Introduction

Phytoliths (Greek, phyto= plant, lithos= stones) or Plant stones are amorphous form of Silicon dioxide (known as “opal”) deposition found in many plants⁴. The term “phytolith” refers only to microscopically recognizable shapes, not amorphous pieces or traces of silica detectable only by microchemical methods that would not be recognized as discrete types¹².

As some plants were found to produce distinguishable or characteristic or “diagnostic” shaped phytoliths, they are well established as useful tools in archaeology, angiosperm taxonomy¹¹ and recently for Nanotechnology¹⁰. The occurrence of silicon as silica bodies or phytoliths in many plants has been correlated with mechanical support, reduction of water loss, protection from herbivores, prevention of the entry of pathogens, protection from metal toxicity, root elongation, increased capture of light for photosynthesis and in the cooling of leaves⁹.

In a recent review, Mazumdar⁹ mentioned that phytoliths in pteridophytes have immense possibility to be useful as taxonomic tools in future for the delineation of family (e.g., Polypodiaceae), genus (e.g., Equisetum) and species (e.g., Isöetes weberi) and also for phylogenetic analysis (e.g., Pteridaceae). But, so far, only 225 species of pteridophytes belonging to 21 families have been studied⁹ out of 12,000 species of pteridophytes distributed world wide².

Recently, Mazumdar and Mukhopadhyay reported the occurrence and morphology of opal phytoliths in 6 species of Thelypteridaceae⁴⁶, 13 species of Lycopods⁵, 4 species of eusporangiate ferns & 23 species of leptosporangiate ferns and one species of Horsetails⁷ and suggested that phytoliths could be an useful tool in the taxonomy of pteridophytes as some plate or sheet like phytoliths having distinct features occur in some species. Among the aquatic pteridophytes phytoliths were reported in a few taxa. Iriarte and Paz (2009) observed the occurrence of angular, polyhedral epidermal cells like phytoliths in Isöetes weberi Herter. They didn’t find any diagnostic form in Marsilea ancylopoda A. Braun. Mazumdar and Mukhopadhyay⁵ observed large plate like phytoliths in Isöetes coromandelina L. Sundue¹³ reported the absence of phytoliths in aquatic ferns Ceratopteris pteridoides (Hook.) Hieron and C. richardii Brongn.

On the other hand, occurrence of phytoliths in Pteris vittata L. (Pteridaceae), commonly known as “Chinese brake fern” is confusing. Sundue¹³ didn’t find phytoliths in P. vittata. But¹, found them from lamina and vein areas of the pinnae.

Objective of the present study was to find the occurrence and morphology of phytoliths in some aquatic ferns and Chinese brake fern.

Materials and Methods

Fresh plants of Azolla pinnata R.Br. (Azollaceae) and leaves of Salvinia molesta Mitch. (Salviniaceae), Marsilea minuta L. (Marsileaceae) and Pteris vittata L. (Pteridaceae) were collected from Golapbag Locality of Burdwan, West Bengal, India. Leaves of Ceratopteris thalictroides (L.) Brongn. (Parkeriaceae) were obtained from the herbarium of Pteridology Laboratory, Botany Department, The University of Burdwan.

Phytoliths were extracted by wet oxidation method of Mazumdar and Mukhopadhyay⁴. All the materials were washed thoroughly several times with tap water and then with distilled water. About 0.2 g of each plant materials were taken in the test tubes and digested with concentrated Nitric Acid in water bath. Digested materials were washed with distilled water at 1700 rpm for 10 minutes and then boiled in 10 % Hydrochloric acid in water bath to remove calcium, then, washed with distilled water many times to remove the acid. The processed materials were then centrifuged with acetone at 1700 rpm for 10 minutes each time and dried with Acetone.

Dried materials were mounted in 10 % glycerin on the glass slide. Photographs were taken with QWin image analyzing software (machine QG2-32, version V 3.2.0) in Leica DM 3000 microscope.

Results and Discussion

Opal phytoliths were detected in sporophytic plant bodies of the studied aquatic ferns Marsilea minuta, Azolla pinnata, Salvinia molesta and Ceratopteris thalictroides. Morphology of phytolith showed wide range of variations, from sheet like to amorphous. Commonly occurring forms were cylindrical or triangular, square or rectangular plates. However these were observed in many ferns during previous works by present authors⁴⁵⁶⁷ and found to be not useful as distinguishing character.

In Marsilea minuta hyaline large plates formed by adjoining smooth rectangular phytoliths with parallel margins resembling epidermal cells (Fig. 1 A, B) were found. In A. pinnata smooth hyaline plates with wavy margins resembling epidermal cells were also observed (Fig. 1 E). Plates like bodies were also found in Salvinia molesta (Fig. 1 F, H) and Ceratopteris thalictroides (Fig. 1 G), but not with distinguishable shapes.

In Pteris vittata variables shaped phytoliths including large epidermal plates with parallel striations (Fig. 1 C) and heavy deposited plates (Fig. 1 D) were found.

Present study reports for the first time the occurrence of phytoliths in aquatic ferns of M. minuta, S. molesta, A. pinnata and C. thalictroides. This study also confirms the early report of the occurrence of phytoliths in P. vittata by Chauhan et al.¹ But presence of phytoliths with blunt ends, smooth sides and denticulate, sparsely denticulate or shallowly lobed margins characteristic of pteridoid ferns¹³ were not observed among the phytoliths extracted during the present study. However, specific chemical test is required to confirm whether extracted bodies from P. vittata are silica phytoliths or not.

Conclusion

Plate like phytoliths observed in M. minuta and A. pinnata are considered here as characteristic at least at the genus level. Further investigation is required for their usefulness at species level. Recently, Mazumdar et al.⁸ have shown that it is possible to make distinction at species level in Equisetum (Equisetaceae) by phytolith characters.

Lithophytic, poikilohydric and epiphytic pteridophytes accumulate high amount of silica for mechanical support and reduction of water loss¹. Occurrence of phytoliths in all the presently studied species indicated importance of silica for aquatic ferns. However, biological and ecological advantages of phytolith formation in aquatic plants is to impart mechanical support or to provide protective measures against insect attack are not clear and might be an interesting field for future research.