. rubra) (Table II).

Differences in the subgenus Decaloba in terms of the colpi sculpture and the number of bacula inserted in the lumen (Fig. 5j-r) were observed from the evaluation of the pollen grains in the electron microscopy. P. rubra showed a reticulated exine with sinuous muri, apparent columelles and few bacula inserted in the lumens (Fig. 5p-r). The pollen grains of P. morifolia also presented reticulum with sinuous muri, without apparent columelles and with many bacula inserted in the lumen (Fig. 5m-o). However, in P. suberosa, the exine of the pollen grains is characterized by presence of reticulum with flat muri and absence of bacula into the lumen (Fig. 5j-1). Another pollen grain characteristic that differentiated these species was the lumen size, which was smaller in P. suberosa when compared to P. morifolia and P. rubra. In addition, the pollen grains of P. rubra and P. morifolia presented three mesocolpus, and P. suberosa presented six mesocolpus.

DISCUSSION

The pollinic characteristics are useful to differentiate and establish relationships between different species (Fazal et al. 2013, Amorim et al. 2014) because they are genetically determined and very constant and specific to each species (i.e., pollen grain size, number of apertures, aperture type and exine ornamentation) and therefore have a high taxonomic value (Mert 2010, Tangarife et al. 2011).

The pattern of clustering based on the pollinic characteristics allowed to classify the species into two subgenera, Passiflora and Decaloba, congruent with the classification of Feuillet and MacDougal (2003) and supported by phylogenetic studies using molecular markers and sequencing technique (Yockteng and Nadot 2004, Hansen et al. 2006, Mäder et al. 2010, Porter-Utley 2014, Milward-de-Azevedo et al. 2014b, Muschner et al. 2012, Krosnick et al. 2013, Ocampo and D’Eeckenbrugge 2017). These authors revealed that the Passiflora and Decaloba subgenera showed monophyletic clades.

In the present study, the subgenus Passiflora was represented by 15 species (P. alata, P. gibertii, P. edulis, P. edmundoi, P. tenuifila, P. galbana, P. muchronata, P. foetida, P. setacea, P. cincinnata, P. malacophylla, P. racemosa, P. maliformis, P. subrotunda and P. coccinea). This subgenus is characterized by leaves entire or lobed; glands on petioles, stipules, and margins of leaves; hypanthium tubular or campanulate; operculum tubular or filamentous; membranous limen, herbaceous vines or woody lianas, free serrate bracts, large colorful flowers, attract as pollinators bees, bats and hummingbirds, are diploid with 2n = 18 and self-incompatible according to described by Feuillet and MacDougal 2003, 2004, Melo and Guerra 2003, Santos et al. 2015, Ocampo and D’Eeckenbrugge 2017 (Table I). However, subgenus Decaloba is represented by P. morifolia, P. rubra and P. suberosa, characterized by the absence of ocelli on the leaf blades, by the absence of bracts, and by capsular fruits. The species present small flowers (< 4 cm diameter) with one or two series of corolla filaments, a folded operculum, usually white or greenish and small fruits, diploid 2n = 12, 24, according to described by Ulmer and MacDougal 2004, Milward-de-Azevedo 2014b (Table I).

In general, it was observed that the majority of the Passiflora species presented apertural membrane strongly ornamented with a large number of densified bacula inserted into the lumen. This characteristic may indicate a mechanism against dehydration, mainly during pollination, since the great majority of the analyzed species presented a relatively large apertural area. Among the species of the subgenus Passiflora, P. edulis was the one that presented pollen grains with large lumens with few bacula inserted inside (Fig. 3r), while in the species of the subgenus Decaloba, P. suberosa was the only one that did not present bacula inserted into the lumen (Fig. 3l).

The pollinic characteristics based on the presence or absence of bacula are important for the taxonomic delimitation of the subgenus Decaloba in the species from southwest Brazil (Milward-de-Azevedo et al. 2004). Indeed, some pollinic characteristics based on the presence or absence of the bacula have been used as a palinotaxonomic resource of great importance for phylogenic studies in the Passifloraceae (Presting 1965).

Regarding the appearance of the exine surface of the Passiflora pollen grains, lipophilic substances were found between the free reticulum columelles, called pollenkitt (Fig. 3g). This substance has important functions during the pollen grain dispersion because it aids in the adhesion of the pollen grains to the body of the pollinating agent. The pollenkitt can also protect against dehydration and UV radiation damage, as well as with the pollen grain recognition system on the stigmatic surface (Pacini and Hesse 2005). Other authors also observed the presence of pollenkitt in the exine of the pollen grains of Passiflora (Souza et al. 2004, Soares et al. 2013, Silvério and Mariath 2014).

The classification of the pollen grain shape according to the PD/ED ratio, it was observed that the majority of the species of Passiflora belonging to supersections: Laurifolia, Stipulata and Passiflora (Feuillet and MacDougal 2003) presented a PD/ED ratio above 0.90 and were therefore classified as oblate-spheroidal, with the exception of P. coccinea (supersection Coccinea), which presented a PD/ED ratio of 0.79 and a suboblate shape. This major difference could lead to the P. coccinea species becoming more distant from other species of the subgenus Passiflora being allocated in Group 2. Previous studies based on taxonomic progress and phylogenetic analyses using molecular markers particularly over the last 11 years by Muschner et al. (2003), Yockteng and Nadot (2004), and Hansen et al. (2006) did not support the classifications into supersections, sections, and series.

In contrast, in the subgenus Decaloba represented by P. morifolia, P. rubra and P. suberosa belonging to two supersections (Bryonioides and Decaloba) according to classification proposed by Feuillet and MacDougal (2003) presented a subprolate pollen grain shape was observed for P. morifolia and P. suberosa, while a prolato-spheroidal shape of the pollen grain was observed in P. rubra, similar to the results described by Milward-de-Azevedo et al. (2010). Some authors have affirmed that the oblate-spheroidal, prolate-spheroidal and spheroidal shapes confer the Passifloreaceae with one of their main palynological characteristics and may help in the taxonomic classification of this group (Mezzonato-Pires et al. 2015b).

Other descriptors of high value diagnostic for studies taxonomic in Passiflora are those related to the pollen grain morphology and the pollen aperture (Dettke et al. 2009, Mezzonato-Pires 2015a,b). The wide morphological diversity of the Passifloraceae has enabled the creation of different types of pollen. Presting (1965) identified typical pollen grains for the species of the Passifloraceae using the reticulum pattern (lumen diameter and presence or absence of bacula) and exine thickness as criteria. On the other hand, Araújo and Santos (2004) chose pollen types based on the operculum variations (elliptic and pontoperculum operculum) and sizes of the reticles lumens. In the present study, the pollen types were typified based on the apertural pattern, shape of the pollen grains and the muri characteristics with the presence or absence of bacula in the lumen.

However, previous studies have shown contradictory results due to the complexity of the pollen grains of the Passifloraceae species, especially in relation to the pollen grain size and the type and number of pollen grains. The pollen aperture in P. alata was described in the literature as being 6-colpate (Dettke and Santos 2009) or 6-syncolpate (Araújo and Santos 2004, Evaldt et al. 2011). For P. edulis,Tangarife et al. (2011) classified the pollen aperture as 6-colporoid, while Dettke and Santos (2009) described it as 6-colpate. Recently, Menzzonato-Pires et al. (2015b) classified the pollen aperture of P. alata, P. racemosa, P. edulis, P. foetida and P. setacea as 6-syncolpate and that of P. mucronata as 6-colpate. The results related to the type of ornamentation and pollen aperture found by the authors were partially similar to those obtained in the present study, except for P. mucronata, with 6-syncolpate aperture. Studies carried out by other authors have shown that there is some divergence in the pollen morphology in the majority of Passiflora species, with differences in the pollen grain number, type of pollen aperture and nomenclatures adopted for the pollen grain structures. This divergence likely occurs because the studies of pollen characterization of Passiflora deal with descriptions based mainly on the external aspect of the exine, which contributes to several interpretations (Detcke 2009, Evaldt et al. 2011).

For the characteristics shape, number and aperture type of the pollen grains of the subgenus Decaloba, there is a consensus in the pollen descriptions for P. rubra (Milward-de-Azevedo et al. 2010, 2014a, Amorim et al. 2014). However, this was not observed for the other two species of the subgenus Decaloba, P. morifolia and P. suberosa, which had pollen grains classified as 6-colporate (Dettke and Santos 2009), 12-colpate (Amela García et al. 2002, Milward-de-Azevedo et al. 2004), oblate (Silvério and Mariath 2014), prolate-spheroidal (Milward-de-Azevedo et al. 2004) and subprolate (Milward-de-Azevedo et al. 2010). The results reported by these authors were similar to those observed in the present work for P. suberosa and P. morifolia in terms of the pollen grains subprolate format and the 12-colporate pollen opening.

However, our results demonstrate relative congruence with most of the taxonomic affinities related to the subgenera Passiflora studied and show that the classification of the Passiflora species can be carried out based on morphological characteristics of the pollen grains, thus identifying morphopollinic patterns (i.e., pollen grain shape, pollen apertures and ornamentation of the exine), which can contribute to the taxonomy of this genus.

Most of the species studied belong to the subgenus Passiflora (2n = 18) area presents self-incompatible, while those of the subgenus Decaloba are self-compatible (2n = 6) (Melo et al. 2016). Although there is no chromosome homology between these subgenera, many interspecific hybrids of Passiflora have been successfully obtained, because in many cases the barriers of interspecific incompatibility are relatively fragile (Meletti et al. 2005, Soares et al. 2015). Thus, the Passiflora species used in the present work have great potential in interspecific hybridization programs with the purpose of introgression of resistance genes in yellow passion fruit (P. edulis), which is the main commercial species in Brazil. Among the interesting characteristics present in some evaluated species are: resistance to foliar diseases (Santos et al. 2015, Jesus et al. 2016, Freitas et al. 2016a) and soil (Freitas et al. 2016b) and the presence of self-compatibility which can increase productivity and decrease variation in fruit size among plants in commercial plantations. In addition, interspecific hybrids among the wild species studied have shown ornamental potential (Bugallo et al. 2011, Santos et al. 2012, 2014, Ocampo et al. 2016, Melo et al. 2016, Coelho et al. 2016).

It is interesting to note that although the pollen morphology analysis may be useful in taxonomic studies of the Passiflora genus, it should only be considered an additional tool for species classification to increase our understanding of the systematics of the genus, for re-evaluating the circumscriptions and arrangements of the infrageneric categories currently established, and for better understanding the phylogenetic lineages of the Passiflora. Futher studies should be carried out under the light transmission electron microscopy and ontogeny of the structures that compose the pollen grain sporoderm of the Passiflora, enabling better understanding of the exine ornamentation and probably highlighting other traits that may be useful to the classification of the taxa.

CONCLUSIONS

The morphology of Passifloraceae pollen grains can be used as a morphological descriptor to establish the taxonomy and phylogenetic relationships of the species of this family. However, future studies involving transmission electron microscopy will be necessary to enable better understanding of the exine ornamentation, probably highlighting other palynological characteristics that can be used in the delimitation of the taxa.

ACKNOWLEDGMENTS

The authors are grateful to the Fundação de Amparo à Pesquisa do Estado da Bahia (FAPESB) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for financial support for research and fellowship (DCR0013/2015) granted to the first author, and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES-PROCAD - 2013 and CAPES/EMBRAPA) and the NAP/MEPA - ESALQ/USP (Centre for the Support of Electron Microscopy Research Applied to Agriculture) by the use of scanning electron microscopy. We also thank the curator and staff of the herbarium (HURB - Universidade Federal do Recôncavo da Bahia).

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  Contribution to the centenary of the Brazilian Academy of Sciences.

Publication Dates

• Publication in this collection
  16 Oct 2017
• Date of issue
  Aug 2018

History

• Received
  03 May 2017
• Accepted
  08 June 2017