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.
Cheilanthoid ferns (Pteridaceae, Cheilanthoideae) are characterized as a cosmopolitan and diversified group inhabiting arid and semiarid environments. Morphological circumscription of the genera of this subfamily has been difficult, generating taxonomic controversies [
The genus Doryopteris s.s. was circumscribed by Yesilyurt et al. (2015) [
General characteristics of the morphology and anatomy of the sporophyte have been described elsewhere [
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 [
The aims of this study were to characterize the anatomical traits of Doryopteris triphylla associated with its xeromorphic condition, analyze the chemical compounds secreted by the glandular trichomes, determine the sporophytic chromosome number, and estimate the corresponding karyotype.
Botanical material was obtained from LIL, MCNS and SI [
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 [
For histochemical tests, cross sections of fresh fronds of Doryopteris triphylla were made and the following reagents were applied: Toluidine blue to detect polysaccharides [
Place | Vouchers |
---|---|
Argentina. Buenos Aires-La Cascada | Morrone, Guissani 6238 (SI) |
Argentina. Entre Ríos-El Palmar | Morrone 5881(SI) |
Argentina. Catamarca-Tintigasta | Prado s.n. (MCNS) |
Argentina. Tucumán-Barrancas Coloradas | Venturi 807 (LIL) |
Argentina. Tucumán-Barrancas del Río Salí | Schreiter 8877 (LIL) |
Argentina. Tucumán-Huasa Pampa | Villa Carenzo, Vaca 2122 (LIL) |
Argentina. Tucumán-Ciudad Universitaria | Villa Carenzo, Legname 1874 (LIL) |
Argentina. Tucumán-Río Loro | Villa Carenzo 1540 (LIL) |
Argentina. Tucumán-San Pedro de Colalao | Delgado, Ríos, Neira 915, 916, 917, 919, 920, 921 (LIL) |
Argentina. Tucumán-Las Higueritas | Legname, Cuezzo 4635C (LIL) |
Argentina. Tucumán-Río Las Juntas | Castillón 3521A, B (LIL) |
[
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) [
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.
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 (
Root. The root has a uniseriate epidermis composed of thin-walled cells; the cortex has 2 - 3 parenchyma layers of irregular and thin wall cells, and 4 - 6 layers of sclerenchyma cells of non-lignified thickened walls that are near the diarch stele (
Petiole. In cross section, the petiole is terete to slightly semi-terete; it exhibits single-layered epidermis covered with a thick cuticle. The cortex has 2 - 3 layers of thick-walled parenchyma cells and 5 - 6-layers of thin-walled parenchyma cells. The vascular bundle is surrounded by 2 - 3 layers of pericycle and endodermis with thickened radial walls (
xylem groups, the larger one oriented to the dorsal side, in an open V arrangement, and the other group, in ventral position, composed of 5 - 10 xylem elements, is observed at the three section levels.
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/mm2 (
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 (
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 (
The secretion products of the glandular trichome are presented in
The results indicate a sporophytic chromosome number of 2n = 60. Chromosome length is 1.59 to 4.38 µm (
Reagent | Target compounds | Reaction | Figure |
---|---|---|---|
Toluidine Blue | Polysaccharides | + | 3(a) |
Ruthenium Red | Pectin | + | 3(b) |
Phloroglucinol | Lignin | - | 3(c) |
Sudan IV | Lipids | + | 3(d) |
Nile Blue | Acid lipids | + | 3(e) |
Neutral Red | Lipids | + | 3(f) |
Ferrum Chloride | Dihydroxyphenols | + | 3(g) |
Vanillin/H2SO4 | Phenols | + | 3(h) |
Vanillin/HCl | Flavonoids | + | 3(i) |
Aluminum Chloride | Flavonoids | + | 3(j) |
length of the short arm (s), length of the long arm (l) and centromeric index (ci) are shown in
Chromosome pair | C (µm) | l (µm) | s (µm) | Ci% | Chromosome type |
---|---|---|---|---|---|
1 | |||||
2 | 3.14 | 1.71 | 1.43 | 45.54 | m |
3 | 1.59 | 0.82 | 0.76 | 47.79 | m |
4 | 3.69 | 2.8 | 0.88 | 23.84 | sm |
5 | 3.43 | 2.7 | 0.73 | 21.28 | sm |
6 | 3.21 | 2.49 | 0.71 | 22,11 | sm |
7 | 3.15 | 2.21 | 0.94 | 29.84 | sm |
8 | 2.78 | 2.17 | 0.61 | 21.94 | sm |
9 | 2.65 | 1.98 | 0.67 | 25.28 | sm |
10 | 2.25 | 1.7 | 0.55 | 24.44 | sm |
11 | 3.26 | 2.74 | 0.53 | 16.25 | st |
12 | 4.04 | 3.45 | 0.59 | 14.60 | st |
13 | 3.96 | 3.28 | 0.68 | 17.17 | st |
14 | 3.9 | 3.35 | 0.55 | 14.10 | st |
15 | 3.64 | 3.07 | 0.52 | 14.28 | st |
16 | 3.62 | 3 | 0.62 | 17.12 | st |
17 | 3.33 | 2.75 | 0.57 | 17.11 | st |
18 | 3.21 | 2.79 | 0.42 | 13.08 | st |
19 | 3.16 | 2.62 | 0.54 | 17.08 | st |
20 | 3.11 | 2.62 | 0.49 | 15.75 | st |
21 | 3.08 | 2.63 | 0.44 | 14.28 | st |
22 | 2.94 | 2.54 | 0.4 | 13.60 | st |
23 | 4.38 | 3.86 | 0.52 | 11.87 | t |
24 | 4.22 | 3.94 | 0.27 | 6.39 | t |
25 | 4.04 | 3,53 | 0.5 | 12.37 | t |
26 | 3.87 | 3.5 | 0.36 | 9.30 | t |
27 | 3,78 | 3.33 | 0.45 | 11.90 | t |
28 | 3.5 | 3.16 | 0.34 | 9.71 | t |
29 | 2.95 | 2.65 | 0.3 | 10.16 | t |
30 | 2.73 | 2.5 | 0.1 | 3.66 | t |
subtelocentric (st) chromosomes vary in size, ranging between 2.25 - 3.43 µm and between 2.94 - 3.26-µm in length, respectively. Telocentric (t) chromosomes form two groups, one containing three pairs of chromosomes of length ranging between 3.5 - 4.38 µm, and the other including moderately smaller chromosomes (2.76 - 3.26 µm).
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 [
The root structure is similar to that of other cheilanthoid ferns, Doryopteris concolor (Langsd. & Fisch.) Kuhn, D. lorentzii (Hieron.) Diels and Trachypteris pinnata (Hook. f.) C. Chr. [
The petiole of D. triphylla exhibited the presence of an additional group of xylem elements in the vascular bundles, which has been also observed in others Doryopteris, such as D. concolor and D. lorentzii [
In the epidermis of D. triphylla, stomata length, width and density have mean values similar to those reported for other cheilantoid ferns: Adiantopsis chlorophylla (Sw.) Fée, Argyrochosma nivea (Poir.) Windham, Myriopteris aurea (Poir.) Grusz & Windham, Cheilanthes buchtienii (Rosenst.) R. M. Tryon, Cheilanthes notholaenoides (Desv.) Maxon ex Weath., Cheilanthes pilosa Goldm., Doryopteris concolor and D. lorentzii [
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 [
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) [
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 [
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) [
Phenolic substances, detected using ferric chloride and Vanillin/H2SO4, 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 [
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 [
Moreover, although D. triphylla and the Acrostichum species analyzed by Marcon et al. (2003) [
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) [
This project was funded by Fundación Miguel Lillo. We thank to all herbaria curators for allowing us to consult material; and Lic. Leila Bordón for designing the figures.
Neira, D.A., Andrada, A.R., de los Ángeles Páez, V., Rodriguez, A.M., Ríos, N.F., Martínez, O.G. and Hernández, M.A. (2017) Anatomical, Histochemical and Cytogenetic Features of Doryopteris triphylla (Pteridaceae). American Journal of Plant Sciences, 8, 907-920. https://doi.org/10.4236/ajps.2017.84061