Introduction

There are approximately 1100 species of pteridophytes within 70 families and 192 genera distributed in India, with more than 349 species occurring in the Western Ghats¹. With such high diversity of pteridophyte species occurring in the biodiversity hot spot of the Western Ghats,numerous of which are endemic, rare and endangered, it is challenging to get the appropriate information required for species identification and discrimination with the desired speed². The easiest and one of the most efficient solution to this is classical taxonomic, and morphological data compendia of the extant fern diversity.

Palynology of ferns has proved to be very useful in the identification and discrimination of various fern taxa³. There have been several Palynology studies of pteridophytes that examined the characteristics of spore samples using basic microscopic staining techniques^{2, 4-9} to the modern use of scanning electron microscope (SEM)^10-16. Palynology data depositories have been very useful for taxonomic purposes in ferns and have been previously used for identifying palynological fern sediments¹⁷, interspecies discrimination^18-19, relatedness and phylogeny of the fern species^20-24.This study aims to investigate the spore morphology and perine ultra structure of selected fern species found in the Western Ghats of India by using SEM. The results of the current study may provide a key for future studies in general fern morphology, palynology, fern identification and discrimination of pteridophytes in the Western Ghats. 

Materials and methods

Forty-five different species of fern spore samples collected in June 2016, from the herbarium collection of Morajkar²⁵,of Kudremukh National Park (13°1′ to 13°29′ N latitude and 75°0′ to 75°30′ E longitude), Western Ghats, thatwere made available at St Aloysius College (Autonomous) Mangalore, Karnataka. These fern samples were considered in the study as Morajkar²⁵ has reported ferns that are rare, endemic and threatened in the Western Ghats. The spore surface was studied using Scanning Electron Microscope (SEM) after treating the spores in ultrasonic wave bath (50-60Hz) and subsequent washing with ethanol in a three-step process as elaborated by Hu et al.²⁶. The spore structures and ornamentation were observed and photographed under a tabletop SEM.Two SEM models were utilized in the study namely, Hitachi TM 3030 and JOEL JFC-1600. For each pteridohyte species, multiple spore samples were examined from two different herbarium accession. The spore appertures, size, perine structures and surface variations were recorded as per Tschudy²⁷. 

Results and Discussion

All 45selected fern spore samples were successfully treated and spore morphology was examined in details (Table1,  Fig 1 and Fig 2 Plate 1 and Plate 2).  The size of the spores were found to be highly variable and ranged from 20 X 20µm to 60 X 60µm with an average mean of 44µm X 38µm. Angiopteris helferiana and Nephrolepis hirsutula had the smallest spore, while Lygodium flexuosum and Osmunda huegeliana were found to have large spores. Similar observations of these fern spore sizes were also made by Makgomol¹¹, Zenkteler², and Shaikh & Madhav²⁸.

Table 1: Characteristic spore morphology of 45 selected pteridophytes examined in the current study.

Plate 1: 1. A. aureum, 2. A. philippense, 3. A. raddianum, 4. A. anceps, 5. A helferiana, 6. A. tripinnata, 7. A. pulchra, 8. A. yoshinagae, 9. B. orientale, 10. B. semicordata, 11. B. subcrenatoides, 12. C. tenuifolia, 13. C. gigantean,  Click here to view plate

Plate 2: 25. L. microphyllum, 26. M. torrensiana, 27. M. speluncae, 28. M. membranaceum, 29. M. punctatum, 30 N. hirsutula, 31. O. chinensis, 32. O. huegeliana, 33. P. calomelanos, 34. P. aquilinum, 35. P. argyraea, 36. P. biaurita, 37. P. camerooniana. Click here to view figure

The SEM results divide the spores of 45 fern species into 2 aperture types, trilete type and monolete type. 26 species of fern spores were found to be trilete, while 19 species had monolete aperture types. The majority of the trilete spores were globose (13), followed by11 species with tetrahedral and two with spheroidal type of spore shape. Spores with monolete apperture type were dominated by ellipsoidal shape constituting 14 species while spheroidal and globose spores were found in two and three species respectively. The apertures of most of the fern spores were found to be in accord with the studies of Vijayakanth & Sathish⁷ and Vijayakanth et al.²⁹ It was also observed that the fern ultrastructure, apperture type and to much extent the shape of the spore were found to be similar within a fern genus.

The perispore forms the outer surface and often the characteristic contours of the spores.The most common ornamentation of perispore in the studied pteridophytespore samples was found to be gammate and psilate, with nine and eightfern species having the respective spore ornamentation. Among all the spores examined, Pteris camerooniana was the only fern with clavate type of perine ornamentation. Echinate type of perine ornamentation was seen in only two fern species namely Bolbitis semicordata and Tectaria coadunate. B. semicordata is known to be endemic to the Western Ghats.Additionally, seven species namely S. tenera, Osmunda huegeliana, Pteris quadriaurita, Bolbitis subcrenatoides, Cyathea nilgirensis, Cyclosorus parasitica, and Tectaria polymorpha are also known to be endemic to the Western Ghats. Perine structure of these species are very important for accurate identification and differentiation in future studies. As noted (Table 1), Perine structure of S. tenera and P. quadriaurita had gammate while T. polymorpha and O. huegelianahadbaculate type of perine ornamentation. Other endemic fern species namelyB.  subcrenatoides, C. nilgirensis, and C. parasitica had scabrate, psilateandstriate type of perine ornamentation respectively.In addition to the perine surface structures fern species namely, Sellaginella tenera and Sellaginella delicatula had winged perispore. Earlier study such byZhou et al.³⁰ has also reported winged perispores in Sellaginella species. The perispore surfaces observed in the current study were mostly in agreement with earlier studies^2,7,17,29, this suggests that perispore morphology is generally consistent within species. A similar observation was also made by Moran et al²³ in his study of perispore structures in Dryopteridaceae.

The spore structure given by Zenkteler², describes the spore of P. aquilinum as tetrahedral and trilete with verrucae and baculate structures depicting an irregularly granulate perine structure. A similar spore structure with an uniform gammate perine structure is observed in the current study. It was noted by Zenkteler² that P. aquilinum had the potential to release large numbers of spores which are known to be toxic and carcinogenic ^31-32 and are dispersed by wind. This fern also known as the bracken fern, grows like a weed that can be an immense threat to the ecosystem.There has been confusion with this fern with P. revolutum ³³ and both have been misidentified with each other.Even thought here are studies that report the distribution of this fern in various regions, such as Eastern Ghats⁷,The Western Ghats ^34-36 and other parts of India³⁷, there are also claims that P. aquilinum is not to be found in the Indian sub continent ³⁸.Hence future comprehensive taxonomic studies with a standard method, along with molecular taxonomy are required in this regard, considering the toxicity and weed ability of this fern species.

Based on the results its found that all the pteridophytes within each family have the same aperture type, with the exception of Dryopterideceae. Both the species of Tectaria were found to have monolete aperture type, while A. tripinnata who belongs to the same family had a trilete spore aperture. In most cases the shape of spore were same within genus, but differed distintictively when perispore surface was compared. These results significantly elaborates on the importance of spore ultra structure and other features in identification and discrimination of pteridophytes. Contrary to this the findings of Yañez et al.,¹⁵ suggested that the spores with similar characteristics in phylogenetically unrelated families do not alow palynological features to have an evolutionary value in determining relationships between groups above the genus level. But the study on spore morphology and charaters by Passarelli et al.,³⁹ suggest that perispore characters have distinct diagnostic value, since different combinations of ornamentation/structure were found to vary considerably among the pteridophytes.He also inferred that, when spore ornamentation is useful complementary feature at the specific level identification and discrimination when used in combination with other morphological traits of fern sporophytes. Additionally, the study undertaken by Chao and Huang ²⁴ to investigate spore morphology evolution in Pteris species, revealed that spore characters, similar to leaf morphologies, reversed several times, but the combination of both characters could be useful in identification and discrimination and to establish more natural relationships within this group of fern species. Hence fern spore morphology is an important source of charateristics with systematic value in fern taxonomy.

Conclusion

The variability found in the spore ultra structure and perispore ornamentation reflects the morphological differences observed in the sporophyte of the selected fern species. This study will be an important source of characteristics with systematic value in fern taxonomy. The spore appertures, size, perine structures and surface variations observed within the selected pteridophytes will help future taxonomic studies of ferns in th Western Ghats of India. The ultara structure of the spores will suppliment other morphological characters and advance molecular tools,to enableprecise and standard taxanomic identification and differentiation in other fern species in question, such as P. aquilinum and P. revolutum debate.

Acknowledgement

Our sincere thanks to Rev Fr Swebert D’Silva SJ, Principal St Aloysius College (Autonomous), Dr Asha Abraham, HOD, Department of Post Graduate Studies and Research in Biotechnology, St Aloysius College and Rev Fr Dr Leo D’Souza SJ, Director, Dr Küppers Laboratory of Applied Biology, St Aloysius College for providing the research facilities.

Conflict of Interest

No potential conflict of interest is reported by the authors

Funding Source

None

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