Tropical Agricultural Research Vol. 18 Variation of Pyrrosia heterophylla (L.) Price and Evidence for Occurrence of Two Other Pyrrosia Species in Sri Lanka R.H.G. Ranil, D.K.N.G. Pushpakumara1 and T. Sivananthawerl1 Postgraduate Institute of Agriculture University of Peradeniya, Peradeniya Sri Lanka ABSTRACT. Pyrrosia heterophylla (L.) Price (Panam Pethi) is a common creeping fern species belonging to the family Polypodiacea found naturally in South India and Seychelles Island and Sri Lanka. It is used in indigenous medicine. Although detailed information is scanty for P. heterophylla, a wide range of morphological variation exists within the species. The objective of this study was to identify variation of P. heterophylla in Sri Lanka. The study was carried out in Kalutara, Kandy, Kegalle, Kurungala, Matale and Ratnapura districts. Variable characters such as habit and habitats of the species, shape, color, venation, thickness, width, length of sterile fronds, length, width and spore arrangement of fertile fronds, scale and cross sections of rhizomes were assessed in 28 samples. Three major habits were identified for the species on the basis of arrangement of fronds on the substrate. The cross sections of rhizome and venation pattern of sterile fronds were not significantly different within and among samples. Leaf shape, leaf length, leaf width and thickness of sterile fronds and length and width of fertile fronds were variable within and among samples. Out of 28 samples, four samples deviated from normal spore arrangement of P. heterophylla. In three samples, a sub marginal spore arrangement was observed whilst the other samples showed interrupted spore arrangement. Out of four samples, three closely resembled P. piloselloides whilst the other sample closely resembled P. niphoboloides. This is the first record of occurrence of P. niphoboloides and P. piloselloides in Sri Lanka, and continuation of the same observations for at least another season and research on cytology of the samples are required for confirmation of these findings. INTRODUCTION Pteridophyte flora is an important component of the biodiversity of Sri Lanka. At present, 343 Pteridophyte species have been recorded from Sri Lanka (Fernando, 2002). Except for few species of Pteridophytes, the majority is distributed in terrestrial habitats. Terrestrial Pteridophytes usually prefer moist and shady environment and thus a majority of them are concentrated in various habitats of tropical rainforests. Although less recognized, Pteridophytes have the ability to provide food, medicine, fiber, craft, building material, abrasive and material for decoration (Winter and Amoroso, 2003). Pyrrosia is a relatively common fern genus in the family Polypodiaceae. It contains about 50 species. Natural distribution of the genus extends from Central Africa 1 Department of Crop Science, Faculty of Agriculture, University of Peradeniya, Peradeniya, Sri Lanka. Ranil, Pushpakumara & Sivananthawerl through South and South East Asia to Oceania and New Zealand (Winter and Amoroso, 2003). Six Pyrrosia species have been recorded from Sri Lanka, namely Pyrrosia heterophylla (L.) Price, P. gardneri (Mett.) Sledge, P. lanceolata (L.) Farwell, P. ceylanica (Giesenh.) Sledge, P. pannosa (Mett.) Ching and P. porosa (Presl.) (Fernando, 2002). P. heterophylla (which was previously called as Drymoglossum heterophyllun (L.) Trimen) has renamed as Pyrrosia heterophylla by Price (Hovenkamp, 1986). It is a local common species which is restricted to South India, Sri Lanka and Seychelles and grows on tree trunks and branches, shrubs and rocks in lower and middle altitude areas (Hovenkamp, 1986). It is a creeping fern, which has a long and slender rhizome usually covered by multi cellular, fine structures called scales. It has dimorphic fronds. The sterile frond is mainly responsible for photosynthesis and shows a wide range of variation in terms of length, width, thickness and shape, whilst the fertile frond is longer, thin and linear and its spores are arranged on the lower surface of the lamina (Santhosh and Madhusoodanan, 1998). P. heterophylla is used in indigenous medicine to prepare oil treatment, and leaves are used as a styptic to arrest capillary haemorrhages and eczema (Jayaweera, 1981). P. heterophylla has first been collected from Sri Lanka by Paul Hermann in the period of 1672-1675 and first described by Linnaeus (1749) in Flora Zeylanica (Sledge, 1960). Later, Thwaites (1864) has described the species in the Plantarum Zeylaniae. The last description of P. heterophylla was compiled by Beddome (1883) in his Handbook to the ferns of British India, Ceylon and Malay Peninsula. Hovenkamp (1986) has compiled a monograph for all Pyrrosia species in the world, which contains description of P. heterophylla. However, such description is mainly based on South Indian materials and only a few samples have been collected before 1900 from Sri Lanka. Santhosh and Madhusoodanar (1998) have prepared a detailed description of P. heterophylla for South India. As in any other Sri Lankan Pteridophytes, detailed information of P. heterophylla is scanty. Information on ecological, morphological and anatomical variation and reproductive biology of P. heterophylla is not well documented, although such information is vital for conservation and sustainable uses of P. heterophylla. Therefore, the objective of this study was to identify the morphology, habitats and host plant variations of P. heterophylla populations in Sri Lanka. MATERIALS AND METHODS Sampling of Pyrrosia heterophylla Review of literature, observation of herbarium samples at the Royal Botanical Gardens, Peradeniya and individuals in Kandy district revealed that the species is distributed in and around Kandy district including Matale and Kegalle. Thus, samples for identification of variation in P. heterophylla were collected from Kandy, Matale, Kegalle Kurunegala, Ratnapura and Kalutara districts on a random basis. Details of each collection positions are given in Appendix 1. Natural forest margins, homegardens and roadsides of each district were visited for collection of samples. After initial observations, undisturbed habitats of natural forests were excluded because the occurrence of P. heterophylla is limited due to low light intensity inside the forest. At each place where P. heterophylla was observed, sample specimens with fertile and sterile fronds were collected for further observation. Variation of Pyrrosia heterophylla (L.) Data collection Data was collected in the field and in the laboratory. Plant habit and its microclimatic environment were identified at each sampling location. Plant habit of each sample was identified on the basis of arrangement of fronds along the rhizome on the substrate. Microclimate of each sampling location was identified using three light levels namely, direct, medium and low sunlight. At each location, the habitat was identified based on existing vegetation as natural forest margins, homegardens or roadsides. Then, the substrates of P. heterophylla were identified based on epiphytic and lithophytic nature. If epiphytic samples were observed host plant species was identified to species level by using preserved specimens of host plants. In the laboratory, a representative specimen from each sample was preserved to be deposited at the National Herbarium, the Royal Botanical Garden, Peradeniya. The remaining specimens were observed for morphological variation as described in Table 1. Initially, character variations were obtained and analyzed and the data were analyzed using hierarchical cluster analysis procedure of SAS (SAS Software, 1999). Out of the 28 collected samples, only 12 samples had fertile fronds, and only these samples were used for the cluster analysis. Further, for cluster analysis, instead of length and width, length to width ratio was used. Table 1. Morphological characters observed to identify variation in Pyrrosia heterophylla. Character Method of observation 1. Rhizome characters (a) Cross section Rhizome was sectioned from 3 places and observed using a dissecting microscope (2×10) and line diagrams drawn. (b) Thickness Three places of rhizome were selected from each samples and thickness measured using a vernier caliper. (c) Scales of rhizome Shape and color and transparency of scales were observed using a light microscope (10×10) and line diagrams drawn. 2. Sterile frond (a) Length, width and thickness Thirty-five leaves were selected from each sample and length, width and thickness measured using a vernier caliper. (b) Shape Shape of sterile fronds was assessed using 1-4 scale (1-rounded, 2-oblong, 3-lanceolate, 4-linear) developed after observation of a large number of samples. (c) Color Color of leaf was measured using a Munsel color chart. (d) Venation Venation pattern of 54 sterile fronds of 28 samples was observed, after obtaining transparent appearance by keeping fronds 10 minutes in boiling water. 3. Fertile frond (a) Length and width (b) Spore arrangement Eighty four fertile fronds were selected from 12 samples and length and width of fertile frond were measured in the same way as sterile fronds. Eighty four fertile fronds were selected from 12 samples and spore arrangement and sequence of the spore development were observed at different maturity levels (from spore development to spore release from sporangia) using a dissecting microscope (2×10). Ranil, Pushpakumara & Sivananthawerl RESULTS AND DISCUSSION Morphological variation of Pyrosia heterophylla Rhizome characters P. heterophylla has a long, creeping, slender rhizome. The diameter of rhizome varied from 0.5-0.9 mm. It was covered with brown color scales. The cross section of rhizome varied from terete to cylindrical shape. Cortical sclerenchyma sheath, and two or three vascular bundles were clearly observed from all samples (Fig. 1). The rhizome was covered by multi cellular, fine structures called scales. Shape of the scale varied from round to linear and their margins varied from dentate to ciliate (Fig. 2). Some scales had long cilia (Fig. 2a). The central part of the scale was blackish brown in color and middle and outer parts were usually brown and light brown, respectively. However, transparent outer parts were observed in scales of four samples. Frond characters Sterile fronds of P. heterophylla were simple and fleshy. Variation of sterile frond characters observed in the study is given in Table 2. Four frond shapes namely, rounded, oblong, lanceolate and linear were identified in sterile fronds (Fig. 3). Fronds had 0.1-0.3 cm long stripes. Veins of sterile fronds were initialed from anastomosis and formed large costal areoles and small marginal areoles (Fig. 4). Free veins of areoles were forked or simple. Midribs were presented in two thirds of the leaf and except midribs other veins were immersed. Color of the sterile frond varied from yellow green to dark green. Fertile fronds of P. heterophylla were simple, longer and linear than sterile fronds. Variation of fertile fronds length and width of samples is given in Table 2. In fertile fronds, apexes were rounded. Length of stripes ranged from 0.3-0.4 cm and showed little variation within and among samples (Fig. 5). Table 2. Variation of frond characters of Pyrrosia heterophylla. Leaf characters Mean (± SE) Range Within plant variation (%) Between plant variation (%) Sterile frond Length (cm) Width (cm) Thickness (cm) Fertile frond Length (cm) Width (cm) 2.67 (± 0.12) 1.43 (± 0.02) 0.12 (± 0.01) 5.62 (± 0.34) 3.45 (± 0.05) 1.30 - 5.80 1.10 - 1.90 0.01 - 0.25 2.30 - 21.00 3.00 - 04.00 45 62 41 23 32 55 38 59 77 68 Note: SE - Standard Error Variation of Pyrrosia heterophylla (L.) Arrangement of sori Sori of P. heterophylla were arranged on lower surface of the fertile frond. At the initial stage spores were white, when maturing, they became brownish. Lower surface of fertile fronds of 24 samples were totally covered by the sori (acrostichoid spore arrangement) (Fig. 5a). Three samples showed the sub marginal spore arrangement where the mid rib was clearly visible (Fig. 5b), whilst another sample showed interrupted spore arrangement where the mid rib was visible in some areas and sori were not uniformly distributed (Fig. 5c). a b c Fig. 1. Cross section of rhizome of Pyrrosia species (×100). Note: a - cortcal sclerenchyma sheath, b - vascular bundle and c - epidermis. a b c Fig. 2. Shapes of rhizome scales of Pyrrosia species (×100). Note: a - triangular, b - rounded and c - linear. Ranil, Pushpakumara & Sivananthawerl a b c d Fig. 3. Shape of sterile fronds of Pyrrosia species (×1). Note: a - rounded, b - oblong, c - lanceolate, d - linear. b a Fig. 4. Venation pattern of sterile fronds of Pyrrosia species (×2). Note: a - stipe, b - areoles. Fig. 5. Three spore arrangements of Pyrrosia species (×1). b a c Note: a - acrostichoid, b - sub-marginal, c - interrupted spore. Variation of Pyrrosia heterophylla (L.) Habits and habitats of Pyrrosia heterophylla Three major plant habits were observed on the basis of arrangement of fronds along the rhizome on substrates. They were: leaves arranged along the rhizome and one leaf partially covered by another leaf (Habit I); leaves arranged on both side of the rhizome and they grow parallel to the substrate (Habit II); and leaves arranged one side of the rhizome and grows away from the substrate (Habit III) (Fig. 6; Table 3). A majority of the plants occurred in homegardens (Table 3). Most of the ferns preferred homegarden as habitats, because of high moisture and shady environment. In comparison with roadside habitats, homegardens are cool and shaded. Thus, a majority of P. heterophylla was observed in homegardens. Except in forest boundaries, P. heterophylla was rarely observed inside natural forests. Variation of host plants of P. heterophylla is given in Table 4. Host plants ranged from small ornamental shrubs of one meter height such as Acalypha wilkisiana to tall tree of over 20 m height such as, Mangifera indica, Artocarpus heterophyllus and Peltophorum pterocarpum (Table 4). It grows well even on tree trunks, which have smooth bark such as Areca catechu as well as rough bark (Mangifera indica) and grows on both monocots (Caryota urenus) and Dicots (Citrus aurantum). Table 3. Different habitats, plant habits and light levels of observed samples. Light Level Plant habit Habitat Low Medium High Total I II III Total Homegarden 1 11 5 17 - 5 12 17 Roadside 7 2 2 11 4 6 1 11 Total 8 13 7 28 4 11 13 28 Fig. 6. Variation of plant habits of Pyrrosia heterophylla (×0.5). Habit I Habit II Habit III Note: Habit I - leaves arranged along the rhizome; Habit II - leaves arranged both sides of the rhizome; Habit III - leaves arranged one side of the rhizome. Ranil, Pushpakumara & Sivananthawerl Table 4. Variation of host plant of Pyrrosia heterophylla. Host plants Common name Habit of host plant Occurrence (%) Codiaeum verigatum Croton (E) Shrub 17 Peltophorum pterocarpum Pare mara (S) Tree 11 Mangifera indica Mango (E) Tree 11 Artocarpus heterophylla Jak fruit (E) Tree 11 Acalypha wilkisiana Acalypa (E) Shrub 11 Pithecelobium dulci Andara (S) Shrub 7 Gliricidia sepium Gliricidia (E) Shrub 7 Areca catechu Arecunut (E) Tree 7 Myristica fragrans Nutmeg (E) Tree 4 Dracaena fragrans Dracaena (E) Shrub 4 Crescentia cujete Rum tree (E) Shrub 4 Citrus aurantum Sour orange (E) Tree 4 Caryota urenus Kitul (S) Tree 4 Note: E - English name; S - Sinhala name Cluster analysis The dendrogram developed from cluster analysis using all observed variables is shown in Figure 7. It is clear from Figure 7 that all samples can be divided into three significant clusters at the distance of 1.0054. Sample 26 is clustered and separated from all other samples. Length and width of fertile fronds of sample 26 is comparatively high (Table 5). The interrupted spore arrangement was unique to this sample and rhizome scale had transparent outer parts. Such characters are deviations from normal characters of P. heterophylla (Table 5). According monograph of world Pyrrosia species, this sample closely resembled P. niphoboloides, which has been restricted to North West and East Madagascar (Hovenkamp, 1986). P. niphoboloides had not previously been recorded in Sri Lanka. Three samples (3, 5 and 6) are also grouped into a separate cluster and are different from all others, due to the sub marginal spore arrangement. They are different from are P. heterophylla due to transparent outer part of scales. Literature revealed that sub marginal spore arrangement and transparent scales are more close to P. piloselloides than P. heterophylla. P. piloselloides has also not previously been recorded in Sri Lanka. This is the first record in Sri Lanka for the occurrence of P. niphoboloides and P. piloselloides. However, it is important to note that this identification of P. niphoboloides based on only one sample is questionable. Hence, observation of samples for at least one more season is necessary for the confirmation of these findings. Such information will provide consistency of characters observed in identification of new species. Further, investigations on cytological molecular and biochemical information will support the conformation of such occurrences in Sri Lanka. Variation of Pyrrosia heterophylla (L.) Fig. 7. Dendrogram of hierarchical cluster analysis for all variables. Note: sn - sample number. Table 5. Significant differences in spores arrangement, scale color and ratio of length to width of fertile fronds of three Pyrrosia species. Characters P. heterophylla P. piloselloides P. niphoboloides Spores arrangement Acrostichoid Sub marginal Interrupted Scale color Light brown Transparent Transparent Ratio between length and width of fertile fronds 1.56 0.92 5.45 CONCLUSIONS P. heterophylla shows a wide range of variation for characters of sterile and fertile fronds, habit and habitats. Cluster analysis revealed that P. heterophylla samples could be separated into three groups. Majority of the samples belong to P. heterophylla whilst one sample was suggested as P. niphoboloides and three samples as P. piloselloides. This is the first record of the occurrence of P. niphoboloides and P. piloselloides in Sri Lanka. Ranil, Pushpakumara & Sivananthawerl ACKNOWLEDGEMENTS Authors wish to thank Mr. W.P. de Winter, Editor of Plant Resources of Southeast Asia, Cryptogams, Netherlands and Mr. Tom Stuart, Coordinator of American Ferns Society, USA for their generosity in providing current information with respect to identification of Pyrrosia spp. Special word of thanks to Mr. Janaka Kandepola for preparation of illustrations. REFERENCES Beddome, R.H. (1883). Handbook to the Ferns of British India, Ceylon and the Malay Peninsula. Thacker Spink and Company, Calcutta, India. pp. 410-413. Fernando, B. (2002). Ferns of Sri Lanka. Fern Society of Sri Lanka, David Garage Road, Katuneriya, Sri Lanka. Hovenkamp, P. (1986). A monograph of the fern genus Pyrrosia (Polypodiaceae). Leiden Botanical Series. No: 9. pp. 281-310. Jayaweera, D.H.A. (1981). Medicinal plant (indigenous and exotic) used in Sri Lanka. Part IV. The National Science Council of Sri Lanka, Colombo, Sri Lanka. pp. 220-221. Linnaeus, C.V. (1749). Flora Zeylanica, Holmiae. Santhosh, N. and Madhusoodanan, P.V. (1998). Fern Flora of South India: Taxonomic Revision of Polypodioid Ferns, Daya Publishing House. Delhi, India. pp. 113-116. Sledge, W.A. (1960). The Polypodiaceae and Grammitidaceae of Ceylon. Bulletin of the British Museums (Natural History). British Museum, London, 2(5). SAS (1999). SAS institute Inc., Software/Release 8, Cary, NC, USA. Thwaites, G.H.K. (1864). Enumeratio Plantarum Zeylaniae. London. Winter, W.P. and Amoroso, V.B. (2003). Plant resources of Southeast Asia, cryptogams: Ferns and fern allies. Backhuys Publications, Leiden. No: 15(2). Variation of Pyrrosia heterophylla (L.) APPENDICES Appendix 1. Locations of samples of Pyrrosia species collected for the study. Sample No. Species Districts Location 1 P. heterophylla Kandy University premises, University of Peradeniya 2 P. heterophylla Kandy University premises, University of Peradeniya 3 P. piloselloides Kandy University premises, University of Peradeniya 4 P. heterophylla Kandy Udispattuwa, Teldeniya 5 P. piloselloides Kandy University premises, University of Peradeniya 6 P. piloselloides Kandy Horticultural Research and Development Institute, Gannoruwa. 7 P. heterophylla Kandy University premises, University of Peradeniya 8 P. heterophylla Kandy Kandy to Colombo road, Kadugannawa 9 P. heterophylla Kegalle Weradeniya, Nelumdeniya 10 P. heterophylla Kegalle Mangala gama, Molagoda 11 P. heterophylla Ratnapura Elapatha, Amuwala 12 P. heterophylla Ratnapura Elapatha, Amuwala 13 P. heterophylla Ratnapura Gurupaskada watta, Amuwala 14 P. heterophylla Ratnapura Ilanga watta, Amuwala 15 P. heterophylla Ratnapura Ilanga watta, Amuwala 16 P. heterophylla Ratnapura Arambe gedara, Amuwala 17 P. heterophylla Kandy Sinhapitiya, Gampola 18 P. heterophylla Kandy University premises, University of Peradeniya 19 P. heterophylla Kurunegala Godawela, Polgahawela 20 P. heterophylla Kurunegala Kiribamuna, Ibbagamuwa 21 P. heterophylla Kurunegala Walakumbura, Alauwa 22 P. heterophylla Kandy University premises, University of Peradeniya 23 P. heterophylla Matale Galwadukumbura, Kawdupelella 24 P. heterophylla Matale Galwadukumbura, Kawdupelella 25 P. heterophylla Matale Aranwatta, Alupothuwala 26 P. niphoboloides Kalutara Gonaduwa, Moronthuduwa 27 P. heterophylla Kandy Udurawana, Wattegama 28 P. heterophylla Kandy Getambe, Peradeniya INTRODUCTION MATERIALS AND METHODS CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES << /ASCII85EncodePages false /AllowTransparency false /AutoPositionEPSFiles true /AutoRotatePages /All /Binding /Left /CalGrayProfile (Dot Gain 20%) /CalRGBProfile (sRGB IEC61966-2.1) /CalCMYKProfile (U.S. Web Coated \050SWOP\051 v2) /sRGBProfile (sRGB IEC61966-2.1) 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