MORPHOLOGY AND PECULIAR FEATURES OF SPORES OF FERN SPECIES OCCURRING IN POLAND El ż

The morphology of fern spores collected from natural sites in Poland was examined under light microscopy. Spore samples represented 44 species in 18 genera and in 13 families. Only spores of Ophioglossaceae were obtained from the herbarium of the Adam Mickiewicz University in Poznań while the remaining samples were obtained from living plants. Spore size ranges between 20 to 75 μm and the spores of Osmunda regalis and Polypodium interjectum were found to have remarkably large dimensions. The spores are ellipsoidal, tetrahedral and spherical/globoid in shape. Their apertures are monolete or trilete types. The exine surface patterns are baculate, cristate, granulate, reticulate, tuberculate and verrucate. Pictures of the analyzed spores are collected in a contrasting (size, colour) table to make it easier to distinguish between species. The peculiar characters of fern spores are described after a review of major articles concerning the allergenic features of fern spores with special attention to Pteridium aquilinum whose spores and vegetative tissues revealed mutagenic and carcinogenic activity.


INTRODUCTION
Studies to determine spores of fern species native to Poland have not been extensive (P i ę k o ś 1974, P i ę k o ś 1975a, 1975b, P i ę k o ś -M i r k o w a , 1979; K a ł u z i n s k a , 1994).In the accessible literature there are some valuable morphological studies of living and fossil fern spores (T y r o n and L u g a rd o n , 1991).In such specialist, multithematic works however, which contain data relating to 1480 taxa, it is difficult to find the necessary information needed to identify fern genus or even species with the speed that is sometimes desired.A solution could be provided by compendia of the local pteridoflora, with a comparative combination of pictures belonging to native fern spores.The data included in such a compendium would enable interspecific differences between size, shape and surface sculpture of fern spores to be observed quickly and efficiently.
Information concerning morphological data of fern spores may be useful not only to botanists, but also for palynologists and allergologists.Monitoring atmospheric fern spore loads has been initiated in airstream over Edinburgh UK (C a u l t o n et al. 1995).When the fern stands are 1.6 km away from the city center in a long-term volumetric spore trap placed on a roof-top (21m above the ground) 2-3 spores on m 3 was found.Other analysis revealed that fern spores made up 6.2-8.6% of the total airspora sampled (L a c e y and M c C a r t n e y , 1994).Very light spores overcome long distances and numerous fern species inhabited urban-areas eg.Gdańsk (B u l i ń Detailed study indicated that fern spore diurnal patterns showed a late morning (09.00 and 11.00 hrs.) the peak period of dissemination, highly correlated with local meteorological conditions.The seasonal distribution of airborne fern spores includes late summer and early autumn.They are not abundantly represented in the atmosphere and are counted together with pollen grains and fungal spores.Between August and September the daily mean concentrations exceeded 750 spores on m -3 of air 1 m from the stand of ferns (L a c e y and M c C a r t n e y , 1994; S i m a n et al. 1999).With regard to personal observations some species disseminate spores much earlier e.g.Osmunda regalis spores are ready to disperse at the beginning of June (from 2-3 rd to 12 th of June).Moreover, Matteuccia struthiopteris a typical rheophyte -at the end of February/ beginning of March (very often during snow coverage).Dispersed spores of some fern species have consequences for human health through inhalation and ingestion, especially in areas where e.g.Pteridium aquilinum (bracken fern) population has increased.In England newspapers regularly print information to warn readers about the peak of the dissemination of spores e.g.Pteridium aquilinum which are known to contains toxic and carcinogenous substances, (P a g e , 1997; S i m a n et al. 1999).The most alarming information about the carcinogenic activity of the extracts from spores and vegetative tissues of bracken has been obtained from experiments that have demonstrated the induction of adducts in the DNA of cultured human (K562) cells in vitro.But the possible carcinogenic effects of spores other than bracken so far are unknown (S i m a n et al. 2000).Using extracts from spores of five fern species for the nasal test: Anemia phyllitidis, Dicksonia antarctica, Pteridium aquilinum, Pteris vittata and Sadleria pallida, it was shown that damage was induced after 2 h of exposure in atopic adult volunteers (G e l l e r -B e r n s t e i n et al. 1987).In Poland such an analysis concerning our most frequent native fern species has not yet been undertaken.

MATERIALS AND METHODS
The majority of spores were collected from living plants growing in the field, only spores of Ophioglossaceae were obtained from the herbarium of the Adam Mickiewicz University in Poznań.Sites of fern spore collection are listed in Table 1.Spore size ranges were measured in 30 samples of each species for the length of the polar and equatorial axes.The size classes followed E r d t m a n (1957): very small <10μm; small 10-25μm; medium 25-50μm; large 50-100μm; very large 100-200μm.The descriptive terms of the spore surface followed T r y o n and L u g a r d o n (1991).The main morphological microcharacters of spores of 44 species of ferns are summarized in Table 1 which contains their description, useful to distinguish between the taxa.Spore shape and sculpturing terminology is based on T r y o n and L u g a r d o n (1991).For slide preparation, spores were mounted in glycerol, viewed under 250 x magnification and photographed under a Jenamed Carl Zeiss Jena microscope with a Practica camera.
The preparation of fern spores from the herbarium specimens for LM observations was made according the method improved by H u et al. (2010).Usually spores collected from the herbarium are covered by debris (scales and glandular hairs) which affects the analysis of their surface.Therefore, Hu proposed a new technique for the removal of contamination from spores using an ultrasonic wave (50-60Hz, 90W) bath with 70% of ethanol for 10 min.before the standard fixation (H u et al. 2010).
Thick-walled fern spores are stable in a vacuum which enables SEM analysis of Pteridium aquilinum spores without dehydration techniques.Their structural characters provide stable and highly evident criteria for species determination.

RESULTS AND DISCUSSION
In the present study, the spore morphology of homosporous fern species was investigated by light microscopy.This contribution is a part of a broader study on fern spores obtained from species native to Polish pteridoflora.The group covered in this study consists of 44 taxa from approximately 53 mentioned in the Polish Check List (M i r e k et al. 2002).
The size of the majority of spores was rather medium (between 25-40 μm) in length or diameter; only spores of Osmunda regalis and Polypodium interjectum had a wide range in dimension.The size of spores may correlate with ploidy level and the number of spores per sporangium.Three spore size groups, which contained small, medium and large spores, were distinguished (Table 1).
Under LM, the spores revealed differences in their morphological characters (e.g.size, shape, colour, and the surface pattern of ornamentation).The aperture types of the spores were trilete and monolete (triradiate or simple leasural) and they were correlated with spore shape.When the shape was globose/spheroidal or tetrahedral, the aperture was trilete, and when it was ellipsoidal in the polar view, the aperture was monolete.The trilete spores have radial symmetry and were flat or convex at the distal face, while monolete, ellipsoidal or reniform spores have bilateral symmetry (Fig. 2).
The colour of trilete spores of Ophioglossaceae (a rare and threatened species in Poland) is white to light yellow, with small spheroid-tuberculate elements on the surface.Monolete spores, medium sized in the majority, ranged from light to dark brown, except for the bright yellow dye of Polypodium vulgare and P. interjectum spores.Only two species have chlorophyll--bearing spores, which were manifested as the clear green colour of Osmunda and the greenish-brown of Matteuccia struthiopteris (Fig. 2).
The sporoderm ornamentation depends on the exospore and perispore multifoliate substructure.Notably, an exospore larger than the perispore forms characteristically long, undulate folds or wings contiguous to the lower lying strata.
Under SEM analysis, tetrahedral spores of P. aquilinum (relatively small) were not distinguished from other species by any special morphological character (Fig. 1 A The seasonal distribution of airborne fern spores includes late summer and early autumn.They are not abundantly represented in the atmosphere and are counted together with pollen grains and fungal spores. Positive skin test reactions to aqueous extracts from fern spores were observed in a group of patients suffering from hay-fever (G e l l e r -B e r n s t e i n et al. 1987).Common household decorative ferns, such as Adiantum capillus-veneris, Cyrtomium falcatum and Pteris vittata, induced milder skin reactions, but Dryopteris setigera induced a much stronger one.When spore extracts of Acrostichum aureum were used in nasal provocation tests, 60-70% of patients suffering from allergic health problems reacted to this factor (B u n n a g et al. 1989).
Exposure to Lycopodium spores caused a case of asthma in a dental technician, as reported by K ob a y a s h i (in S i m a n et al. 1999).The published literature contains some information relating to the adverse effects of fern spores (skin rashes, respiratory problems, sneezing and watering of the eyes) in fern spore collectors and herbarium curators.A preventative measure against spore inhalation is to wear a face mask so as to avoid contact with dangerous particles (S i m a n et al. 1999).
, B).Their apertures are commisures, linear, narrow, and trilete.The exine is thick and densely structured.Ornamentation is similar on the distal and proximal face.The surface is irregularly covered by mild, small verrucae and bacula, without any sharp structures.The spores are heteropolar with a rounded distal face and a flattened proximal face, while their ambs are rounded to triangular.Pteridium aquilinum has the potential to realase large numbers of relatively small spores which are dispersed by wind between August and October.Bracken spore density was mainly measured in the proximity of fern populations where spore concentrations are often very high.One person may inhale 50,000 bracken spores in 10 min.(D e l a C r u z etal.2010).According to literature data, fern spores may contain a variety of carcinogens such as: ptaquiloside (norsesquiterpene glucoside); L-ecdysone and 20-hydroxy ecdysone; shikimic acid; aquilide A and quercetin, whose presence and activity have also been analysed in bracken spores (S i m a n etal.1999  and literature cited there).The DNA-damaging potential of Pteridium aquilinum spores and aqueous extract of fronds was demonstrated when mice were force--fed with bracken and were seen to have developed lung, intestinary or mammary tumours and died (S im a n etal.2000).Spores of other homosporous ferns, Anemia phyllitidis, Pteris vittata and Sadleria pallida, also induce DNA adducts, i.e. covalent modifications of the normal bases present in the DNA and detected by 32 P-post-labelling analysis (S i m a n etal.2000).

Table 1 .
The main morphological characters of spores of 44 species of ferns occurring in Poland.