The epiphytic fern Microgramma squamulosa occurs in the Neotropics and shows dimorphic sterile and fertile leaves. The present study aimed to describe and compare qualitatively and quantitatively macroscopic and microscopic structural characteristics of the dimorphic leaves of M. squamulosa, to point more precisely those characteristics which may contribute to epiphytic adaptations. In June 2009, six isolated host trees covered by M. squamulosa were selected close to the edge of a semi-deciduous seasonal forest fragment in the municipality of Novo Hamburgo, State of Rio Grande do Sul, Brazil. Macroscopic and microscopic analyzes were performed from 192 samples for each leaf type, and permanent and semi-permanent slides were prepared. Sections were observed under light microscopy using image capture software to produce illustrations and scales, as well as to perform quantitative analyses. Fertile and sterile leaves had no qualitative structural differences, being hypostomatous and presenting uniseriate epidermis, homogeneous chlorenchyma, amphicribal vascular bundle, and hypodermis. The presence of hypodermal tissue and the occurrence of stomata at the abaxial face are typical characteristics of xeromorphic leaves. Sterile leaves showed significantly larger areas (14.80cm2), higher sclerophylly index (0.13g/cm2) and higher stomatal density (27.75stomata/mm2) than fertile leaves. The higher sclerophylly index and the higher stomatal density observed in sterile leaves are features that make these leaves more xeromorphic, enhancing their efficiency to deal with limited water availability in the epiphytic environment, compared to fertile leaves.
El helecho epífito Microgramma squamulosa se encuentra en el Neotrópico y tiene hojas estériles y fértiles dimorfas. El objetivo de este estudio fue describir y comparar cuantitativa y cualitativamente la organización estructural de las hojas de la M. squamulosa, investigando las características morfológicas y anatómicas, y señalando los factores que contribuyen a la adaptación al ambiente epífito. Los análisis macroscópicos y microscópicos se realizaron a partir de 192 muestras de hojas de cada tipo. Láminas permanentes y semi-permanentes fueron preparadas y las secciones fueron observadas en el microscopio de luz. Hojas fértiles y estériles no mostraron diferencias estructurales cualitativas, son hipostomáticas, presentan epidermis uniseriada, haz vascular anficrival e hipodermis. La presencia de hipodermis y aparatos estomáticos en la nsuperficie abaxial de la epidermis son típicas de hojas xeromórficas. Hojas estériles presentaran áreas significativamente mayores, tasa más alta de esclerofilia y mayor densidad de estomas que hojas fértiles. La mayor tasa de esclerofilia y mayor densidad de estomas son características que hacen las hojas estériles más xeromórficas, aumentando su eficiencia para hacer frente a la disponibilidad de agua limitada en el ambiente epífito, en comparación con las hojas fértiles.
Studies about leaf anatomy are an important tool to identify differences between plant organs and adaptive strategies developed in many different environments. Hietz & Briones (1997) studied the adaptations to epiphytism of Pleopeltis mexicana (Fée) Mickel & Beitel. (Polypodiaceae), Phlebodium areolatum ]]>
Elaphoglossum glaucum T. Moore (Dryopteridaceae), and found an association between water availability and stomatal density. Ribeiro et al. (2007) analyzed the leaf anatomy of Anemia tomentosa (Savigny) Sw. var. anthriscifolia (Schrad.) Mickel and Anemia villosa Humb. ]]>
et al. (2006) with species of Dryopteris Adans. (Dryopteridaceae) and by Hernández et al. (2008) with species of ]]>
Cheilanthes Sw. (Pte- ridaceae). Tejero-Díez et al. (2010) compared anatomically the Polypodium plesiosorumsensu Moran (Polypodiaceae) group, in order to identify taxonomic characteristics. However, most studies have been focused on epiphytic angiosperms (Zanenga-Godoy & Costa 2003, Proença & Sajo 2007, Noguera-Savelli & Jáu- regui 2011). ]]>
Polypodiaceae shows pantropical distribution, with about 12 000 species (Smith et al. 2008). Microgramma C. Presl sensu Tryon & Tryon (1982) comprises of 30 species (Salino et al. 2008). Their leaves are glabrous or scaly, ]]>
Microgramma squamulosa Kaulf. de la Sota is an epiphytic fern occurring in Peru, Bolivia, Brazil, Argentina, Paraguay and Uruguay (Tryon & Stolze 1993), both in human-modified environments (Gonçalves & Waechter 2003) and in natural forests (Kersten & Silva 2002). This species presents a long rhizome from which arise the petioles of the fertile and sterile ]]>
et al. 2007, Suffredini et al. 2008).
Hirsch & Kaplan (1974) studied the organography and development of the shoot systems of M. squamulosa and M. vacciniifolia (Langsd. ]]>
M. squamulosa has been studied by Suffredini et al. (1999). Jaime et al. (2007) described the diagnostic characteristics of M. squamulosa to contribute to its taxonomic identification and to the control of ]]>
M. squamulosa have been characterized in the pharmacognostic study conducted by Suffredini et al. (2008). However, these studies did not analyze the structural differences between dimorphic leaves.
Epiphytic species of ]]>
M. squamulosa, and ii) to point more precisely those characteristics which contribute to adaptations in the epiphytic environment.
Material and methods
Study area: Fertile and sterile leaves of M. squamulosa were collected in the municipality of Novo ]]>
et al. 1986) located in an Area of Special Environmental Interest. The predominant regional climate, according to Köeppen, is classified as Cfa type, being humid-temperate, with rainfall throughout the year (Moreno 1961).
Sampling: In June 2009, six isolated host trees (phorophytes) covered by an extensive M. squamulosa rhizome (Fig. 1A) were selected next of the edge of the forest fragment. For each leaf type (Fig. 1B, 1C), 192 samples were collected, 32 from each phorophyte. The leaves were collected from the internal area of the phorophyte ]]>
2/s. Voucher material was deposited in the Herbarium Anchieta of the Universidade do Vale do Rio dos Sinos (PACA 108023), in São Leopoldo, RS, Brazil.
Macroscopic and microscopic ]]>
For macroscopic analyses, 120 leaves of each type were used. They were scanned and their images were digitalized using a desktop scanner connected to a computer. The leaves were dehydrated in an oven at 65ºC until mass was constant. The sclerophylly index was calculated according to Rizzini (1976). The other 72 leaves of each type were used for microscopic analyses. An area of 25mm2 in the midline portion of the leaves was selected and fixed in ]]>
®). The semi-permanent slides of paradermal sections were obtained after dissociation (Franklin 1946) of other 36 leaves of each type, which were later stained with 0.05% toluidine blue (Sakai 1973), mounted on 50% glycerin and luted with clear nail polish (Purvis et al. 1964). Slides were mounted with epidermis samples of the two faces of the leaves to classify the leaves according to the occurrence of ]]>
The permanent and semi-permanent slides were digitalized using a photomicroscope (Olympus CX 41) coupled to a DC 3000 camera (MicrometricsTM) and software Micrometrics SE Premium® 2.9. The sections were described according to Van Cotthem (1970), Ogura (1972), White (1974), Sen & Hennipman (1981) and Hernández
et al. (2006).
After digitizing the macroscopy and microscopy images, thicknesses (leaf blade, epidermis, hypodermal tissue, midrib and sclerified layer), areas (leaf blade, vascular bundle and stoma), leaf width and length, and stomatal density (Fig. 2A, 2C) were obtained through the Micrometrics SE ]]>
® 2.9 software, using the method adapted from Godoi et al. (2010) and Santos et al. (2010). The thickness of the hypodermal tissue was calculated as the mean of the abaxial and adaxial hypodermal thicknesses of each leaf. Stomatal density was analyzed after the software provided a random definition 1mm2 areas for each paradermal section, and one quadrant per section was examined for each leaf (Fig. ]]>
).
To compare quantitative parameters between fertile and sterile leaves, the SPSS 17.0 was used. The Shapiro-Wilk test was used to confirm normal data distribution. As the hypothesis of normal distribution was rejected, the Mann-Whitney test was used, and the level of significance was set at 5%.
]]>
Results
The fertile and sterile leaves presented no qualitative structural differences. The cross sections of the leaf types showed uniseriate epidermis in both faces. Stomata were at the same or a little higher level than the other epidermal cells (Fig. 3A, 3B). The ]]>
Fig. 3A, 3B).
The midrib had an amphicribal ]]>
Fig. 3A). A sclerified layer, whose cells had a brownish content, was seen outside the endodermis surrounding all the vascular bundle (Fig. 3C).
The epidermal cells had sinuous ]]>
Fig. 3D) and abaxial (Fig. 3E) faces. Front views showed that fertile and sterile leaves presented stomata only on the abaxial face, characterizing them as hypostomatous. The stomatal complexes were anomocytic, with two to five cells surrounding the guard cells (Fig. 3E).
The comparative ]]>
2), higher sclerophylly index (0.13g/cm2) and higher stomatal density (27.75stomata/mm2) than fertile leaves (
Table 1).
Discussion
Ferns show adaptations which permit their survival under extreme environmental conditions (Page 2002). In ]]>
M. squamulosa such as the occurrence of stomata on the abaxial face were evidenced. According to Dickison (2000), hypostomatous leaves lose less water than plants that have stomata on the adaxial face because this face of the epidermis is directly exposed to sunlight, which significantly increases the temperature and elevates the level of transpiration. Furthermore, the distribution of stomata is related with the thickness of the leaf, i.e. thinner leaves tend to be hypostomatous and present a lower intercellular resistance than thicker leaves (Parkhurst 1978).
The anomocytic stomatal complex of M. squamulosa was also found in various Polypodium species studied by Tejero-Díez et al. (2010), another genus of Polypodiaceae. However, this is not a pattern of this family, since Tejero-Díez et al. ]]>
The chlorenchyma was the most abundant tissue of the mesophyll of M. squamulosa. De la Sota (1963) observed that M. megalophylla ]]>
Epiphytes are exposed to higher light incidence and to a higher water deficit. Plants under these conditions tend to present high stomatal density (Larcher 2000). The greater number of stomata, the higher is the efficiency during gas ]]>
M. squamulosa presented more stomata than the fertile leaves, what may contribute to a major xeromorphism.
This study ]]>
M. squamulosa were wider, resulting into greater areas, although the fertile were longer. Previously, Sehnem (1970) and Jaime et al. (2007) reported different sizes of both leaf types, although no statistical comparisons were made by these authors. Fertile leaves measured 3 to 10cm in length, and were up to 1.5cm wide, whereas sterile leaves were about 15cm long and 2cm wide. In the present study, both foliar types exhibited dimensions close to those described by the ]]>
The V-shaped xylem observed in M. squamulosa was also registered by Ribeiro ]]>
et al. (2007) for Anemia villosa Humb. & Bonpl. ex Willd. (Anemiaceae). In species of the Polypodium plesiosorum group, Tejero- Díez et al. (2010) described the xylem as C, X and T-shaped.
The sclerified layer ]]>
M. squamulosa surrounding the cells of the vascular bundle was also seen in the rhizome of this species by Suffredini et al. (2008). The species also has phenolic compounds (Jaime et al. 2007), which are responsible for the brownish color of the support tissues and parenchymal cells close to the vascular bundle (Ogura 1972), as found in this study. ]]>
The presence of hypodermal tissue in M. squamulosa leaves is a typical characteristic of xeromorphic leaves; it is responsible for water storage and also participates in plant support (Kraus 1949, Esau 1965). Tejero-Díez et al. (2010) observed the presence of a hypodermis in the leaves of Polypodium ]]>
species (Polypo-diaceae). In epiphytic angiosperms, Noguera-Savelli & Jáuregui (2011) also reported the occurrence of hypodermis in only four of 11 analyzed species of Orchidaceae. The authors observed that the hypodermis was disposed in different ways, indicating variability in the arrangement in different epiphytic species.
The ]]>
M. squamulosa. The xeromorphic characteristics of epiphytic plants are an adaptation to the environment where water is supplied in pulses (Benzing 1990). In addition to the hypodermis, the Microgramma species present specialized mechanisms for light collecting ]]>
et al. 2009). Hietz & Briones (1997) studied the relation between water availability and the distribution of epiphytic ferns and verified that even species that developed into humid forests show adaptations to xeromorphism.
Fertile and sterile leaves of M. ]]>
are classified as sclerophyllous by Rizzini (1976) because they have a sclerophylly index below 0.6g/cm2. Despite the equal classification, the sterile leaves showed sclerophylly indexes statistically higher than the fertile leaves. Sclerophyll leaves are adapted to environments where water is supplied in pulses because they can reduce their excessive water loss (Sobrado & Medina 1980) and are also more resistant to herbivory (Edwards et al. 2000).
Leaf dimorphism is frequent in epiphytes, although little is known about the variables which control or induce appearance of fertile leaves. These variables include climatic sazonality or are related to other exogenous factors, to the age of the plants and to their distribution on different zones of the phorophyte, or even, fertile leaves can appear apparently, spontaneously (de la Sota 1971). In the present study, there was found ]]>
M. squamulosa. These sterile leaves may invest more in biochemical defenses against herbivores and, therefore, have a higher specific leaf mass (Mehltreter 2008). The higher leaf mass resulted in the higher sclerophylly index, which combined with the higher stomatal density can diminish water loss.
Acknowledgments
The authors are thankful to the Universidade Feevale for support and to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the master scholarship to the first author.
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*Correspondencia: Ledyane Dalgallo Rocha: Programa de Pós-Graduação em Qualidade Ambiental, Universidade Feevale, RS 239, 2755 - Novo Hamburgo-RS, 93352-000, Brazil; ledyane@gmail.com Annette Droste: Programa de Pós-Graduação em Qualidade Ambiental, Universidade Feevale, RS 239, 2755 - Novo Hamburgo-RS, 93352-000, Brazil; annette@feevale.br Günther Gehlen: Programa de Pós-Graduação em Qualidade Ambiental, Universidade Feevale, RS 239, 2755 - Novo Hamburgo-RS, 93352-000, Brazil; guntherg@feevale.br Jairo Lizandro Schmitt: Programa de Pós-Graduação em Qualidade Ambiental, Universidade Feevale, RS 239, 2755 - Novo Hamburgo-RS, 93352-000, Brazil; jairols@feevale.br 1. Programa de Pós-Graduação em Qualidade Ambiental, Universidade Feevale, RS 239, 2755 - Novo Hamburgo-RS, 93352-000, Brazil; ledyane@gmail.com, annette@feevale.br, guntherg@feevale.br, jairols@feevale.br
Received 08-XII-2011. Corrected 04-VIII-2012. Accepted 03-IX-2012.