Ultrastructure and pollen morphology of Bromeliaceae species from the Atlantic Rainforest in Southeastern Brazil

Pollen grain morphology of Bromeliaceae species collected in areas of the Atlantic Rainforest of southeastern Brazil was studied. The following species were analyzed: Aechmea bambusoides L.B.Sm. & Reitz, A. nudicaulis (L.) Griseb., A. ramosa Mart. ex Schult.f., Ananas bracteatus (Lindl.) Schult.f., Billbergia distachia (Vell.) Mez, B. euphemiae E. Morren, B. horrida Regel, B. zebrina (Herb.) Lindl., Portea petropolitana (Wawra) Mez, Pitcairnia fl ammea Lindl., Quesnelia indecora Mez, Tillandsia polystachia (L.) L., T. stricta Sol., T. gardneri Lindl., T. geminifl ora Brongn. and Vriesea grandifl ora Leme. Light and scanning electron microscopy were used and the species were grouped into three pollen types, organized according to aperture characteristics: Type I – pantoporate pollen grains observed in P. petropolitana, Type II – 2-porate pollen grains, observed in the genera Ananas, Aechmea and Quesnelia, and Type III – 1-colpate pollen grains, observed in the genera Billbergia, Pitcairnia, Tillandsia and Vriesea. Pollen data led to the construction of an identifi cation key. The results showed that the species analyzed can be distinguished using mainly aperture features and exine ornamentation, and that these characteristics may assist in taxonomic studies of the family.


INTRODUCTION
Bromeliaceae Juss comprises nearly 3.172 species, distributed in 58 genera (Luther 2008).It is considered the fourth-richest Angiosperm family in terms of the number of species in the Atlantic Forest (Stehmann et al. 2009), and the southeastern of Brazil represents a center of endemism for many of its taxa.
The traditional infrafamiliar taxonomy of Bromeliaceae (Smith and Downs 1974, 1977, 1979) relies on the characteristics of the position of the ovary, as well as types of habit, fruits and seeds in order to recognize the subfamilies Bromelioideae, Pitcairnoideae and Tillandsioideae.However, phylogenetic studies based on morphological and molecular data only consider Bromelioideae and Tillandsioideae to be monophyletic, while Pitcairnoideae is considered to be polyphyletic (Terry et al. 1997, Givnish et al. 2007, 2011).
Taxonomic disagreements regarding Bromeliaceae at the generic and infrageneric levels are widely spread among the three subfamilies and are worsened by the limited knowledge of many morphological VANESSA J.D. SILVA, ESTER M. RIBEIRO, ANDREA P. LUIZI-PONZO and ANA PAULA G. FARIA structures (Faria et al. 2004).The fl oral morphology is usually poorly preserved in herbarium collections, and therefore, it is also poorly studied.This limits the utilization of many characteristics that could potentially be diagnostic.Benzing (1994) and Schulte and Zizka (2008) mentioned the importance and the necessity of more studies focusing on the detailed analysis of underemployed characteristics, such as the morphological variation of stigmas and petal appendages, as well as the pollen morphology.In this context, pollen morphology has provided important information for the taxonomy of Bromeliaceae, aiding in a better defi nition of the generic and specifi c delimitation (Moreira et al. 2005).
Mez (1891-1894, 1896, 1934-1935) made the fi rst attempt to apply pollen morphology to the systematics of Bromeliaceae.Based on the types of aperture, the author classifi ed the subfamilies Pitcairnoideae and Tillandsioideae as stenopalynous, due to having pollen grains that were predominantly monocolpate, while Bromelioideae was considered euripalynous and had different types of aperture (including the tribes Poratae and Sulcatae), in addition to inaperturate grains (tribe Integrae).Other important palynological studies concerning Bromeliaceae include the studies by Ehler and Schill (1973), Erdtman and Praglowski (1974), Wanderley and Melhem (1991), Halbritter (1992), Sousa et al. (1997), Melhem et al. (2003), Souza et al. (2004) and Moreira et al. (2005).However, knowledge regarding the palynology of Bromeliaceae is still scarce, especially when considering the species' richness within the family.
The present work describes the pollen morphology of bromeliad species from the Atlantic Rainforest in southeastern Brazil, with the aim to provide additional morphological characteristics to support the taxonomy and systematic of the family.

MATERIALS AND METHODS
Pollen grains were obtained from herbarium specimens deposited in the CESJ herbarium (acronyms follow Index Herbariorum; Thiers 2014 continuously updated).The species analyzed were gathered from portions of the Atlantic Rainforest in the states of Minas Gerais and Espírito Santo, southeast of Brazil.Whenever possible, more than one exsiccate was examined, in order to compare all possible variations within a single species.The best quality material was indicated as "standard" (marked with an asterisk in the examined material list below), and the other materials from the same species were referred to as "comparison" materials.
The Pollen grains were prepared using acetolysis (Erdtman 1960) and in addition to the Wodehouse (1935) modifi ed methodology.Fragile pollen grains were also prepared by lactic acetolysis (ACLAC 40%) following Raynal and Raynal's (1971) protocol.They were studied under light microscopy (LM).For scanning electron microscopy (SEM), the untreated pollen grains were mounted on a stub with adhesive carbon double face tape and sputtered with a 20 nm thick gold coat.Three slides containing acetolyzed pollen grains were prepared for each specimen.Whenever possible, the largest diameter of 25 pollen grains on polar view of each specimen was measured, as well as the equatorial and polar diameters on equatorial view; a micrometer ocular and a BX-41 Olympus microscope was used.To evaluate the measurements, Microsoft Excel software was applied in order to calculate the arithmetic mean (X), size range (X min -X max ), standard error (Sx), standard deviation (S), variability (V), and confi dence interval.If it was not possible to take 25 measurements for a specimen, only the arithmetic mean was calculated.
The photomicrographs were taken using a Canon camera adapted to a Zeiss Primo-Star light microscope.The terminology follows Punt et al. (2007), and Barth and Melhem (1988).
The pollen grains of B. zebrina show wide colpus, with an ornamented margin.Under SEM, an undulate tectum is observed, mixed with a small reticulum.Tillandsia geminifl ora has folded apertures with fused ends.Vriesea grandifl ora has polygonal lumina that are variable in size, containing pila.Under SEM, the reticulum decreases towards the poles and the colpus exhibit a median constriction.Aechmea species presented heterobrochate exine with higher lumina.Sousa et al. (1997) also described differences in the type of exine (semi-tectate, tectate-perfurate, rugulate, reticulate and microreticulate) for species of Aechmea from northeastern Brazil, strengthening the morphological diversity of the pollen grains within this genus.Wanderley and Melhem (1991) analyzed 12 species of the Vriesea genus and found morphological characteristics similar to those found in V. grandifl ora, such as grains 1-colpate, heterobrochate exine and lumina with few pila.
Portea petropolitana was distinguished from all other species by presenting pantoporate pollen grains.Smith and Dows (1979) considered this characteristic as diagnostic for the genus, and Halbritter (1992) confi rmed this apertural pattern for species of Portea.Pantoporate grains, however, are also observed in some Aechmea species native to northeastern Brazil, which is related to the Gravisia complex (Read and Luther 1991).Mez (1891Mez ( -1894Mez ( , 1896Mez ( , 1934Mez ( -1935) ) fi rst suggested the morphological proximity between Portea and Aechmea species originally described within the genus Gravisia, while Faria et al. (2004) confi rmed this close relationship using phylogenetic studies.
The Billbergia species have been reported in the literature to have 1-colpate pollen grains, varying from reticulate to heterobrochate (Ehler and Schill 1973, Wanderley and Melhem 1991, Halbritter 1992, Melhem et al. 2003).These characteristics were confi rmed for the genus in our study.Related to the colpus, only B. zebrina showed ornamentation in the margins.B.A. Moreira (unpublished data), described the pollen morphology of this species, presenting a well-ornamented colpus and a psilate-perforate exine.Our study corroborates the colpus ornamentation, but indicates differences in the characterization of the exine, which is characterized here as microreticulate.Erdtman and Praglowsky (1974) and Halbritter (1992) analyzed pollen grains of Pitcairnia species and observed the 1-colpate aperture pattern and exine with reticulate ornamentation in P. fl ammea pollen grains, as well as for other taxa of the genus.Our study corroborates the aperture and ornamentation pattern of this specie.
The basic 1-colpate aperture pattern of the genus Tillandsia is confi rmed for the species analyzed in the present study.Wanderley andMelhem (1991) andSouza et al. (2004) analyzed the pollen grains of Tillandsia species and indicated differences in the exine structure, as the main palynological characteristic.The results obtained in this study corroborate the morphological variety of the patterns of exine ornamentation, which varied from psilate-perfurate in T. geminifl ora, and microreticulate in T. stricta and T. gardneri, to heterobrochate in T. polystachia.
This study revealed some useful taxonomical characteristics that could be used to identify species, to delimit groups of species, and for future taxonomic and phylogenetic studies of Bromeliaceae.VANESSA J.D. SILVA, ESTER M. RIBEIRO, ANDREA P. LUIZI-PONZO and ANA PAULA G. FARIA the Programa de Capacitação em Taxonomia (PROTAX), process number 562218/2010-6.The authors would also like to thank the Bryophyte Laboratory (Universidade Federal de Juiz de Fora), the Núcleo de Microscopia e Microanálise da Universidade Federal de Viçosa and Fátima Salimena (curator of CESJ herbarium), for the logistic support.