Anatomical , Histochemical and Cytogenetic Features of Doryopteris triphylla ( Pteridaceae )

Doryopteris triphylla (Pteridaceae-Cheilanthoideae) grows in xeric habitats in Brazil, Paraguay, Uruguay and Argentina. The aim of this study was to characterize D. triphylla anatomically, histochemically and cytogenetically. For anatomical characterization, rhizomes, roots, petioles and leaves were made and then stained using Safranine-Astra Blue for further observations. Leaf blades were also cleared. For histochemical analysis, leaf cross sections were stained with different reagents to identify glandular trichomes compounds. For cytogenetic characterization, a karyogram was performed using laboratory cultivated roots. Results show a dictyostelic rhizome covered with scales with apical secreting gland; diarch roots; petiole cross-sections show thick cuticle, uniseriate epidermis, parenchymatic cortex cells with thick walls and a vascular bundle with two xylem groups; and hypostomatic fronds with glandular trichomes. Histochemical studies of secretion products of the glandular trichomes were positive for polysaccharides, pectins, lipids, acid lipids, dihydroxyphenols, phenols and flavonoids. Cytogenetically, D. triphylla is described as a diploid species (2n = 60), with chromosomes gradually decreasing in size. The apical glands in scales of rhizomes, the presence of two xylem groups in the vascular bundle in the petiole and the glandular trichomes on the abaxial surface are new contributions to the species. The type of chemical products secreted by glandular leaf trichomes and karyotype estimation is shown for the first time in this species.


Introduction
Cheilanthoid ferns (Pteridaceae, Cheilanthoideae) are characterized as a cosmopolitan and diversified group inhabiting arid and semiarid environments.
General characteristics of the morphology and anatomy of the sporophyte have been described elsewhere [13] [14] [15].In general, there are few records of histochemical studies in ferns, particularly in Doryopteris; Salatino and Prado (1998) [16] reported the presence of glycosylated flavonoids in Doryopteris ornithopus (Mett.)J. Sm.
Reports on the karyotype of Doryopteris are limited to chromosome counts.
The two basic chromosome numbers in the family Pteridaceae are x = 29 and 30 [17].Moran and Yatskievych (1995) [18] indicate a base number of x = 30 for Doryopteris; most of the species are diploid (n = 30, 2n = 60) and a great part of the cytogenetic analyses are based on gametophytic chromosome counts [19] [20].The phenomenon of interspecific polyploidy in the genus was mentioned for D. nobilis L., with n = ca 60 for individuals from Paraguay [20] and in D. palmata L., with n = 60 for material from the Galapagos Islands [18].Karyotype analyses were performed for other ferns, such as some species of Acrostichum L., Lycopodium L. and Woodwardia Sm. [21] [22] [23], and of Polypodium L. [24].The aims of this study were to characterize the anatomical traits of Doryopteris triphylla associated with its xeromorphic condition, analyze the chemical com- pounds secreted by the glandular trichomes, determine the sporophytic chromosome number, and estimate the corresponding karyotype.

Anatomical Studies
To study the characteristics of the abaxial and adaxial epidermis of the leaf blades, as well as of the reflexed margin, the material was subjected to the clarification technique of Dizeo de Strittmatter [26] and Astra blue staining [27].For anatomical studies, cross free hand sections of rhizome, root, petiole, leaf blade and rachis were made.Sections were bleached in 1:1 commercial sodium hypochlorite: water, rinsed with distilled water and stained with safranin-Astra blue.In all cases, slides were mounted in a water/glycerin solution (1:1).Cross sections of petiole were made at three levels: basal (next to the rhizome), intermediate, and apical (next to the leaf blade).Stoma types were determined using the classification of Van Cotthem (1970) [28]; stoma length and width were measured and stoma density was calculated as the number of stomata per mm 2 .
Length of glandular trichomes and paraphyses was measured, with 10 repetitions; mean and standard deviation (sd) were calculated.Mean thickness of cell wall of the rhizome scales was measured.The anatomical descriptions of the rhizome were based on Metcalfe and Chalk (1972) [29].

Cytogenetic Studies
For cytogenetic studies, roots of plants cultivated in the laboratory were used; they were pretreated with 0.002 M 8-hydroxyquinoline at 4˚C for 24 h.They were fixed with Farmer solution (ethanol:acetic acid 3:1).They were rinsed with distilled water, then hydrolyzed in 1N HCl at 60˚C for 20 minutes, then rinsed in distilled water again and finally mounted and squashed with a drop of 2% propionic hematoxylin.Counts were made using seven metaphase plates.The karyogram was performed on a metaphase plate with well dispersed chromosomes, which were classified using the nomenclature proposed by Levan et al. (1964) [36].

Morphology and Anatomy
Rhizome.Short rhizome of 2 -5 mm in diameter, dictyostelic; from outside to inside, it is composed of a single-layer epidermis, and cortex of sclerenchyma tissue; vascular bundles are surrounded by 2 -3 layers of pericycle and endodermis with Casparian band in the radial walls (Figure 1 Leaf blade.In the abaxial epidermis, cells are rectangular, with sinuous walls and smooth cuticle.Three types of stomata are observed: anomocytic, diacytic and polocytic (52%, 36% and 12%, respectively); mean length of stomata is 42.6 µm (sd 0.42 µm) and mean width is 36.1 µm (sd 3.95 µm).Stomata are evenly distributed and at the same level of or slightly above epidermis cells; the recorded density is 127 stomata/mm 2 (Figure 2 In the sori, paraphyses are observed among sporangia; paraphyses are mostly originated in the receptacle, a few of them seem to originate from the base of the sporangium foot.These paraphyses are bicellular glandular trichomes composed of a foot and a head, 48.66 µm (sd 5.22 µm) in length (Figure 2(j)).

Histochemical Analysis
The secretion products of the glandular trichome are presented in Table 2 and Figure 3.

Cytogenetic Studies
The results indicate a sporophytic chromosome number of 2n = 60.Chromosome length is 1.59 to 4.38 µm (Figure 4).Total length of the haploid chromosome complement is 100.06 µm.The estimated karyotype formula is 2 m + 7 sm+ 12 st + 9 t (Figure 4).Values of total length of each chromosome (c),

Discussion and Conclusions
The anatomy of the dictyostelic rhizome of Doryopteris triphylla does not contribute with traits for species identification.This structure has also been observed in other Doryopteris species [37], in other Pteridaceae species and in different families of ferns [38].Rhizome scale with a glandular trichome on the apex is a morphological trait that has not been described in floristic studies mentioning this species [13] [15].This trait may have been unnoticed because trichomes often fall off easily when scales are adult, as observed in some species of Pteris: P. ciliaris Eat., P. cretica L., P. denticulata Sw., P. ensiformis Burm.f. and P. multifida Poir.[39].
In cross section of the leaf blade, the thickness of the palisade parenchyma is markedly lower than that of the spongy parenchyma; this trait was indicated for other Pteridaceae species occurring in exposed sites, such as Adiantopsis chlorophylla, D. concolor, D. lorentzii and Trachypteris pinnata [37] [40] [41] [45].
In sori, glandular paraphyses occurring with sporangia are mentioned for the first time in this species.These sterile structures differ from the trichomes on the abaxial side of the lamina on being of larger size.Thus, we agree with Wagner (1964) [46] that paraphyses tend to provide some sort of protection to the developing receptacle and young sporangia from external effects, and that they are usually found in ferns of sunny, dry or exposed environments as is the case of Doryopteris triphylla.
Histochemical tests allowed us to detect and locate in situ the principal metabolites present in trichome secretions.Our results indicate that the content is of complex nature, including polysaccharides, lipids, phenols and flavonoids.The presence of non-cellulosic polysaccharides such as pectin was demonstrated using Rutheniun red in the cell wall of the secreting gland and in the site of attachment of the stalk with the lamina.Pectin might be related to translocations of secondary metabolites [47].
Positive reactions for lipids were obtained in the glandular head using Sudan IV and Neutral red, and for acid lipids in all the trichome with Nile blue.According to Werker (2000) [48], lipid metabolites would play a protective role.
Phenolic substances, detected using ferric chloride and Vanillin/H 2 SO 4 , were found in trichomes.Flavonoids were the only type of phenolic compounds histochemically identified in these trichomes using Vanillin/HCl and aluminum chloride.Those compounds are present in glandular trichome secretions in numerous species [49] and are important for plant protection against visible and ultraviolet light, and play a significant role in plant chemical defense against the attack of herbivores, bacteria and fungi [50] [51].The histochemical analysis of glandular trichomes shows a subcuticular chamber, which is interpreted as the site of accumulation of chemical substances before their release.
The revision of cytogenetic records in the literature reveals a noticeable lack of works aimed at establishing the inter-and intra-chromosomal relationships that provide the parameters of a karyotype.The comparison of the karyotype estimated for D. triphylla with the few karyotypes described for other fern genera shows some similarities.The karyotype formula of D. triphylla exhibits metacentric and submetacentric chromosomes, a characteristic shared with others species of Acrostichum [21], Lycopodium [22] and Woodwardia [23].Likewise, D. triphylla is diploid and shows a karyogram whose chromosome length (1.59 µm to 4.38 µm) is very similar to that observed in diploid species of Polypodium (2.2 µm to 4.5 µm) [24].The comparison also shows that the length of the haploid chromosome complement of D. triphylla is similar to the length observed in diploids of Woodwardia (ca. 100 µm) [23].However, chromosome length of D. triphylla and Polypodium is similar, but in Polypodium, most of the chromosomes are telocentric and the remaining chromosomes of the complement are acrocentric.Murray (1985) [24] and Marcon et al. ( 2003) [21] did not mention metacentric or submetacentric chromosomes for that genus.
Moreover, although D. triphylla and the Acrostichum species analyzed by Marcon et al. (2003)  [21] are diploid, with 2n = 60, and similar in terms of chromosome morphology, chromosome length of Acrostichum (approximately 5.0 µm to 8.0 µm) is almost twice that observed in D. triphylla, which is reflected in its haploid chromosome complement, of nearly 100 µm in D. triphylla and 192 µm in Acrostichum.
We conclude that Doryopteris triphylla is a typically xeromorphic fern, since it exhibits sclerenchyma tissue in root, rhizome and petiole, glandular trichomes in frond, sinuous thickened walls in rectangular epidermis cells; thick cuticle and rhizome scales with glands.All of these traits were indicated by Hevly (1963) [43] for ferns occurring in xeric habitats.Anatomical characters, trichome secretion products, chromosome counts that confirm the basic number established for the genus (x = 30) and karyogram contribute with novel information for Doryopteris, which may help understand the phylogenetic relationships within the genus.
Photographs of observations were taken with an Olympus Q-color digital camera attached to an Olympus BX43 microscope, 7.1 MP Canon Powershot camera attached to a Zeiss Axiostar Plus microscope, Olympus-U-CMA D3 camera mounted on Olympus CX41 microscope, and a Nikon camera mounted on Nikon SMZ 800 stereoscopic microscope.Observations of fluorescent stained sections were made with an Olympus BX43.U-TVO.5xc-3 epifluorescence microscope, using a UV 365 nm filter.
(d)).The indument is composed of bicellular glandular trichomes, consisting of a unicellular foot and head 63.43 µm (sd 5.91 µm) long, and located in the abaxial epidermis (Figure2(e)).Epidermis cells on the adaxial surface and reflexed margin exhibit sinuous and thick walls (Figure2(f) Figure 2(g)).In cross section, the leaf blade is dorsiventral and hypostomatic; both epidermis are single-layered and have a thick cuticle; the palisade parenchyma is composed of 1 -2 cell layers occupying 1/3 of lamina thickness, whereas the spongy parenchyma with big intercellular spaces (Figure 2(h)), is 5 -6-layered and occupies 2/3 of the lamina thickness; vascular bundle surrounded by a conspicuous endodermis with Caspary bands (Figure 2(i)).The pseudoindusium is subterminal, multilayered, with tracheids in the proximal portion, and two cell layers in the recurved distal portion (Figure 2(j)).

Table 2 .
Histochemical identification of compounds in glandular trichome of Doryopteris triphylla.

Table 3 .
Cytogenetical analysis.Values of total length of each chromosome (C), length of the short arm (s), length of the long arm (l) and centromeric index (Ci) are shown.