OPCE-STR.vp Acta Bot. Croat. 69 (2), 163–171, 2010 CODEN: ABCRA 25 ISSN 0365–0588 Cryoseston in Stara Planina (Balkan) Mountains, Bulgaria JAROMÍR LUKAVSKÝ*, VLADISLAV CEPÁK Academy of Sciences of Czech Republic, Institute of Botany, Department of Plant Ecology, Centre for Bioindication and Revitalisation, Dukelská 135, CZ-379 82 Tøeboò, Czech Republic. Numerous snow fields persisted in the Stara Planina (Central Balkan) Mountains, some with a very light tint, but with dirty surfaces, at the end of May 2009. The cryoseston com- munity was quite different in individual snow fields. We found Chlorophyta: zygospores of Chloromonas nivalis and Chloromonas rostafiñski, Chromophyta: Chromulina ele- gans, Bacillariophyceae: Aulacoseira granulata var. angustissima, Aulacoseira italica, Cyclotella menenghiniana, Navicula nivalis, Hantzschia amphioxys, Fungi: Chionaster nivalis and Selenotila nivalis. Alpine species such as Chlamydomonas nivalis were not found in the snow fields studied, even though they were located in the alpine zone. Key words: Cryoseston, Stara Planina (Balkan), Bulgaria Introduction A luminary of cryoseston research, KOL (1968) described 78 taxa classified as cryo- seston, 63 of which were Chlorophyta, 6 Cyanobacteria, 1 Xanthophyceae, 1 Dinophyceae and 7 Fungi. Many of the described taxa were synonymous, representing different stages of a single life cycle, or were observed incompletely, and they have been progressively elimi- nated through cultivation and molecular biology (HOHAM 1973, 1979; HOHAM and MULLET 1977; HOHAM and BLIN 1979; KOMÁREK and NEDBALOVÁ 2007). The last authors listed 47 Chlorophyta in the cryoseston. In Bulgaria, cryoseston was recorded for the first time by KOL (1956) in the Rila and the Pirin Mts. These reports were confirmed by WODENITSCHAROFF (1962) in the Rila Mts., and a new locality was found by LUKAVSKÝ et al. (2009) in the Vitosha Mountains. Although the Stara Planina (Balkan) Mountains have the same geographic attributes that enable the oc- currence of cryoseston in other mountains in Bulgaria, snow algae have not previously been recorded there. ACTA BOT. CROAT. 69 (2), 2010 163 * Corresponding author, e-mail: lukavsky@botany.cas.cz In memory of Stefan Petkov 1866–1951, luminary of Bulgarian phycology Stefan Petkov, Bulgarian botanist, born on 5th of June 1866 in Lovech, Ottoman Empire; died on 8th of December 1951 in Sofia, Bulgaria. He was a lecturer in Sofia University »St. Kliment Ohridski«, the author of the first bibliography of the Bulgarian flora. From 1907, he was a member of Bulgarian Literary Society, today the Bulgarian Academy of Sciences. U:\ACTA BOTANICA\Acta-Botan 2-10\300 Lukavsky.vp 11. listopad 2010 11:28:35 Color profile: Disabled Composite 150 lpi at 45 degrees Recently, extreme habitats (e.g. snow, polar soils, thermal springs) have received in- creasing attention as a potential source of algae with unusual physiological characteristics and biochemical pathways and products (ØEZANKA et al. 2007, 2008). The isolation of new algal strains with such characteristics is the goal of this project. Material and methods Localities Snow fields were sampled in the central Stara Planina (Balkan) Mts., between the Mazalat chalet to the Dermenka chalet, during the last week of May 2009. The snow fields were tens of square meters in size. GPS positions and altitudes of the sites are given in table 1. Sampling and sample processing Snow samples were collected in plastic bags of volume ca 50 mL and transported in thermos bottles to the laboratory of the Institute of Plant Physiology in Sofia. Samples were melted and centrifuged 20 min. at 3000 g, and living material was examined using a NU2 microscope (C. Zeiss, Jena, D) with 63/0.8 and HI 100/1.35 objectives, and photographed with a DP10 digital camera (Olympus, Japan). The samples were also inoculated into Z nu- trient solution, according to Zehnder in STAUB (1961), to be isolated into unialgal culture. All data in this paper were derived only from samples collected in the field. Results and discussion In snow samples from the Stara Planina Mts., we found and determined the following organisms, (Figs. 1–55): Chlorophyta, Chlorophyceae Chloromonas nivalis (Chodat) Hoham and Mullet 1978 (syn. Scotiella nivalis, Chloromonas cryophila), Figs. 1–4, 10, 11. Cysts ellipsoid, 26–31 ´ 12–15 mm, a smooth, thick cell wall, with 6 longitudinal, twisted ribs on surface, joined into prominent thickenings at each pole (Fig. 2). Cell volume 164 ACTA BOT. CROAT. 69 (2), 2010 LUKAVSKÝ, J., CEPÁK, V. Tab. 1. Localities studied in central Stara Planina (Balkan) Mountains at end of May 2009. No Location GPS Altitude, m a.s.l. 1 near Chalet Taza N 42° 44' 19.6" E 025° 04' 16.3" 1994 2 main chain N 42° 44' 02.8" E 025° 03' 77.4" 2037 3 main chain N 42° 44' 04.6" E 025° 03' 17.5" 1993 4 main chain N 42° 44' 06.4" E 024° 59' 02.1" 1882 5 main chain N 42° 43' 64.3" E 024° 56' 35.1" 2133 6 under peak Botev N 42° 43' 02.0" E 024° 56' 30.4" 2201 7 Peak Botev N 42° 43' 02.8" E 024° 55' 05.3" 2376 8 Botev Shelter N 42° 43' 21.0" E 024° 54' 32.8" 2057 9 Near peak Zhaltets N 42° 43' 45.4" E 024° 53' 02.3" 2108 U:\ACTA BOTANICA\Acta-Botan 2-10\300 Lukavsky.vp 11. listopad 2010 11:28:35 Color profile: Disabled Composite 150 lpi at 45 degrees filled with amorphous green chloroplast, grains, and yellow-red oil drops; green cells were seen rarely. Dominated in fields 5 and 8. Flagellates not observed. The characteristic big oil drops at each cell pole (LUKAVSKY et al. 2009) were lacking but the size range and external ornamentations of the cells confirm our identification. Resting stages of several snow algae in the genus Chloromonas were formerly described as vegetative cells, and given the genus name Scotiella Fritsch. The life history of Chloro- monas nivalis was described in detail by HOHAM and MULLET (1977, 1978) and KAWECKA (1981). A characteristic feature of the cysts of some biflagellate volvocalean algae, which allows identification to species level, is the number and shape of ribs, or shape and length of spines. KOMÁROMY (1982) with respect to cyst morphology distinguished five groups, where Chloromonas nivalis stands apart. This alga dominated in spring as well as in sum- mer snow collected in the Vitosha Mts. (LUKAVSKÝ et al. 2009). It is recognised as a charac- teristic organism of middle altitude, open localities (HOHAM and MULLET 1977, KOMÁREK and NEDBALOVÁ 2007). Chloromonas rostafiñski (Starmach et Kawecka) Gerloff et Ettl (nom. dubium Chlamydomonas flavo-virens, syn. Chlamydomonas rostafiñski), Figs. 5–9, 35, 36, 41–55 Cysts ellipsoidal, 15 ´ 25 mm, cell wall thick, uniformly covered with blunt protrusions or spines (Figs. 52, 53), cell content green – orange (Fig. 43), chloroplast without pyrenoid. Flagellates egg-shaped 12 ´ 8 mm, papilla lacking, chloroplast cup-shaped without pyre- noid (Figs. 5, 36), flagella not observed. Dominant in fields 5 and 6. Chloromonas rostafiñski was discovered by Rostafiñski and published as Chlamydo- monas flavo-virens, but without the diagnosis and description required by the Botanical Code. A valid description of this organism (but from a different locality) was provided by STARMACH and KAWECKA (1965) as Chlamydomonas rostafiñski, from yellowish-green snow in the alpine zone of the Polish Tatra Mts. It has been recorded in the High Tatra Mts. (KAWECKA et al. 1979, KAWECKA 1984, LUKAVSKÝ 1994) and Vitosha Mts. (LUKAVSKÝ et al. 2009). The cell wall of resting cells is covered by numerous longitudinal ribs which show indentations of different length, and hence the papillae-like structures visible by light mi- croscopy are formed (Figs. 45, 49, 52, 53, 55). These non-motile cells are believed to be formed from vegetative cells through gradual development of cell wall ornamentation (KAWECKA and ELORANTA 1986). Sexual reproduction was described by KAWECKA (1984) as isogamy. A small joined cell, and probably the sporangium of a parasitic Chytrid at- tached to a big spore can be seen in figure 51. More research on this species in the Stara Planina Mts. is clearly necessary. Chromophyta, Chromophyceae Chromulina elegans Doflein Cells spherical (rarely with pointed posterior), diameter 6–8 mm, 1 flagellum 6–8 mm in length. One cup-shaped chromatophore, yellow-orange, without pyrenoid or stigma, a few small drops or grains in protoplasm. Present in field 9. This alga was described by Doflein from a peat-bog in Germany. NOVIS (2002) tenta- tively identified it in cryoseston collected on Mt. Philistine, Arthur´s Pass national Park, ACTA BOT. CROAT. 69 (2), 2010 165 CRYOSESTON IN STARA PLANINA (BALKAN) MTS., BULGARIA U:\ACTA BOTANICA\Acta-Botan 2-10\300 Lukavsky.vp 11. listopad 2010 11:28:35 Color profile: Disabled Composite 150 lpi at 45 degrees New Zealand, at 1600–1700 m. a.s.l. LUKAVSKÝ et al. (2009) reported the species from snowfields in the Vitosha Mountains. It is similar, also to Chromulina ettlii Hindák, which was described from brownish snow in the High Tatra Mts (HINDÁK 1969). The last had cells spherical, 7–9 mm in diame- ter, flagellum of 12–15 mm long, chromatophore one, cup-shaped, yellow-brown, lacking pyrenoid, contaractile vacuoles 1–2. Another similar species, Chromulina chionophila Stein was reported from NY USA, in coniferous canopy as yellow-green snow (HOHAM 1975), but this species has 2–3 chromatophores per cell and it is flattened in cross section. Some species ascribed to the genus Chromulina, originally defined as uniflagellate, have 166 ACTA BOT. CROAT. 69 (2), 2010 LUKAVSKÝ, J., CEPÁK, V. Figs. 1–27. Cryoseston of snow fields in the Stara Planina Mountains, collected in May 2009. 1–4, 10, 11 – zygospores of Chloromonas nivalis; 5 – zoospore, 6–9 – cysts of Chloromonas rostafiñski; 12–21 – Chionaster nivalis, variability of spores, 14, 15 – cf. C. bicornis; 22–27 – Selenotila nivalis, variability of thalli. Origins of samples: snow field No. 1. (12, 22–24, 27); No. 2. (13, 21); No. 4. (1–4); No. 5. (25, 26); No. 6. (7–11); No.7. (5, 6). U:\ACTA BOTANICA\Acta-Botan 2-10\300 Lukavsky.vp 11. listopad 2010 11:28:42 Color profile: Disabled Composite 150 lpi at 45 degrees since been shown to possess a second smaller flagellum not visible by LM (NOVIS 2002). Ultrastructural studies would be required to determine this material. Bacillariophyceae The centric diatoms Cyclotella menenghiniana (Fig. 28), Aulacoseira italica and A. granulata var. angustissima (Figs. 33, 34) were found in fields 1 and 8 (Tab. 2), but only as empty frustules. Similarly, the pennate diatom cf. Navicula nivalis (Fig. 29, cf. also Achnanthes coarctata) was also only observed as empty frustules. The only living diatom species was Hantzschia amphioxys (Figs. 30–32) from snow fields 2 and 3. ACTA BOT. CROAT. 69 (2), 2010 167 CRYOSESTON IN STARA PLANINA (BALKAN) MTS., BULGARIA Figs. 28–55. Cryoseston of snow fields in the Stara Planina Mountains, collected in May 2009. 28 – Cyclotella meneghiniana; 29 – cf. Navicula nivalis; 30–32 – Hantzchia amphioxys; 33 – Aulacoseira granulata var. angustissima; 34 – Aulacoseira italica; 35, 36 – zoospores of Chloromonas rostafiñski; 37–40 – zoospores »small«; 41–44, 46–48, 50, 51, 54, 55 – cysts of Chloromonas rostafiñski; 49, 52, 53 – empty cell walls, 51 – sporangium of a Chytri- diomycet?. Scale bar = 10 mm. Origins of samples: snow field No. 1. 49–55; No. 2. 29, 30; No. 3. 31, 32, 37–40; No. 5. 41–48; No. 6. 33, 34; No. 8. 28, 35, 36. U:\ACTA BOTANICA\Acta-Botan 2-10\300 Lukavsky.vp 11. listopad 2010 11:28:46 Color profile: Disabled Composite 150 lpi at 45 degrees Navicula nivalis Ehr. 1854 (syn. Navicula mutica var. nivalis (Ehr.) Hustedt 1911 is a cosmopolitan of high mountains from aerial biotopes such as moss mats, wet rocks (KRAMMER and LANGE-BERTALOT 1986). The other species, from our samples can be ubiquists transported by wind. Samples were not sufficient for the creation of permanent preparations, and further studies and determinations are necessary to distinguish the genera Navicula and Achnan- thes (the last has raphae only on one valve). Surprisingly no Bacillariophyceae were listed by KOL (1968), but later 33 taxa were determined in Antarctic cryoseston (ZIDAROVA 2008). Fungi Chionaster nivalis (Bohl.) Wille, Figs. 12–21. Cells colourless, 2–3 (rarely more) projections up to 25 mm long arise from a triangular centre (diameter 12–14 mm; e.g. Fig. 17) filled with numerous small spheres. Rarely, there were only 2 projections (Figs. 14–15); this form has been described as Chionaster bicornis Kol. The variability in morphology and the number of projections is very wide in our mate- rial; the specimen closest to typical Chionaster (sensu Kol) is Fig. 17. Reproduction was not observed. Dominant in field 2. Notes: This species was already recorded in Bulgaria, in the Pirin Mts. by KOL (1956), the Vitosha Mts. by LUKAVSKÝ et al. (2009), and the Rila and Pirin by LUKAVSKÝ (unpub- lished). According to the list of genera of fungi (KIRK et al. 2001), Chionaster Wille (1903) 168 ACTA BOT. CROAT. 69 (2), 2010 LUKAVSKÝ, J., CEPÁK, V. Tab. 2. Cryoseston of central Stara Planina (Balkan) collected at end of May 2009. Species distribu- tions in individual snow fields. GPS positions and altitudes see Tab. 1. d = dominate, x = present, r = rare, ZSP = zoospores. Species No of locality 1 2 3 4 5 6 8 9 Chromophyceae Chromulina elegans x Bacillariophyceae, Centrales x Cyclotella meneghiniana x Aulacoseira granulata var. angustissima x Aulacoseira italica x Bacillariophyceae, Pennales Hantzschia amphioxys x x cf. Navicula nivalis x Chlorophyceae Chloromonas nivalis, x x x d Chloromonas rostafiñski d d x ZSP- Chl. rostafinski x d ZSP »small«, diameter 8–10 mm x x x x Fungi Chionaster nivalis x d x x Selenotila nivalis x r x x x x x U:\ACTA BOTANICA\Acta-Botan 2-10\300 Lukavsky.vp 11. listopad 2010 11:28:46 Color profile: Disabled Composite 150 lpi at 45 degrees is a »nomen dubium«, and its reproduction was never observed. It is probably a spore of some yet unidentified fungus from Hyphomycetes. Selenotila nivalis Lagerh. (syn. Selenozyma), Figs. 22–27. Cells cylindrical-clavate, 30 ´ 3–5 mm, arranged into irregular clusters, colourless, filled by small globes, reproducing through budding. Dominant in fields 7 and 9, present in all sampled snow fields. There is a wide variability in morphology of thalli. Notes: Selenotila nivalis is an invalid taxon according to KIRK et al. (2001). Sometimes it is included in the genus Selenozyma (ARX et al. 1977). Similarly to Chionaster nivalis, this species often occurs with snow algal populations. Species richness and composition We have determined 13 taxa together, including Fungi. The species composition and general distribution pattern of snow algae in the Stara Planina Mts. was similar to that in the Bohemian Forest (LUKAVSKÝ 1993). Only low–moderate altitudes species (Chloromonas nivalis and C. rostafiñski) were found, despite the large areas of snow habitats with open exposure. In contrast, the cryosestons of the Rila and Pirin Mts. were dominated by giant cells of cf. Chlamydomonas nivalis, accompanied by only a few other species, e.g., Koliella viretii (Chod.) Hind. (KOL 1956, WODENITSCHAROFF 1962, LUKAVSKÝ unpublished data). The Vitosha and the Giant Mts, however, contained a combination of ecotypes: Chlamydo- monas nivalis was present but not dominant (KOCIÁNOVÁ et al. 1989, LUKAVSKÝ et al. 2009). In the Stara Planina we found a single »giant spore« resembling Chlamydomonas nivalis (Fig. 7), but it was brown rather than the typical brick-red and we believe that this is a cell of Chloromonas rostafiñski. Surprisingly, some common genera such as Koliella, Raphidonema, Stichococcus (KOMÁREK et al. 1973, HOHAM 1973, HOHAM and DUVAL 2001, NOVIS 2002, LUKAVSKÝ et al. 2009) were absent from our samples. All our snow samples were heavily contaminated by dust and pollen grains, so nutrients should be present in excess. Perhaps the reason for the absence of the species is that Stara planina Mts. are high and long, but narrow and chain-like mountains, not huge and compact complexes as the Rila, the Pirin or the Vitosha Mts. The former are accordingly more influenced by their surrounding, and the snow fields melted too early. Bacillariophyceae were present in a few fields (Tab. 2.) but usually as dead valves. Liv- ing cells of Hantzschia amphioxys (Figs. 31–32) were found only in fields 3 and 4. Living diatoms are known as components of the cryoseston, for example on Livingstone Island, Antarctica, where ZIDAROVA (2008) determined 33 taxa of Bacillariophyceae, 2 Ochrophy- ceae (Chromophyta) and 4 taxa of Chlorophyta. We can conclude that the Stara Planina Mts. provide suitable localities for cryoseston, particularly Volvocales, and Fungi, but so far not in such density as to prominently colour the snow. Acknowledgements This work was supported by a grants of the Ministry of Education, Youth and Sports of the Czech Republic (MSMT 1M0571), and of AV0Z60050516 of Academy of Sciences. We thank P. Novis for valuable comments on the manuscript, staff members of the Institute of Plant Physiology and Genetics of BAS Sofia, G. Gaceva and V. Titlová for technical assistance. ACTA BOT. 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CROAT. 69 (2), 2010 171 CRYOSESTON IN STARA PLANINA (BALKAN) MTS., BULGARIA U:\ACTA BOTANICA\Acta-Botan 2-10\300 Lukavsky.vp 11. listopad 2010 11:28:46 Color profile: Disabled Composite 150 lpi at 45 degrees