OBSERVATIONS ON AEROPHYTIC CYANOBACTERIA AND ALGAE FROM TEN CAVES IN THE OJCÓW NATIONAL PARK

This study, carried out in 2010–11, focuses on species composition and distribution of cyanobacterial and algal communities colonizing ten caves (Biała, Ciemna, Koziarnia, Krakowska, Łokietka, Okopy Wielka Dolna, Sąspowska, Sypialnia, Zbójecka and Złodziejska Caves) in the Ojców National Park (South Poland). A total of 85 taxa were identified, 35 of them belonging to cyanobacteria, 30 chlorophytes, and 20 belonging to other groups of algae. Aerophytic cyanobacteria dominated in these calcareous habitats. Nine species, Gloeocapsa alpina, Nostoc commune, Chlorella vulgaris, Dilabifilum arthopyreniae, Klebsormidium flaccidum, Muriella decolor, Neocystis subglobosa, and Orthoseira roseana, were the most abundant taxa in all the caves. The investigated microhabitats offer relatively stable microclimatic conditions and are likely to be responsible for the observed vertical distribution of aerophytic cyanobacteria and algae.


INTRODUCTION
Caves are unique in terms of specific natural characteristics such as: microclimate, temperature, humidity and others.According to M u l e c and K o s i (2009), caves have a special place in human history and many caves are inscribed on the United Nations Educational, Scientific and Cultural Organization (UNE-SCO) World Heritage List.Recently, many caves have experienced intensified tourist visits and to make them more attractive for visitors, artificial illumination was installed which changed physico-chemical conditions in the caves.However, most caves, at least in Europe, are damp and the walls at the entrance are covered with green algal gelatinous mass (P o u l i č k o v á and H a š l e r , 2007).At the entrance of limestone caves and on the surfaces around electrical lights, cyanobacteria compete for light with other algae, bryophytes and ferns, but in the deepest recesses of the caves they are usually the only phototrophs (R o u n d , 1981).Most caves represent stable environments characterized by uniform temperatures throughout the year, high humidity and low natural light (H e r n á n d e z --M a r i n é and C a n a l s , 1994; D u c a r m e et al.  (2008,2009,2010,2011).
The aim of this study was to present the spatial distribution of aerophytic algae and cyanobacteria growing in ten caves in the Ojców National Park differing in location, morphology and environmental conditions as part of an extensive study of caves in the Polish Jura.

STUDY AREA
The caves are one of the most characteristic elements of the Ojców National Park and its surroundings.The presence of caves in this area has been noticed for a long time (C h a r d e z and D e l h e z , 1981; S z e l e r e w i c z and G ó r n y , 1986; B i s e k et al. 1992; G r a d z i ń s k i et al. 1995a, b, 1996, 1998, 2007; G r a d z i ń s k i and S z e l e r e w i c z , 2004; G ó r n y and S z e l e r e w i c z , 2008).These caves were created by underground waters dissolving Jurassic marine limestones (M i c h a l i k and P a r t yk a , 1992).Some of the caves exceed 300 m in depth.Ten caves: Biała, Ciemna, Koziarnia, Krakowska, Łokietka, Okopy Wielka Dolna, Sąspowska, Sypialnia, Zbójecka and Złodziejska Caves, were studied in the Park (Fig. 1; Table 1).The most attractive caves from the natural and environmental point of view, having corridors longer than 50 m, include the following: Biała, Ciemna, Koziarnia, Krakowska, Łokietka, Okopy Wielka Dolna, Sąspowska and Zbójecka Caves (P a rt y k a , 1997).Only the Łokietka and Ciemna Caves (among the studied ones) are open to tourists.

MATERIALS AND METHODS
Samples for algological analysis were taken from the ten caves of the Ojców National Park in the spring, summer and autumn of 2010 and 2011.Algal crusts were scraped from the walls and ceiling, using a scalpel, into labelled plastic bags and analysed under a light microscope (Jenamed 2) or inoculated for culturing on agar plates (Figs 4-6).Additionally, in the two caves (Łokietka and Ciemna) open to tourists, samples were also collected from around artificial light sources, at different distances from the source, and from sites of most intensive growth.Scraped material was used directly for observation under the light microscope.Two sampling zones were distinguished in each cave: A -light zone, comprising the entrance (usually lit by sunlight and well-oxygenated); and B -dark zone comprising the chambers illuminated only by weak natural daylight or artificial light.
Collected organisms were cultured on standard Bristol agar medium, at 20 o C under a 12/12h light/dark cycle at 3000 μEm -2 s -1 provided by 40W fluorescent tubes.For transmission electron microscopy (TEM), cells were fixed as previously described (Massalski et al. 1995).Ultra-thin sections were cut with glass knives on a Reichert-Jung ultramicrotome.Observations and photographs were made with a TESLA BS 500 electron microscope.Cells for TEM were double fixed with glutaraldehyde in phosphate buffer and postosmicated with osmium tetroxide in the same buffer, then they were dehydrated in series of alcohols, embedded in the synthetic epoxy resin "SPURR" and polymerized at 70 o C for 18 h.For scanning electron microscopy (SEM), the samples were treated with 36% HCl, washed several times with distilled water and boiled in concentrated H 2 O 2 with KClO 3 in order to remove organic matter.Cyanobacteria and algae were identified according to: A n a g n o s t i d i s and K o m á r e k (1988); B r o w n and B o l d (1964); B r o w n and L e a n (1969); E t l l and G ä r t n e r (1988,1995); H o f f m a n n (1986,2002); K o m á r e k and A n ag n o s t i d i s (1986,1989); K r a m m e r and L a ng e -B e r t a l o t (1986,1991).

RESULTS
A total of 85 aerophytic algae and cyanobacteria species were identified in the ten caves in the Ojców National Park (Fig. 2; Table 2).Overall, cyanobacteria were dominant and represented by 35 species (40% of the total), green algae 30 species (32.7%), and 12 diatoms species (27.2%).The frequently encountered species among cyanobacteria were as follows: Aphanocapsa parietina Näg., Calothrix fusca (Kutz.)Born.& Flah., Chroococcus tenax (Kirchn.)Hieronymus and Nostoc commune Vauch.ex Born.& Flah.They occurred both in the light (A) and dark (B) zones of the studied caves.Microscopic observations revealed that cyanobacteria are arranged in patina assemblages which are blue, brown, green, or gray, and are arranged mosaic-like inside the caves.Among the cyanobacteria present, the following were dominant only in the caves open for tourism, i.e. the Łokietka Cave and the Ciemna Cave: Gloeocapsa alpina (Näg.)F. Brand, Nodularia harveyana Thuret ex Born.& Flah.and Tolypothrix epilithica Näg.The most rare green algae species were: Bracteococcus minor (Chodat) Petrová (Fig. 3), Desmococcus olivaceum (Pers.ex Arch.)Laudon, Muriella decolor Vischer, Scotiellopsis terrestris (Reisigl) Punčochářová & Kalina and Thelesphaera alpina Pascher.In the Zbójecka and Ciemna Caves, green algae such as: Chlorella vulgaris Beijerinck, Coleochlamys perforata (Lee & Bold) Ettl & Gärtn.and Klebsormidium montanum (Skuja) Watanabe, were encountered on well-illuminated surfaces.Diatoms were present in the zone of both direct and diffuse sunlight, but they usually preferred the wet habitats (permanently wet or periodically sprinkled with water dripping and sipping from the ceiling and walls).Among the aerophytic diatoms, the most frequently encountered taxa were: Diadesmis contenta (Grun. in Van Heurck) D.G. Mann in Round, Hantzschia amphioxys (Ehr.)Grunow, Luticola nivalis (Ehr.)D.G. Mann, Orthoseira roseana (Rabenh.)O'Meara, and Pinnularia borealis Ehr.The most species of cyanobacteria and algae (in Biała Cave -11 species, Ciemna Cave -16, Koziarnia Cave -9, Krakowska Cave 20, Łokietka Cave -12, Okopy Wielka Dolna -18, Sąspowska Cave -13, Sypialnia Cave -23, Zbójecka Cave -25, Złodziejska Cave -13) were found on the rocky surfaces and in the indentations close to the entrance (the A bright zone).There were also species found only in the places within direct sunlight: Chroococcus cf.Gradziński et al. (1995aGradziński et al. ( , b, 1996Gradziński et al. ( , 1998Gradziński et al. ( , 2007)) (1981), P e n t e c o s t , (1992) as well as P e n t e c o s t and Z h a n g ( 2001), the type of substratum is an important factor determining the species composition, distribution and structure of algal communities.It was observed likewise that cyanobacteria such as: Anabena cf.oscillarioides Bory de Saint-Vin., Gloeocapsa biformis Erceg., Nostoc punctiforme (Kütz.ex Hariot) Hariot, and some aerophytic diatoms (Caloneis silicula (Ehr.)Cl., Grunowia tabellaria (Grun.)Rabenh.and Hantzschia amphioxys (Ehr.)Grun.) were dependant on temperature, light and humidity.These cyanobacteria and algae prefer humid places during their development, but they also display considerable resistance to drying as well as to low air temperature during winter.The adaptation mechanism of algae living in low temperature is not yet precisely known (M u l e c and K o s i , 2008, 2009).It can be therefore concluded that the environmental conditions in the ten caves of the Ojców National Park are stable and conducive to consequent development of cyanobacteria, green algae and aerophytic diatoms.
2004; P o u l i č k o v á and H a š l e r , 2007; L a mp r i n o u et al. 2009).According to L a m p r i n o u et al. (2012), a typical cave is described as having three major habitat zones based on light penetration and intensity: the entrance-, transition-, and dim light zone.Moreover, cave characteristics, such as dimensions, morphology, location, orientation and rocky substrate, can play an important role for the biocommunity structure.All caves belong to habitats of extreme conditions characterized by a low content of nutrients (P e d e r s e n , 2000; M u l e c et al. 2008).However, many groups of organisms prefer such conditions for the colonization and growth.The following occur most frequently in caves: liverworts, mosses, some ferns, flowering plants, algae and cyanobacteria (D o b a t , 1970; K u e h n et al. 1992; S a n c h e z et al. 2002; M u l e c , 2005; M u l e c et al. 2008; M u l e c and K o s i , 2009).Algae often play a key role in the food webs and in the colonization processes of rocky habitats (sediments, rocky surfaces, artificial material) as well as they produce colourful effects on the caves walls (G o l u b i č , 1967).These processes are favoured by usually stable environmental conditions prevailing in caves.All these factors make caves attractive for cosmopolitan species, which consequently eliminate the native components (P i p a n , 2005).Although infrequently, but new algal species have also been identified in caves (J o n e s , 1964; H e r n a n d é z --M a r i n é and C a n a l s , 1994).Aerophytic algae and cyanobacteria are usually observed in cave entrances illuminated by direct or indirect sunlight and also around the artificial light in caves that are open to tourists (P e n t e c o s t , 1992; M u l e c , 2005; M ul e c et al. 2008; C z e r w i k -M a r c i n k o w s k a and M r o z i ń s k a , 2011).In Poland algological studies in caves have been carried out by the following researchers: S t a r m a c h (1963); M r o z i ń s k a -B r o d a and C z e r w i k --M a r c i n k o w s k a , (2004); C z e r w i k -M a r c i nk o w s k a and M r o z i ń s k a ,

Fig. 2 .
Fig. 2. Participation of particular groups of algae and cyanobacteria in ten studied caves.

Fig. 6 .
Fig. 6.The wall of Ciemna Cave cover with growths of aerophytic cyanobacteria and algae, liverworts and mosses.
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