Acta Botanica Universitatis Comenianae

ACTA BOTANICA UNIVERSITATIS COMENIANAE

Volume 50

2015 COMENIUS UNIVERSITY IN BRATISLAVA The journal was edited with the title / Časopis bol vydávaný pod názvom Acta Facultatis Rerum Naturalium Universitatis Comenianae, Botanica

Editor in Chief / Predseda redakčnej rady Karol Mičieta; [email protected]

Executive Editor / Výkonný redaktor Soňa Jančovičová, [email protected]

Editorial Board / Členovia redakčnej rady Dana Bernátová, Danica Černušáková, Katarína Mišíková, Jana Ščevková

Editor Ship / Adresa redakcie Redakcia Acta Botanica Universitatis Comenianae, Révová 39, SK811 02 Bratislava 1 Tel. ++421 2 54411541 Fax ++421 2 54415603

Published by / Vydavateľ © Comenius University in Bratislava, 2015 © Univerzita Komenského v Bratislave, 2015

ISBN 9788022340823 ISSN 05242371

Acta Botanica Universitatis Comenianae Vol. 50, 2015

AEROBIOLOGICAL ANALYSIS OF AIRBORNE POLLEN AND FUNGAL SPORE FALL IN BRATISLAVA IN 2015

Jana Ščevková●, Jozef Dušička, Karol Mičieta

Comenius University in Bratislava, Faculty of Natural Sciences, Department of Botany, Révová 39, 811 02 Bratislava, Slovakia

Received 9 November 2015; Received in revised from 18 November; Accepted 8 December 2015

Abstract In the atmosphere of Bratislava, the spectrum and quantity of pollen grains and fungal spores were analysed during the vegetation period of the year 2015 using a Burkard volumetric pollen trap. During the study period, the annual total of 27 747 pollen grains, belonging to 28 higher plant taxa and 108 029 spores belonging to six fungal taxa were recorded. The most abundant pollen grains were those of Urticaceae, Cupressaceae/Taxaceae, Betula, Fraxinus, Pinus, Poaceae and Ambrosia taxa, while Cladosporium, Alternaria and Epicoccum were the most abundant fungal spores in the air of Bratislava. The highest monthly total pollen concentration was recorded in April, while the highest total monthly spore concentration was recorded in July. Out of all analysed meteorological parameters, relative humidity and temperature seem to be the most important factors affecting the daily airborne pollen and spore concentrations, respectively.

Key words: higher plants, fungi, aerobiology, pollen calendar

Introduction Hypersensitivity to pollen grains and fungal spores belong to seasonal allergic diseases, occurrence of which relates to a blooming period of allergenic plants and sporulating period of allergenic fungi. In the air of Bratislava, pollen grains and fungal spores with the ability to trigger the symptoms of respiratory allergy are presented throughout the entire vegetation period. Trees bloom in spring, especially in March and April. Grasses bloom in later spring, since the second half of May till the second half of June. Later summer-early autumn (August, September) is the time of year when weeds bloom (Jurko 1990). In Bratislava just like in other European countries, the fruiting period of allergenic fungi lasts from February to October (Corden, Millington 2001; Munuera Giner et al. 2001; Corden et al. 2003; Rizzi- Longo et al. 2009). Information concerning the occurrence of pollen grains and spores in the air is very important for the course of medical treatment and prophylaxis. In Bratislava, continuous monitoring of airborne pollen grains and fungal spores has been conducted since 2002. The research reported in this paper aims to determine the spectrum and quantity of pollen grains and fungal spores in the air of Bratislava in the year 2015 and to determine the beginning, peak, and end dates for the key airborne pollen and spore seasons.

Material and Methods The study was carried out in the city of Bratislava, situated in the southwestern part of Slovakia. The city has a warm and dry continental climate with average temperatures ranging from -1 to -4 ºC in January and from 19.5 to 20.5 ºC in July. The annual rainfall varies from 530 to 650 mm, as average, falling mostly in summer (May-July) (Hrvoľ 2014). The daily mean pollen/spore concentrations were monitored from February to October 2015 using a Burkard 7-day volumetric pollen trap. The sampler is situated on the flat roof of the Department of

● Corresponding author: Jana Ščevková; [email protected]

Botany, Faculty of Natural Sciences, Comenius University in Bratislava at the height of 10 m above ground at the altitude of 183 m a. s. l. Sampling method, slide preparation, and data interpretation were performed according to the standard method adopted by the British Aerobiology Federation (1995). Pollen grains and fungal spores were counted in 12 vertical transects per slide under a light microscope at a magnification of × 400 following the methods outlined by Ščevková et al. (2010). Pollen and spore concentrations were expressed as mean daily pollen/spore concentrations per cubic meter of air. The main pollen/spore seasons (MPS/MSS) of selected pollen/spore types were established according to method by Nilsson, Persson (1981), which defines the main pollen/spore season as the period from which the sum of concentrations reaches 5% of the annual total pollen/spores until the time when the sum reaches 95%. To determine how changes in daily pollen/spore concentrations were affected by meteorological conditions, daily values of mean air temperature (ºC), relative humidity (%) and rainfall (mm) were taken into consideration. Meteorological data were recorded at the meteorological station of the Department of Astronomy, Physics of the Earth and Meteorology of the Faculty of Mathematics, Physics and Infor- matics of Comenius University in Bratislava, Mlynská dolina situated 1 km NW of our sampling site. Spearman’s correlation coefficients were used to establish the relationship between the daily pollen/ spore concentration of selected taxa and meteorological parameters. All data analyses were carried out in Statistica 12.

Results and Discussion

The annual total number of sampled pollen grains and fungal spores in the air of Bratislava was 27 747 and 108 029, respectively. The identified pollen grains belong to 28 vascular plant taxa, including 20 trees and/or shrubs and eight herbaceous species. The observed spores belong to six taxa of patho- genic fungi (Tab. 1). On the basis of the annual total pollen concentrations for the study period, as the most abundant pollen types in the air of Bratislava were identified: Urticaceae (20.9 % of the total), Cupressace/Taxaceae (named in the tables and figures as Cupressaceae, 13.5 %), Betula (9.7 %), Fraxinus (8.7 %), Pinus (8.7 %), Poaceae (7.3 %) and Ambrosia (6.8 %). Pollen types from Aesculus, Castanea, Fagus, Larix, Platanus and Ulmus were represented with a small quantity (annual total pollen concentration <120 pollen grains) in the air of Bratislava (Tab. 1). Based on the aeropalynological monitoring performed in Bratislava during the period 2002–2009 the most abundant pollen types were Betula, Urticaceae, Cupressaceae/Taxaceae, Populus, Pinus, Poaceae and Ambrosia (Ščevková et al. 2010). Variability among pollen seasons in terms of airborne pollen quantity corresponds with the biological rhythms of the plants and meteorological conditions. Ščevková et al. (2010) hints at the possibility of cyclic alteration in pollen grain production, with years of high and low production, respectively. Out of all airborne fungal spores observed in the study area, the most abundant were those of Cladospo- rium (81.8 %), Alternaria (10.1 %) and Epicoccum (6.5 %) (Tab. 1). This is in accordance with the results obtained by Chrenová et al. (2004) who performed the aeromycological survey in the air of Bratislava from February to October 2003. There was a remarkable increase in the number of pollen grains from February to April (Tab. 1, Fig. 1) with the highest concentration of pollen grains recorded in April (7 139 pollen grains, 25.7 % of total); arboreal taxa flowering in spring were the most important contributors. Pollen concentration began markedly to decrease afterwards in May. In our study, there was another increase in number of pollen grains from July to August due to the flowering of herbaceous taxa. In September, the pollen concentration once again started to decrease (Tab. 1, Fig. 1). In February and March, the most abundant pollen types in the air of Bratislava were those of Cupres- saceae /Taxaceae and Alnus (Tab. 1). In April, Betula, Fraxinus and Populus were the most important pollen contributors. In May, a remarkable decrease of pollen amount was closely related to the ending of the main pollen season of most trees. In May, pollen grains of Pinus were the most abundant in the air of Bratislava. The most frequent pollen grains recorded in June were those of Poaceae, while in July and August the dominant pollen types were of the synanthropic and ruderal entities, such as

Urticaceae and Ambrosia. Pollen grains of invasive plant Ambrosia artemisiifolia were abundant also in September (Tab. 1).

Tab. 1. Pollen/spore calendar of the monitored taxa in the air of Bratislava and related monthly pollen/spore concentrations, year 2015

Pollen-producing Percentage FEB MAR APR MAY JUN JUL AUG SEP OCT taxa of total Acer - - 209 ------0.8 Aesculus - - 23 75 - - - - - 0.4 Alnus 52 653 2 ------2.5 Ambrosia - - - - - 2 965 900 17 6.8 Artemisia - - - - - 7 201 26 - 0.8 Betula - 7 2 683 15 - - - - - 9.7 Carpinus - - 204 ------0.7 Castanea - - - - 95 27 - - - 0.4 Chenopodiaceae - - - - 39 71 221 137 1 1.7 Corylus 31 202 ------0.8 Cupressaceae 9 3 269 439 7 - 3 9 8 - 13.5 Fagus - - 81 10 - - - - - 0.3 Fraxinus - 666 1 753 5 - - - - - 8.7 Humulus - - - - - 4 374 25 - 1.5 Juglans - - 193 100 - - - - - 1.1 Larix - 1 1 ------0.1 Pinus - - 91 2 142 155 30 - 4 - 8.7 Plantago - - - 60 253 197 64 36 - 2.2 Platanus - - 107 9 - - - - - 0.4 Poaceae - - 16 520 882 524 23 66 3 7.3 Populus - 529 709 ------4.5 Quercus - - 252 286 - - - - - 1.9 Rumex - - - 37 79 11 - - - 0.5 Salix - 43 325 17 - - - - - 1.4 Sambucus - - - 278 68 - - - - 1.2 Tilia - - - - 171 18 - - - 0.7 Ulmus - 70 45 ------0.4 Urticaceae - - 6 735 874 2 303 1 330 544 13 20.9 Total 92 5 440 7 139 4 296 2 616 3 197 3 187 1 746 34 Spore-producing Percentage FEB MAR APR MAY JUN JUL AUG SEP OCT taxa of total Alternaria 4 65 69 195 821 2 926 2 991 2 959 884 10.1 Cladosporium 27 676 989 7 639 18 918 23 371 20 793 12 227 3 701 81.8 Epicoccum 2 41 52 140 447 1 439 2 219 2 144 524 6.5 Helminthosporium - - - 2 21 47 49 39 9 0.2 Polythrincium - - 1 - 22 54 54 31 6 0.2 Stemphylium - 27 43 108 104 325 438 306 80 1.3 Total 33 809 1 154 8 084 20 333 28 162 26 544 17 706 5 204

For fungal taxa, a remarkable increase in the number of their spores was observed from February to July, with the highest spore concentration recorded in July (28 162 spores, 26 % of the total). Spore concentration began markedly to decrease afterwards in August (Tab. 1, Fig.1).

Fig. 1. Mean monthly distribution of total pollen/spore concentrations (PC/SC) of all analysed taxa in the air of Bratislava, expressed as percentages over the monitored year 2015

Among the selected taxa with the most abundant pollen grains in the air of Bratislava, Urticaceae pollen season was the longest (123 days), while the shortest pollen season was that of Betula (15 days) (Tab. 2). Out of three selected fungal taxa Cladosporium spore season was the longest (135 days) (Tab. 2). The highest peak daily pollen and spore concentration recorded in the season was found for Cupressaceae/Taxaceae (791 pollen m-3) and for Cladosporium (1 649 spores m-3) (Tab. 2).

Tab. 2. Characteristics of the main pollen/spore seasons of the most abundant taxa in the air of Bratislava, year 2015

Taxa Characteristics of the main pollen/spore season Duration Peak value SPI/SSI Pollen-producing taxa Start date End date Peak date (days) (PG/S m-3) (PG/S) Cupressaceae 8 MAR 11 APR 35 791 8 MAR 3 461 Fraxinus 17 MAR 20 APR 35 296 11 APR 2 198 Betula 9 APR 23 APR 15 504 15 APR 2 511 Pinus 3 MAY 4 JUN 33 273 11 MAY 2 208 Poaceae 12 MAY 28 JUL 78 78 29 MAY 1 843 Urticaceae 10 MAY 9 SEP 123 185 3 JUL 5 233 Ambrosia 13 AUG 20 SEP 39 230 30 AUG 1 725 Spore-producing taxa Alternaria 8 JUN 4 OCT 119 330 13 SEP 9 870 Cladosporium 16 MAY 27 SEP 135 1 649 9 JUL 79 572 Epicoccum 8 JUN 3 OCT 118 386 30 AUG 6 329 PG pollen grains, S spores, SPI/SSI seasonal pollen/spore index – sum of daily average pollen/spore concentration recorded during the MPS/MSS

Meteorological factors, particularly mean temperature, relative humidity, and rainfalls, directly influence the release of pollen grains as well as spores and its dispersal throughout the atmosphere. Sunny and warm weather generally corresponds with higher pollen production (Davies, Smith 1973), while cold and humid weather dampen the production of pollen grains as rainfall directly removes pollen grains from the atmosphere (Jones, Harrison 2004). All fungal spores identified in the air of Bratislava represent so-called “dry” types of spores which abundantly occur in the atmosphere on sunny, warm days without precipitation (Timmer et al. 1998). Spearman’s correlation coefficients between the daily mean pollen/spore concentrations of the seven main pollen types as well as three main spore types and the selected daily meteorological parameters were assessed during MPS/MSS. For Cupressaceae/Taxaceae, Betula, Pinus, Urticaceae and Ambrosia,

significant negative correlations were found between the daily pollen concentrations and relative humidity (Tab. 3). The significant and negative correlations were also observed between daily pollen concentration of Cupressaceae/Taxaceae as well as Poaceae and daily rainfall totals (Tab. 3). On the other hand, the correlation between temperature and pollen concentrations of Betula and Ambrosia was significant and positive. Similarly to our results, Bartková-Ščevková (2003), pointed out (based on the records obtained in the years 1995 and 1997) also that relative humidity and air temperature are the most important factors affecting the quantity of Betula and Ambrosia pollen grains in the air of Bratislava. In Bratislava, just like in several other aeromycological surveys (Grin-Gofroń and Mika 2008; De Linares et al. 2010; Boddy et al. 2014), significant positive correlations were observed between temperature and the daily spore concentrations of Alternaria and Epicoccum (Tab. 3).

Conclusion

In 2015, the most abundant pollen types identified in the air of Bratislava were those of Urticaceae, Cupressaceae/Taxaceae, Betula, Fraxinus, Pinus, Poaceae, and Ambrosia. Out of all fungal airborne spores observed in the study area, Cladosporium, Alternaria and Epicoccum were dominant. The highest pollen concentrations were recorded in April, while the highest spore concentrations were recorded in July. Relative humidity and temperature seem to be the most important factors affecting the occurrence of pollen grains and spores, respectively, in the atmosphere of Bratislava.

Tab. 3. Spearman’s correlation coefficients between the daily mean pollen/spore concentrations and daily meteorological variables for the MPS/MSS period in the year 2015 for the eight main pollen types and three main spore types

Pollen-producing taxa d.f. Tmean RH R Cupressaceae 33 ns -0.80** -0.71** Fraxinus 33 ns ns ns Betula 13 0.64* -0.59* ns Pinus 31 ns -0.77** ns Poaceae 75 ns ns -0.69** Urticaceae 121 ns -0.69** ns Ambrosia 37 0.65* -0.77** ns Spore-producing taxa Alernaria 117 0.99*** ns ns Cladosporium 133 ns ns ns Epicoccum 115 0.89*** ns ns * p<0.05, ** p<0.01, *** p<0.001 Tmean mean daily temperature, RH relative humidity, R daily rainfall, d.f. degrees of freedom

Acknowledgements

This study is the result of the project implementation: Comenius University in Bratislava Science Park supported by the Research and Development Operational Programme funded by the ERDF, grant no. ITMS 26240220086. This study was also supported by the Grant Agency VEGA (Bratislava), grant no. 1/0380/13.

References

Bartková-Ščevková, J., 2003: The influence of temperature, relative humidity and rainfall on the occurrence of pollen allergens (Betula, Poaceae, Ambrosia artemisiifolia) in the atmosphere of Bratislava (Slovakia). Int. J Biometeorol, 48: 1-5. Boddy, L., Büntgen, U., Egli, S., Gange, A. C., Heegaard, E., Mirk, P. M., Mohammad, A., Kauserud, H., 2014: Climate variation effects on fungal fruiting. Fungal Ecology, 10: 20-33.

British Aerobiology Federation, 1995: Airborne pollen and spores. A guide to trapping and counting. National Pollen and Hayfever Bureau, Rotherham, UK. Chrenová, J., Mišík, M., Ščevková, J., Mičieta, K., Mlynarčík, D., 2004: Monitoring of microscopic airborne fungi in Bratislava. Acta Facult. Pharm. Comenianae, 51: 68-72. Corden, J. M., Millington, W. M., 2001: The long term trends and seasonal variation of the aeroallergen Alternaria in Derby, UK. Aerobiologia, 17: 127-136. Corden, J. M., Millington, W. M., Mullins, J., 2003: Long-term trends and regional variation in the aeroallergen Alternaria in Cardiff and Derby UK – are differences in climate and cereal production having an effect? Aerobiologia, 19: 191-199. Davies, R. R., Smith, L. P., 1973: Weather and the grass pollen content of the air. Clinical Allergy, 3: 95-108. De Linares, C., Belmonte, J., Canela, M., de la Gauardia, C. D., Alba-Sanchez, F., Sabariego, S., Alonso-Pérez, S., 2010: Dispersal patterns of Alternaria conidia in Spain. Agricultural and Forest Meteorology, 150: 1491-1500. Grinn-Gofroń, A., Mika, A., 2008: Selected airborne allergenic fungal spores and meteorological factors in Szczecin, Poland, 2004-2006. Aerobiologia, 24: 89-97. Hrvoľ, J., 2014: Extrémne teploty vzduchu na stanici Bratislava, Mlynská dolina za obdobie 1983-2012. In: Čelková, A. (ed.), 21st international poster day transport of water, chemicals and energy in the soil-plant-atmosphere system, p. 93-101, Bratislava. Jones, A. M., Harrison, R. M., 2004: The effects of meteorological factors on atmospheric bioaerosol concentrations – a review. Science of the Total Environment, 326: 151-180. Jurko, A., 1990: Sezonalita kvitnutia rastlín a peľové alergény v našej vegetácii. Biológia, 45: 367-374. Munuera Giner, M., García, J. S. C., Camacho, C. N., 2001: Airborne Alternaria spores in SE Spain (1993-98). Grana, 40: 111-118. Nilsson, S., Persson, S., 1981: Tree pollen spectra in the Stockholm region (Sweden), 1973-1980. Grana, 20: 179-182. Rizzi-Longo, L., Pizzulin-Sauli, M., Ganis, P., 2009: Seasonal occurrence of Alternaria (1993-2004) and Epicoccum (1994- 2004) spores in Trieste (NE Italy). Ann Agric Environ Med, 16: 63-70. Ščevková, J., Dušička, J., Chrenová, J., Mičieta, K., 2010: Annual pollen spectrum variations in the air of Bratislava (Slovakia): years 2002-2009. Aerobiologia, 26: 277-287. Timmer, L. W., Solel, Z., Gottwald, T. R., Ibanez, A. M., Zitko, S. E., 1998: Environmental factors affecting production, release, and field populations of conidia of Alternaria alternata, the cause of brown spot of citrus. Phytopathology, 88: 1218-1223.

Abstrakt Počas vegetačnej sezóny roku 2015 bolo pomocou Burkardového volumetrického peľového lapača analyzované druhové spektrum a kvantita peľových zŕn a hubových spór v ovzduší Bratislavy. Sumárne za celé analyzované obdobie bolo zaznamenaných 27 747 peľových zŕn, patriacich 28 taxónom vyšších rastlín a 108 029 spór patriacich šiestim taxónom húb. V ovzduší Bratislavy boli najhojnejšie zastúpené peľové zrná taxónov Urticaceae, Cupressaceae/Taxaceae, Betula, Fraxinus, Pinus, Poaceae a Ambrosia; z húb patrili medzi najhojnejšie spóry taxónov Cladosporium, Alternaria a Epicoccum. Najvyššia sumárna mesačná koncentrácia peľových zŕn bola zaznamenaná v apríli a najvyššia sumárna mesačná koncentrácia hubových spór v júli. Zo všetkých analyzovaných meteorologických činiteľov mala naj- väčší vplyv na kvantitu peľových zŕn v ovzduší relatívna vlhkosť vzduchu, pričom kvantitu hubových spór v ovzduší najviac ovplyvňovala teplota vzduchu.

Jana Ščevková, Jozef Dušička, Karol Mičieta: Aeopalynologická analýza výskytu peľových zŕn a hubových spór v ovzduší Bratislavy v roku 2015

Acta Botanica Universitatis Comenianae Vol. 50, 2015

REJUVENATION EFFECT AT OLD VICIA FABA L. SEEDS BY AN ENHANCEMENT OF THE DNA REPAIR DURING AN EXPERIMENTAL STORAGE

Gustáv Murín, Karol Mičieta

Comenius University in Bratislava, Faculty of Natural Sciences, Department of Botany, Révová 39, 811 02 Bratislava, Slovakia

Received 12 November 2015; Received in revised form 8 December; Accepted 15 December

Abstract Plant seeds have for a long time been used as a suitable model for the study of aging at the basic cytogenetic level. This report is covering of 25 years of our experiments with aging focused for an enhancement of DNA repair in plant seeds using a method of a “storage effect”, developed in past by several authors. This method is based at prolongation of G-1 phase of pre-replication DNA repair from hours to days. It offers simple and effective enhancement of DNA repair from damage from different kinds of xenobiotics. Our experiments were focused to a damage caused by mutagens at old seeds with a possible recovery.

Key words: aging, storage effect, mutagenicity, seeds, rejuvenation

Introduction We are reporting here a summary of results from our series of experiments lasting 25 years with a final effect of rejuvenation. Our idea was to use the method of a “storage effect” for seeds of V. faba L. with the synergic effect of their age and mutagen treatment by non-alkylating agent MH or by alkylating agent MMS, both as S-dependent mutagen with the significant impact to the mutagenicity. With higher damage the possibility to observe significant results in DNA repair is higher too. Storage of the mutagen treated old seeds in defined conditions for the 8-days appear to be optimal with a significantly lower frequency of chromosome aberrations in root tips of seeds observed. This prolongation of G-1 phase causes a decrease in damage not only from mutagen treatment, but also from impact of aging. It resulted in the condition of seeds as if they are younger (from 12 years old to the level of 2 years old). Effect of rejuvenation of old seeds by this combined method was observed for the first time. Aging is in humans frequently connected with dietary habits and other aspects of life style. However, plants do not have these aspects of their life and display the effects of aging as well. Consequently “plants present challenges to general theories of biological aging” as stated by Thomas (2002). The search for universal principles of aging in plant models has been conducted for more than a century since a very first report of de Vries in 1901, although partly misinterpreted (see Priestley 1985). One of the continuous attempts of these experiments for such a long period has been to contribute to the elucidation of the general problem of aging (cf. Murín 2001; Murín, Mičieta 2009) based on general theories from Strehler (1962) up to Valleriani and Tielborger (2006). The dependence between the age and instability of the genetic apparatus of the cell is generally known (Kirkwood 1988; Slagboom, Vijg 1989) and was observed in plant seeds as well. Accordingly, Osborne et al. (1984) regard seeds as a unique and attractive system for studying the repair of DNA damage that occurs during the aging process. Cartledge and Blakeslee published the results of storing Datura seeds in soil for 22 years; in this case, the aberration rate was many times lower than in seeds stored in a laboratory (Cartledge, Blakeslee 1935). Interesting is use of material from special volcanic deposits for buried seeds populations for

 Corresponding author: Gustáv Murín; [email protected]

20 years (Ishikawa-Goto, Tsuyuzaki 2004) and experiments to prolong time of the storage of barley seeds (Hordeum vulgare ssp. Vulgare) up to 72 years (Parzies et al. 2000). Another approach is to compare sensitivity of old and young seeds of Allium fistulosum L. to the differently polluted environments, and this study found a strong influence to their chromosomal instability (Bezrukov, Lazarenko 2002). Other authors still work on the optimal balance between time, temperature and moisture of stored seeds (Vertucci et al. 1994; Čupić et al. 2005) with better results in buried than after ripened seed lots (Martinkova, Honek 2005). Large group of authors pointed at the reduction of chromosome aberrations in seeds after an aerated hydration (Burgass, Powell 1984; Eeswara et al. 1998; Thornton, Powell 1992; Thornton et al. 1993) connected with their higher moisture contents (Villiers 1974; Ward, Powell 1983; Powell et al. 2000). For example Ward and Powell (1983) reported evidence for the activation of repair processes in seeds during periods of partial seed hydration and later Burgass and Powell (1984) described experiment where seeds with low vigor as a result of aging showed a large improvement in seed quality following a 2 hrs soak in water, reflected in an increased rate of germination and higher emergence in soil when dried to their initial moisture content after treatment retained these beneficial effects. Opposite conditions are causing their deterioration in dry storage (Villiers, Edgecumbe 1975). Villiers (1974) first among them indicated conditions close to the “storage effect” when reporting that two weeks storage of seeds fully imbibed but unable to germinate allowed a high germination capacity to be maintained for long periods, together with a very low incidence of chromosome aberrations. These reports were confirmed also for appearance and repair of apurinic/apyrimidinic sites in DNA during early germination of Zea mays (Dandoy et al. 1987). Simultaneously, the same observations were made by Gichner and Velemínský (1973) after mutagen treatment of the seeds. The time between the start of imbibition of seeds and the first wave of the semiconservative DNA replication in their cells is very important for the viability of seeds. If this “window” of G-1 phase of first mitotic cycle is prolonged by method of “storage effect” from hours to days, the decrease or increase of genetic damage depended on the conditions of this prolongation. Gichner and Gaul (1971) first observed in barley (Hordeum vulgare L.) a drastic decrease of the height of seedlings that survived storage at 13% – 20% water content (w.c.) during above mentioned prolongation of G1 phase. In a series of experiments, Murata et al. (1982) reported 12% – 18% water content for barley as a condition when germination of seeds was delayed and reduced, parallel with an increase of a frequency of aberrant ana-telophases. Results obtained by these authors inspired our experiments with artificial aging of Vicia faba seeds developed from the “storage effect“ observed by Velemínský and Gichner in series of their experiments (see McLennan 1987). This method was improved for Vicia faba L. in our own experiments (Murín 1993; Murín, Mičieta 1996; Murín, Mičieta 1997a-c; Murín, Mičieta 1998; Murín 2001; Murín, Mičieta 2001; Murín et al. 2007; Murín, Mičieta 2009). It leads us at the end to the combination of impact of mutagen treatment and impact of aging by using old seeds for these experiments. Synergic effect of the damage to the DNA of old seeds than showed more significant repair after storage effect as if old seeds were under storage effect method without mutagen treatment. We observed that damage after treatment with the non-alkylating agent MH was repaired during the storage of damaged seeds at 50% w.c. (Murín 1993). Contrary to the effect of storage on the frequency of chromatid aberrations, no significant differences in the distribution patterns of chromatid aberrations occurred. The explanation for results obtained after MH-treatment followed by 50% w.c. storage could be that although the DNA damage caused by the action of the mutagen is non-randomly distributed in the karyotype of the injured cells, the repair of this damage occurs at the same level for all chromosome segments regardless of their sensitivity to a particular mutagen and the experimental protocol used (Murín, Mičieta 1996). Consequently, our research indicates that while DNA damage is selective and in some segments preferentially involved, the repair of this DNA damage is not selective for a particular chromosome or chromosome segment. Regarding our previous results with treatment by a non-alkylating agent, MH, our results from storing seeds at 50% w.c. in relation to the repair of MMS-induced biological damage showed a decrease in both the frequency of CAs and SCEs (Murín, Mičieta 1994a). Later experiments with the same method of storage showed that DNA damage from both these S-dependent mutagens in DNA double-strand

10 breaks was repaired during the storage of damaged seeds at 50% water contents (Murín et al. 1992; Murín, Mičieta 1994b). Data previously available demonstrated that recovery from MH and MMS-induced chromosomal damage in V. faba due to seed storage at 50% water content is accompanied by unscheduled repair DNA synthesis (Murín 1990). These data support the assumption that this recovery from the clastogenic effects of MH and MMS is related to the degree of excision repair of DNA damage during the experimental storage of seeds (for more see Vonarx et al. 1998). Our tested hypothesis that the strong reduction in germination of old seeds is caused by changes in the content of free endogenous cytokinins was not confirmed by thin-layer chromatography (TLC) and gas-liquid chromatography (GLC) with 1, 2, 4, 5, 7 and 8 year old seeds probably by a methodological inaccuracy (Murín et al. 1994). A study of the darkening of V. faba L. seeds in the course of aging yielded results that the following general conclusions are possible: In the course of aging, the proportion of dark seeds in a given harvest increases; seed lots each year show a shift of color types according to Fisher-Saller's scale up to the darkest ones (Murín 1988b). Darkening of seeds was manifested by the loss of germinating capacity and an increase in the rate of aberrant chromosomes in anaphase cells of root tips, the decrease in viability tested by germinating capacity being more marked up to the mortality of V. faba L. seeds after nine years of storage (Murín 1988b). The difference in the physiological and genetic damage examined between different (dark-light) seed groups within a given harvest, i.e. light seeds of a younger harvest (the same applying for dark seeds) is more as compared with the difference between harvests generally several years apart, the phenomenon being more marked in terms of physiological than genetic damage (Murín et al. 2007). The aging of seeds could also be explained by the fact that in a seed lot there occurs a gradual and irreversible increase in the proportion of less viable seeds (darker seeds in V. faba) up to a certain limit (9 years in V. faba), when viable individuals (light seeds) still remain in the seed lot. Our hypothesis of the seed samples viability decreasing by years and their testa getting darker, if stored in less favorable conditions (high temperature), was confirmed too. The mitotic cycle was partially extended in the course of aging of V. faba seeds. Chromosome aberration frequency was highest in the first mitosis, decreasing in subsequent mitoses probably due to DNA repair mechanisms (Murín 1988a). With the increasing age of V. faba L. cv. Inovec seeds, their germination rate and root growth rate decreased and the chromosome aberration frequency increased. The germination rate and chromosome aberration frequency of V. faba L. seeds proved more sensitive to aging than those of V. sativa L. cv. Vígľašská hnedá. V. faba L. seeds remained viable in the laboratory (at a room temperature of about 20 oC) at the most for 8 – 9 years. The conditions during storage affected the germination rate and chromosome aberration frequency. A marked variability was manifested in old V. faba seeds, the physiological manifestations in the set examined was more uniform than chromosome aberration frequency. In old V. faba L. seeds, fragments of the chromatid type prevailed (Murín 1988a). Older seeds treated with MMS showed the synergic effect of the damage of chromosomes, M.C., and the viability of the seeds generally. However, the storage effect had made an impact in this area as well, and after eight days of storage we observed a 3-4 times lower frequency of chromosome aberrations and a significantly higher viability of seeds. As 12-years old seeds after storage indicated a viability comparable with that of 2-years old seeds that had not been stored, our method points to the possibility of rejuvenation (Murín, Mičieta 1998). All these results indicated that the “storage effect” could be a general phenomenon, which by means of prolonging the period between the mutagenic treatment and the onset of DNA synthesis, is favorable for DNA repair, as manifested in the whole set of parameters evaluating genotoxicity and viability of the seeds examined (Murín, Mičieta 1997b). Apart of the important contribution to the general aspects of aging it has also a practical outcome in case of industrially stored seeds (Murín et al. 2003). In the experiments presented in this report we were focused on the hypothesis that “storage effect“ adjusted in DNA repair-supportive conditions will have a significant impact on the old seeds too and thus could be the tool for decreasing the impact of aging process in these particular objects.

Material and methods Seeds used For our experiments we had a large scale of seeds Vicia faba L. cv. Inovec stored at 4 oC where their lifespan is maximum 12 years. In one experiment we used also seven-year-old of rearranged ACB karyotype obtained from Institute of Plant Genetics, Gatersleben, Germany, and stored in the same condition.

Treatment and washing conditions It is well know that while germinating scale during the aging of the seeds is a sensitive parameter of their age, frequency of chromosome aberrations is unusually low. A possible explanation is that during the imbibition of the old seeds those with high frequency of chromosome aberrations are eliminated from germination. Therefore we used mutagen to reach a significant level of chromosomal damage that can be then a target of the repair process caused by “storage effect”. Before mutagen treatment the seed- coat of dry seeds was penetrated in order to obtain a higher uniformity of the soaking and a greater synchronization of mitotic activity (Thomas, Davidson 1981). Vicia faba seeds were then treated for 5 hrs with doses of 0.2, 0.4 and 0.6 mM of maleic hydrazide (MH, Merck) or 3 mM and 6 mM of methyl methanesulphonate (MMS, Merck) in distilled water at pH 4.8. After the mutagen treatment, seeds were washed for 2 hrs in tap water for elimination of residuals of mutagen.

Imbibition and germination The imbibition of seeds was optimized in plastic jars allowing continual air bubbling of distilled water to avoid possible influence of hypoxia. Before imbibition seeds were treated by 5% chloramine B (30-min treatment followed by washing with distilled water) to prevent microbe contamination of the seedlings. The seeds germinated in wet sawdust in the desiccators at laboratory room temperature allowing satisfactory respiration of seeds.

Storage conditions To obtain specific water content after treatment and washing, seeds were re-dried to 50% w.c. (2 hrs at 37 oC in a thermostat with a fan). Seeds were then stored for 0 or 8 days at 25 oC above 600-ml sterile water at room temperature in the desiccators. Following treatment, washing and re-drying, one half of the seed samples were allowed to germinate immediately and the roots were fixed after two recovery times (48 hrs and 72 hrs). The second half of seed samples was allowed to germinate after 8 days of storage. According of our experience (Murín, Mičieta 1997c), prolongation of the period of storage for more days has no greater effect, i.e. “storage effect“ is limited probably due the limited sources of repair enzymes stored in dormant seeds.

Control of water content For measuring a level of w.c. of seeds during experiments extra samples of ten seeds were weighed before and after drying (8 hrs at 105 oC) to determine their water content according to formula 100 – (Yx100/X) = w.c., where X = weight before and Y = weight after drying.

Cytological evaluations For cytological evaluation we chose ana-telophases, in accordance with other authors (Bezrukov, Lazarenko 2002). These mitotic figures are simpler to evaluate and thus allowing us to make experiments with large number of samples under different doses of the mutagen and recovery times. Mutagen-treated roots of seed samples were fixed in ethanol: acetic acid (3:1), squashed and stained by aceto-orcein. On average, 200 ana-telophases (50 in control) per recovery time were evaluated on the occurrence of fragments (F), bridges (B) or both (F+B).

Measuring of unscheduled DNA synthesis (UDS) Roots grown for 80 hrs were treated with mutagen for 5 hrs, washed 2 hrs and exposed to 3H-TdR for additional two hours periods in 7, 24, and 32 hrs after the treatment was initiated. As the replicative

DNA synthesis was suppressed by HU, an enhanced 3H-TdrR incorporation into nuclear DNA (determined microautoradiographically) was thought to be due to unscheduled DNA synthesis induced by the mutagen (Murín, Mičieta 2002).

Statistical methods First we calculated Pearson's chi-squared test statistic (Agresti 2002) for null hypothesis about the equality of several proportions (probabilities of success, probability of aberration) in R statistical soft- ware with interpretation for probabilities of aberrations close to zero. As a post-hoc test, we performed an exact test of a simple null hypothesis about the probability of success in a Bernoulli experiment. Confidence intervals (CI) are obtained by a procedure first given in Clopper and Pearson (1934). This guarantees that the confidence level is at least α = 0.05 also for probabilities close to zero and one unlike in the classical asymptotic Wald test and CI (Agresti 2002) very often used in applications.

Results

The mitotic cycle of old seeds and its manifestation in chromosome aberrations We conducted the first experiment in this series on the assumption that in a set of seeds of the same age, the first mitosis appears in the cells of more vigor seeds (Tab. 1). Our findings showed that the chromosome aberration frequency in root-tip cells increased simultaneously with a delay in the onset of seed-germination, although the difference in value between 72 and 96 hrs of germination was insignificant. In the next experiment, therefore, we germinated seven-year-old of rearranged ACB karyotype seeds and continuously examined root-tips over 96 h, confirming the relationship to aging that we had assumed (Tab. 2). Tab. 1. Chromosome aberration frequency in 5 years old V. faba L. seeds with different germination onset

Time of Aberration n F B FB Germination % 56 h 500 12 12 7 3.1+0.56 72 h 500 47 17 10 7.4+1.28 96 h 500 48 15 18 8.1+1.72 n = No. of ana-telophases; F = fragments, B = bridges, FB = fragments + bridges

Tab. 2. Chromosome aberrations in 7 years old V. faba L. seeds with different germination onset

Root length Aberrations n F B FB cm % 3 1000 10 16 1 2.7+0.77 1 700 19 15 1 5.0+1.11 n = No. of ana-telophases; F = fragments, B = bridges, FB = fragments + bridges

On the base of previous findings a large set of five-year-old seeds was examined for variations in mitotic cycles and the rate of chromosome aberration. After 96 hrs of germination in moistened sawdust 6,750 anaphases from 77 seeds were evaluated in their root-tip cells. Figs. 1–2 show the range of variation among the seeds we investigated. Fig. 1 illustrates the degree of reproduction, the rate of cell-growth, and chromosome aberration frequency. Seedlings with 7 mm roots showed extreme aberration frequencies (dropping from 41% to the lowest value). Interestingly, marked differences were observed between minimal and maximal values of both the growth and aberration rates (7–72 mm, 0–41 %), as well as an accumulation of low-level aberration frequencies (up to 4%), while the distribution of root-lengths of seedlings with these extreme values graduated without extremes. The growth of each root in constant time depends on the elongation rate of cells, the onset of the first mitoses, and the number of subsequent mitoses through which root-cells pass. The longest roots we studied displayed very low chromosome aberration frequencies, while the shorter roots, having undergone fewer mitotic cycles, exhibited a higher rate of aberrations. Thus, the question arises as to whether chromosome aberration frequency actually decreases in the course of the 2nd and 3rd mitotic

13 cycle. The values we established with regard to root-lengths gave us a baseline for the number of mitotic cycles each respective seedling underwent; subsequently, we were able to obtain information from one-year-old broad bean seeds with regular measurements of root increments during a known interval (approx. 12 hrs) of the mitotic cycle in V. faba (Murín 1961). At periodic intervals, we evaluated the frequency of aberration in the roots of all seedlings within a given mitotic cycle (Fig. 2); in this way we were able to confirm the reduced frequency of chromosome aberration in subsequent mitotic cycles (from 12 to 1%). Contrary to data reported elsewhere (Dubinin et al. 1965), we discovered that the aberration frequency of roots in the first mitotic cycle was so heterogeneous that comparison was insignificant with the aberration frequency in roots in the second mitotic cycle.

Number of seeds examined seeds of Number

% resp. mm 10

10 20 30 40 50 60 70 Empty columns: root length, blackSeeds columns: examined chromosome aberrations in % Fig. 1. Comparison of number of chromosome aberrations to the root length relevant to the stage of a mitotic cycle Explanation: empty columns: root length in mm, black columns: chromosome aberrations in %

5 Chromosome aberrations in in % aberrations Chromosome

1. M.C. 2. M.C. 3. M.C. 4. M.C. Root length - mitotic cycle

Fig. 2. Comparison of number of chromosome aberrations in % to the mitotic cycle

The frequency of chromosome aberrations after MH and MMS treatment After series of preliminary experiments (see Murín, Mičieta 2001) with storage effect we selected the highest MH dosage used in these experiments for studying the effects of a non-alkylating agent on root-tip cells, since the resulting high frequency of chromosome aberrations provides a unique opportunity for observing repair activities during storage. Tab. 3 shows the dose-dependent effect of MH-treatment (0.2, 0.4 and 0.6 mM) on the frequency of induced chromosome aberrations in V. faba L. root-tip cells during different recovery times. Tab. 4 shows the frequency of chromosome aberrations induced by 0.6 mM MH in V. faba root-tip cells resulting from 0, 14, and 28 days of seed storage at 50% w.c.. It indicates that long-term storage leads to a significant reduction in the frequency of chromosome aberrations. However, since the effective- ness of storage is limited, prolonging the storage interval from 14 to 28 days produced less impressive results. The first 14 days of storage reduced the yield of chromatid aberrations by 2.32+0.2% per day. The following two-week storage interval (under identical conditions) resulted in a 1.96+0.14% reduction of aberrations per day. This may indicate limitations in repair capacity depending on the time of storage.

Tab. 3. Dose-dependent effect of MH-treatment (0.2, 0.4 and 0.6 mM) on V. faba L. root tip cells. 200 metaphases (50 in control) per recovery time were evaluated

Recovery time 32 hrs 48 hrs 56 hrs 72 hrs 80 hrs Control 2.0+1.3 5.0+0.6 1.0+0.8 3.6+1.6 1.6+0.4 0.2 - 33.6+1.8 34.9+7.4 47.3+1.7 40.0+1.1 0.4 54.2+0.8 59.5+2.7 58.7+1.8 52.0+8.0 50.2+2.1 0.6 70.9+2.0 70.0+5.3 76.0+6.5 77.5+1.5 78.0+1.1

Tab. 4. Influence of 0, 14 and 28 days seed storage at 50 % water content at the frequency of metaphases with chromosome aberrations induced by 0.6 mM MH on V. faba L. root tip cells. 200 metaphases (50 in control) per recovery time were evaluated

Recovery time 32 hrs 48 hrs 56 hrs 72 hrs 80 hrs 0 68.3+1.4 67.8+6.2 73.1+5.5 70.4+2.8 76.2+4.9 14 30.6+0.3 38.9+1.1 43.9+1.3 46.4+3.2 32.8+3.8 28 6.8+2.3 7.4+1.9 13.3+5.4 17.4+8.9 10.4+2.2

Differently aged seed sets were treated also by mutagen MMS for synergic effect and consequently subjected to experimental storage with the aim of decreasing the effects of both aging and mutagen treatment. According to our long term experience, old seeds are always showing a significant decrease in their viability demonstrated in a germination rate while the difference in aberration rate is rather low. Therefore we used MMS treatment of old seeds to obtain a high aberration base for possibility of consequent significant decrease during “storage effect“ (Fig. 3). This presumption was confirmed, when after 8-days of storage we found 3–4 times lower frequency of chromosome aberrations (Fig. 4, Tabs. 5–6). Even 12-year old seeds after storage showed viability comparable with 2-year old seeds without storage.

48 h

72 h 70

50 Chromosomal aberration (%) aberration Chromosomal 30

0 mM 3 mM 6 mM 0 mM 3 mM 6 mM 0 mM 3 mM 6 mM 12 years old 6 years old 2 years old

Fig. 3. Chromosomal aberrations after different concentrations of MMS treatment at differently aged V. faba L. seeds after 0 days of storage

48 h

72 h 70

50 Chromosomal aberration (%) aberration Chromosomal 30

0 mM 3 mM 6 mM 0 mM 3 mM 6 mM 0 mM 3 mM 6 mM 12 years old 6 years old 2 years old

Fig. 4. Chromosomal aberrations after different concentrations of MMS treatment at differently aged V. faba L. seeds after 8 days of storage

Tab. 5. Results after 48 hrs of recovery time for 0 and 8 days storage of differently aged and mutagen treated Vicia faba L. seeds evaluated by Pearson's chi-squared test and 95% Clopper and Pearson (1934) confidence interval for proportion (LB – lower bound, UB – upper bound, p-value – p-value of Pearson's chi- squared test (p-value < 0.025 is significant), s – significant, ns – non-significant, α = 0.05)

storage/days age/yrs mM #cells #aberrations proportion LB UB p-value s/ns 0 - - 1505 438 29.10 26.82 31.47 <0.0001 s 8 - - 2335 95 4.07 3.30 4.95 - 2 - 1275 79 6.20 4.94 7.66 <0.0001 s - 6 - 1610 237 14.72 13.02 16.55 - 12 - 955 217 22.72 20.10 25.51 0 2 - 455 60 13.19 10.22 16.65 <0.0001 s 0 6 - 710 217 30.56 27.19 34.10 0 12 - 340 161 47.35 41.94 52.81 8 2 - 820 19 2.32 1.40 3.59 <0.0001 s 8 6 - 900 20 2.22 1.36 3.41 8 12 - 615 56 9.11 6.95 11.66 - - 0 1280 121 9.45 7.91 11.19 <0.0001 s - - 3 1270 155 12.20 10.45 14.13 - - 6 1290 257 19.92 17.77 22.21 0 - 0 480 85 17.71 14.40 21.42 <0.0001 s 0 - 3 505 129 25.54 21.79 29.58 0 - 6 520 224 43.08 38.77 47.46 8 - 0 800 36 4.50 3.17 6.18 0.50812 ns 8 - 3 765 26 3.40 2.23 4.94 8 - 6 770 33 4.29 2.97 5.97 - 2 0 420 11 2.62 1.09 4.15 0.00053 s - 2 3 415 29 6.99 4.54 9.44 - 2 6 440 39 8.86 6.21 11.52 - 6 0 510 52 10.20 7.57 12.82 0.00004 s - 6 3 550 76 13.82 10.93 16.70 - 6 6 550 109 19.82 16.49 23.15 - 12 0 350 58 16.57 12.68 20.47 <0.0001 s - 12 3 305 50 16.39 12.24 20.55 - 12 6 300 109 36.33 30.89 41.78 0 2 0 150 3 2.00 0.41 5.73 <0.0001 s 0 2 3 155 26 16.77 11.26 23.60 0 2 6 150 31 20.67 14.49 28.03 0 6 0 210 49 23.33 17.79 29.65 0.00011 s 0 6 3 250 67 26.80 21.41 32.75 0 6 6 250 101 40.40 34.26 46.77 0 12 0 120 33 27.50 19.75 36.40 <0.0001 s 0 12 3 100 36 36.00 26.64 46.21 0 12 6 120 92 76.67 68.07 83.90 8 2 0 270 8 2.96 1.29 5.75 0.31635 ns 8 2 3 260 3 1.15 0.24 3.33 8 2 6 290 8 2.76 1.20 5.36 8 6 0 300 3 1.00 0.21 2.89 0.20490 ns 8 6 3 300 9 3.00 1.38 5.62 8 6 6 300 8 2.67 1.16 5.19 8 12 0 230 25 10.87 7.16 15.63 0.33738 ns 8 12 3 205 14 6.83 3.78 11.19 8 12 6 180 17 9.44 5.60 14.69

Tab. 6. Results after 72hrs of recovery time for 0 and 8 days storage of differently aged and mutagen treated Vicia faba L. seeds evaluated by Pearson's chi-squared test and 95% Clopper and Pearson (1934) confidence interval for proportion (LB – lower bound, UB – upper bound, p-value – p-value of Pearson's chi-squared test (p-value < 0.025 is significant), s – significant, n – nonsignificant, α=0.05)

storage/děsy age/yrs mM #cells #aberrations proportion LB UB p-value s/ns 0 - - 1275 352 27.61 25.17 30.15 <0.0001 s 8 - - 1360 40 2.94 2.11 3.98 - 2 - 840 47 5.60 4.14 7.37 <0.0001 s - 6 - 920 112 12.17 10.13 14.46 - 12 - 865 238 27.51 24.56 30.62 0 2 - 390 36 9.23 6.55 12.55 <0.0001 s 0 6 - 470 101 21.49 17.86 25.48 0 12 - 415 215 51.81 46.88 56.71 8 2 - 450 11 2.44 1.23 4.33 0.00232 s 8 6 - 460 6 1.30 0.48 2.82 8 12 - 450 23 5.11 3.27 7.57 - - 0 860 84 9.77 7.87 11.95 <0.0001 s - - 3 920 142 15.43 13.16 17.93 - - 6 855 166 19.42 16.81 22.23 0 - 0 400 74 18.50 14.81 22.66 <0.0001 s 0 - 3 470 124 26.38 22.45 30.61 0 - 6 405 154 38.02 33.28 42.95 8 - 0 460 10 2.17 1.05 3.96 0.24236 ns 8 - 3 450 18 4.00 2.39 6.25 8 - 6 450 12 2.67 1.39 4.61 - 2 0 270 9 3.33 1.19 5.47 0.02919 ns - 2 3 290 14 4.83 2.36 7.29 - 2 6 280 24 8.57 5.29 11.85 - 6 0 300 24 8.00 4.93 11.07 0.01205 s - 6 3 330 36 10.91 7.55 14.27 - 6 6 300 47 15.67 11.55 19.78 - 12 0 290 51 17.59 13.20 21.97 <0.0001 s - 12 3 300 92 30.67 25.45 35.88 - 12 6 275 95 34.55 28.93 40.17 0 2 0 120 4 3.33 0.92 8.31 0.00058 s 0 2 3 140 10 7.14 3.48 12.74 0 2 6 130 22 16.92 10.92 24.49 0 6 0 140 24 17.14 11.30 24.42 0.03224 ns 0 6 3 180 34 18.89 13.45 25.38 0 6 6 150 43 28.67 21.59 36.61 0 12 0 140 46 32.86 25.16 41.30 <0.0001 s 0 12 3 150 80 53.33 45.02 61.51 0 12 6 125 89 71.20 62.42 78.95 8 2 0 150 5 3.33 1.09 7.61 0.52084 ns 8 2 3 150 4 2.67 0.73 6.69 8 2 6 150 2 1.33 0.16 4.73 8 6 0 160 0 0.00 0.00 2.28 0.11777 ns 8 6 3 150 2 1.33 0.16 4.73 8 6 6 150 4 2.67 0.73 6.69 8 12 0 150 5 3.33 1.09 7.61 0.13942 ns 8 12 3 150 12 8.00 4.20 13.56 8 12 6 150 6 4.00 1.48 8.50

In our following experiments Vicia faba L. seeds were treated with MMS and stored at 50% w.c. for 0, 14 and 28 days. At this level of moisture, the frequency of chromosome aberrations diminished in proportion to the length of storage. This reduction tended to be the case where higher dosages of mutagens were measured, the longest recovery times were recorded, and the length of storage was 28 days (i.e. 7.8 times fewer CAs than at the beginning of storage), while on average, from all recovery times the frequency of CAs for 6 mM dose decreased 4.2 times (2.05 times for 4 mM dose). In 6 mM MMS- treated seeds the reduced frequency of CAs continued concurrently with storage-time, while in the case of 4 mM MMS, the storage effect was observed mostly for the first 14 days (Tabs. 7–8).

Tab. 7. The frequency of chromosome aberrations in course of seed storage (in % of aberrant metaphases induced by 6 mM MMS treatment) Treatment conditions: seeds were treated for 5 hrs, then washed for 2 hrs at 25 oC, redried to 50 % water content and stored at 25 oC, 100 metaphases (50 in control) were scored for each recovery time. SEM = standard error

Storage (days) Treatment Recovery time 56 h 72 h 80 h Σ+SEM Σ+SEM Σ+SEM 0 control 3.9+1.4 1.9+0.2 2.1+1.0 6 mM 78.3+3.5 69.3+8.2 57.6+6.6 14 control 1.1+0.1 1.8+0.6 2.2+0.2 6 mM 39.9+1.0 23.6+4.4 21.3+2.0 28 control 5.4+1.0 1.2+0.9 2.0+1.1 6 mM 19.6+0.1 21.4+0.5 7.7+2.2

Tab. 8. The frequency of chromosome aberrations in the course of seed storage (in % of aberrant metaphases induced by 4 mM MMS treatment) Treatment conditions: seeds were treated for 5 h, then washed for 2 hrs at 25 oC, redried to 50 % water content and stored at 25 oC, 100 metaphases (50 in control) were scored for each recovery time

Storage (days) Treatment Recovery time 72 h 80 h Σ+SEM Σ+SEM 0 control 5.1+1.0 3.1+1.8 4 mM 50.7+4.5 4.9+4.0 14 control 3.2+2.0 3.5+0.8 4 mM 19.4+3.7 10.6+6.3 28 control 3.2+0.1 4.4+0.3 4 mM 25.9+1.6 15.7+5.7 SEM = standard error

The appearance of unscheduled DNA synthesis (UDS) Our goal was to confirm the existence of UDS in experimental storage where evidence of DNA damage and its repair had been found in the form of aberrations at the chromosomal level as presented above. Roots grown for 80 hours were treated with 0.6 mM MH (5 hrs), washed (2 hrs) and exposed to 3H-TdR for additional two-hour periods in 7, 24, and 32 hrs after the treatment was initiated. As the replicative DNA synthesis was suppressed by HU, an enhanced 3H-TdrR incorporation into nuclear DNA (determined microautoradiographically) was thought to be due to unscheduled DNA synthesis induced by the mutagen. A significantly higher incorporation of 3H-TdrR into DNA of MH-treated roots occurred seven hours after the action of MH. The high amount of silver grains per nucleus found immediately after treatment fell after 24 hrs to a controlled level, but labeled nuclei still accounted for 9%. Within 32 hrs we observed no differences in the frequency of grains in comparison with the control group (Table 9). Thus, in growing roots UDS seems to occur up to 24 hrs after MH treatment.

After treatment with MH (0.6 mM, 5 hrs) and washing (2 hrs) of seeds, half of the seed sample was labeled with 3H-TdR, the other half was dried to 50% w.c. and stored for 7 days. After storage the seeds were soaked in distilled water for 24 hrs and then labeled in the same way described above. In contrast to growing roots, UDS in the embryonic cells of seeds after storage and 24 hrs of germination was still fairly high (compare Tabs. 9–10). UDS in DNA of stored embryos was as high as before storage. Only the percentage of labeled nuclei was about twice as low after storage (cf. Tab. 10).

Tab. 9. UDS in growing roots (80 hrs) treated with 0.6 mM MH (5 hrs) and 2 hrs washing The data indicate 3H-TdR incorporation into nuclear DNA at different intervals after onset of MH treatment. HU was applied 2 hrs before and during 3H-TdR labelling (2 hrs). Results are expressed as means + SEM

Time after onset of MH % labelled cells after MH Silver grains per nucleus treatment (hrs) treatment Control MH-treatment 7 4.90+0.7 16.90+1.1 54.90+3.1 24 3.00+0.4 3.60+2.2 9.00+6.7 32 1.90+0.5 2.30+0.2 2.02+0.4

Tab. 10. Influence of storage with 50 % water content on UDS in DNA of MH-treated seeds The data indicate 3H-TdR incorporation into nuclear DNA before and after 7 days of storage. HU treatment as in Table 10. Results are expressed as means + SEM

% labelled cells after MH Storage (days) Silver grains per nucleus treatment Control MH-treatment 0 3.2+0.3 9.6+0.5 26.2+7.5 7 3.3+0.2 10.5+0.4 12.2+3.1

Labeling with 3H-TdR was done immediately after MMS treatment and washing (a), after redrying to 50% w.c. (b) or after 3, 5, 7 and 14 days of storage. Dried and stored samples were exposed to 3H-TdR after 7 hrs soaking (the same interval as in sample a). No significant difference in the incorporation of 3H-TdR into DNA was observed between samples before (a) and after (b) redrying. In stored samples we consistently noted the incorporation of 3H-TdR into nuclear DNA of MMS-treated seeds as measured by the number of silver grains per nucleus and by the percentage of labeled nuclei (Tab. 11).

Tab. 11. Influence of storage with 50 % water content on UDS in DNA of MMS-treated seeds The data indicate 3H-TdR incorporation into nuclear DNA before and after 7 days of storage. Results are expressed as means + SEM

% labelled cells after MMS Storage (days) Silver grains per nucleus treatment Control MMS-treatment 0a 3.30+1.0 12.4+1.1 31.1+2.3 0b 2.10+0.6 11.4+0.7 39.4+4.4 3 1.60+0.3 10.1+0.2 45.1+7.2 5 3.70+0.7 12.7+0.7 39.8+4.7 7 1.20+0.0 9.60+1.2 32.6+4.0 14 2.40+0.2 8.30+0.7 37.1+12.3

Three to fourteen days’ storage of 50% water-saturated V. faba seeds exposed to MH or MMS resulted in a recovery from mutagen-induced chromosomal damage and a significantly higher incorporation of 3H-TdrR into nuclear DNA. This finding supports the hypothesis that recovery from MH- and MMS- induced chromosomal damage is mediated by excision repair during seed storage.

Summary of results Realizing that aging of seeds is closely connected with the effect of chromosome aberrations, with their higher frequency with age and in first mitoses, we have observed three basic characteristic of the experimental set: 1. The frequency of chromosome aberrations significantly increased with the age of the seeds. 2. With a higher dose of mutagen the yield of chromosome aberrations was higher. 3. A combination of the old seeds and mutagen treatment has a synergic effect. We were testing three parallel hypotheses: 1. The storage of seeds has significant positive impact (decreased frequency of chromosome aberrations) at all tested possibilities. More effective is a storage for less than 14 days, preferably 8 days. 2. This impact is higher for older seeds. 3. This impact is more significant for highest dose of mutagen. This aim was successfully reached in all seed sets, evaluated parameters and recovery times. These observations were confirmed by occurrences of UDS showing that storage effect is connected with DNA repair.

Discussion Navaschin (1933) was the first to observe the peculiar connection between the age of seeds and the frequency of chromosome aberrations in root tips on germination. Since then numerous investigations have been devoted to the problem. Basic observations in various plant species were later supplemented by attempts to demonstrate a relationship between aging of seeds and their sensitivity to the action of chemicals (e.g. Avanzi et al. 1969). Interesting results were obtained about the different moisture of stored seeds in the course of the presumed repair mechanisms in plant cells and the corresponding effects of alkylating agents (Gichner, Velemínský 1973). Villiers already in 1973 stated in his theory of seed aging that the primary cause of failure of old seeds to begin cell extension in germination is damage of their membrane systems. And “once hydrolytic enzymes are released from their normal cellular compartments, the original damage would become greatly amplified, as general lysis of the cellular structure would rapidly follow” (Villiers 1973). Villiers (1974) also suggested that “in dry tissues enzyme-controlled turnover and repair may be temporarily suspended” and that “this may be an important factor in the loss of seed viability in storage”. In the same year Floris and Anguillesi (1974) reported that in the course of aging a decrease in the activity of enzymes such as catalase, peroxidase, cytochrome oxidase and decarboxylase occurs in the seeds. Floris with colleagues (Grilli et al. 1995) later described the level of Poly(A)Polymerase as a significant marker of viability of seeds during their aging. A loss of protein synthesizing capacity and an increase in membrane permeability were observed during germination of old seeds, resulting in a decrease in the content of sugars and other metabolic products. Also the production of the major organic volatiles, ethanol and acetaldehyde, during imbibition depends greatly on the age of the seeds (Górecki et al. 1992). Interesting is a hypothesis suggested by Rieger and Michaelis (1959) who found Vicia faba L. seeds susceptible to the action of ethanol or other “automutagens“ which could accumulate in the course of aging as a products of anaerobic respiration. Finally we may summarize that “storage effect” effectively eliminates not only action of mutagens but also action of “automutagens“ that accumulate during the aging process. The experimental delay of G1 phase in first wave of DNA synthesis of old seeds during the imbibition causes a significant enhancement of pre-replicative DNA repair which should be a possible way towards the rejuvenation generally.

Acknowledgements Our gratitude goes to Dr. Stanislav Katina from Department of Applied Mathematics and Statistics (Faculty of Mathematics, Physics and Informatics, Comenius University, Bratislava, Slovakia) for significant help with statistical evaluation of experiments. The author´s thank also to prof. Robert Murray Davis, USA, for his revision of the text.

References

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Abstrakt Semená rastlín sú dlhodobo využívané ako vhodný model pre štúdium starnutia na základnej cyto- genetickej úrovni. Naša štúdia vychádza z 25 rokov našich experimentov so starnutím zameraných na zvyšovanie DNA reparácií pri semenách rastlín pomocou metódy „skladovacieho efektu“, vy- vinutého v minulosti viacerými autormi. Táto metóda je založená na predĺžení G-1 fázy tak, že sa predlžuje predreplikačná DNA reparácia z hodín na dni. Ponúka tak jednoduchý a účinný prostriedok DNA reparácií poškodení spôsobených rôznymi druhmi xenobiotík. V nasledovných experimentoch sme sa zamerali na poškodenie DNA spôsobené mutagénmi pri starých semenách a možné nápravy tohto poškodenia.

Gustáv Murín, Karol Mičieta: Omladzovací efekt pri starých semenách Vicia faba L. zvýšením DNA opráv počas experimentálneho skladovania

Acta Botanica Universitatis Comenianae Vol. 50, 2015

BRYOPHYTES OF SELECTED VILLAGES IN SLOVAKIA

Katarína Mišíková, Lucia Kokešová, Katarína Godovičová

Comenius University in Bratislava, Faculty of Natural Sciences, Department of Botany, Révová 39, 811 02 Bratislava, Slovakia

Received 18 November 2015; Received in revised form 1 December 2015; Accepted 8 December 2015

Abstract In ten selected villages in Slovakia, 81 bryophytes (Marchantiophyta, Anthocerotophyta, Bryophyta) were found out. Species richness varied from 17 to 57 species on individual localities. The highest species number was observed in the village Párnica situated in the northern part of central Slovakia with more humid and colder climate, which probably positively affects the occurrence of several bryophytes. The lowest species numbers were observed in the lowland villages of southern Slovakia. In agricultural landscape, the highest species diversity is often related to the cemeteries and parks. In relation to the ecological groups, the most species occured on bare damp soil or on concrete and stony walls. In rural environment, the poly- and euhemerobic species and bryophytes less sensitive to SO2 pollution are prevalent as sensitive ones. From phytogeographical viewpoint, temperate bryophytes are abundant.

Key words: rural environment, agricultural landscape, Marchantiophyta, Anthocerotophyta, Bryophyta

Introduction The occurence and ecology of bryophytes in rural environment and agricultural landscape is known only scarcely in Slovakia. Bryological data are published only from few villages such as Marianka (Malé Karpaty Mts.) (Mišíková 2013), Borský Svätý Jur (Borská nížina Lowland) (Dobiašová 2014) and Moravský Svätý Ján (Borská nížina Lowland) (Mišíková, Kubinská 2009). Bryophytes of cemeteries and historical parks in several villages in southern Slovakia were studied by Mišíková, Jurčišinová (2013) and Jurčišinová (2014). Several synanthropic bryophyte communities occurring on anthropogenic sites were mentioned by Peciar (1985). An important topic studied by several authors in Europe (e.g. Zechmeister, Moser 2001; Zechmeister et al. 2002; Whitehouse 2001; Andriušaitytė, Jukonienė 2013) is the bryophyte flora of agrocoenoses. Up to now, in Slovakia agricolous bryophytes were studied by Janovicová, Kresáňová (2000), Kresáňová (2002) and Kresáňová et al. (2005). The aim of the presented paper is to provide information concerning bryophyte diversity, ecology, hemeroby and phytogeography in rural environment.

Material and Methods Bryological research was carried out in ten selected villages in years 2009–2014 (Fig. 1), a brief description of the villages is given in Tab. 1. Samples were collected in following habitats – rural environment (RU), cemeteries and parks (CE), extensively managed fields (FI), meadows (ME). Altitude and geographical coordinates were measured using a GPS device (Garmin eTrex) approximately in the centre of villages. The nomenclature of liverworts follows the study by Söderström et al. (2002), nomenclature of bryophytes is based on the work by Hill et al. (2006). Threat categories follow Kubinská et al. (2001), phytogeographical analysis, degree of hemeroby (HB) and air quality (AP) are listed according to Düll (2010).

 Corresponding Author: Katarína Mišíková; [email protected]

Frequency is calculated by formula: Fr [%] = (n1/N) ˟ 100, if n1 is number of sites, where has been the species recorded and N is the total sites number. Herbarium specimens are deposited in the SLO herbarium (Herbarium of the Department of Botany, Faculty of Natural Sciences, Comenius University in Bratislava).

Tab. 1. Description of the selected villages

Village Altitude GPS Year of Phytogeographical number Village (m a.s.l.) Population coordinates bryological observation area (Futák 1980) Podbranč – 614 48°43'59"N 1. 400 2009, 2010 Malé Karpaty Mts. (Podzámok) (Anonymus 1) 17°27'48"E 655 48°15'44"N 2. Borinka 235 2010, 2012 Malé Karpaty Mts. (Anonymus 2) 17°05'11"E Horná 1891 48°14'35"N Podunajská nížina 3. 145 2014 Kráľová (Anonymus 3) 17°54'55"E Lowland 4311 48°13'35"N Podunajská nížina 4. Močenok 140 2014 (Anonymus 4) 17°55'51"E Lowland Kalná nad 2043 48°11'59"N Podunajská nížina 5. 160 2014 Hronom (Anonymus 5) 18°31'10"E Lowland 522 48°11'40"N Podunajská nížina 6. Horná Seč 160 2014 (Anonymus 6) 18°32'41"E Lowland 385 48°17'13"N Podunajská nížina 7. Drženice 225 2014 (Anonymus 7) 18°41'48"E Lowland 825 49°11'43"N 8. Párnica 460 2009, 2014 Západné Beskydy Mts. (Anonymus 8) 19°11'59"E 567 48°11'45"N Krupinská planina 9. Príbelce 300 2009, 2012 (Anonymus 9) 19°15'18"E Plateau Modrý 1560 48°14'26"N Krupinská planina 10. 240 2009, 2012 Kameň (Anonymus 10) 19°20'03"E Plateau

Fig. 1. Studied villages 1. Podbranč, 2. Borinka, 3. Horná Kráľová, 4. Močenok, 5. Kalná nad Hronom, 6. Horná Seč, 7. Drženice, 8. Párnica, 9. Príbelce, 10. Modrý Kameň

Results Overall, 81 bryophytes were found in ten villages, out of them 5 liverworts, one hornwort and 75 mosses (Tab. 2). Two species are redlisted – Anthoceros agrestis (LR: nt) and Didymodon vinealis (LR: nt) (Kubinská et al. 2001). Seven species occured in all evaluated villages (Tab. 2). These taxa are the most frequent bryophytes which are often found on anthropogenic sites in rural and urban environmnet. Species richness varied from 17 to 57 species on individual localities (Fig. 2). Species diversity is related to the geographical position and climate of the village – the most bryophytes were detected in the village Párnica in the north of central Slovakia (51 species), the least (17 species) in the village Horná Kráľová in the south of Slovakia.

In view of the ecological groups, the most species were observed on the ground (B – 49 species, Tab. 2, Fig. 3); they prefered humid places without competition of vascular plants (B1 – 30 species). Epixylic species were found only occasionally (D – 4 species), as the dead wood represents a rare substrate in rural environment.

Tab. 2. List of recorded species

Species *villages **Fr (%) ***Habitats ****Ecological group ANTHOCEROTOPHYTA Anthoceros agrestis 8 10 FI B1 MARCHANTIOPHYTA Frullania dilatata 9 10 CE C2 Lophocolea bidentata 8 10 ME B2 Marchantia polymorpha 8 10 RU B1 Riccia glauca 8 10 FI B1 Riccia sorocarpa 2, 8 20 FI B1 BRYOPHYTA Abietinella abietina 1, 8, 9, 10 40 ME CE B2 Amblystegium serpens 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 100 RU CE ME A1 A2 B1 B2 C1 C2 D Atrichum undulatum 2 10 CE B1 Barbula convoluta 6, 8, 9 30 RU CE B1 Barbula unguiculata 1, 2, 5, 6, 7, 8, 9 70 RU CE FI B1 Brachythecium albicans 1, 2, 6, 9, 10 50 RU CE ME B1 B2 Brachythecium rivulare 8 10 RU A3 Brachythecium rutabulum 2, 3, 4, 5, 6, 8, 9, 10 80 RU CE ME A1 B1 B2 D Brachythecium salebrosum 1, 2, 4, 5, 8, 9, 10 70 RU CE ME B2 Bryoerythrophyllum recurvirostrum 1, 8 20 RU CE A1 Bryum argenteum 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 100 RU CE ME FI A1 B1 Bryum caespiticium 3, 4, 5, 8, 10 50 CE RU A1 B1 Bryum capillare 1, 2, 3, 5, 6, 7, 8, 9, 10 90 CE RU ME A1 B1 Bryum dichotomum 1, 2, 3, 5, 8, 10 60 RU CE ME FI B1 Bryum klinggraeffii 5 10 RU FI B1 Bryum moravicum 8 10 RU CE C1 C2 Bryum rubens 2, 8 20 FI RU B1 Bryum ruderale 2, 8 20 FI RU B1 Bryum violaceum 2, 6 20 FI RU B1 Calliergonella cuspidata 6, 7, 8, 10 40 CE ME B2 Calliergonella lindbergii 10 10 CE B2 Ceratodon purpureus 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 100 RU CE FI ME A1 B1 D Cirriphyllum piliferum 1, 2, 3, 8, 9, 10 60 CE ME B2 Climacium dendroides 10 10 CE B2 Dicranella staphylina 2, 8 20 FI B1 Didymodon fallax 2, 3, 5, 8, 9 40 RU CE A1 B1 Didymodon rigidulus 1 10 RU CE A1 Didymodon vinealis 2 10 CE A1 Encalypta vulgaris 6 10 CE A1 Fissidens bryoides 6 10 RU B1 Fissidens taxifolius 2, 7, 8 30 RU CE B1 Fontinalis antipyretica 8 10 RU A3 Funaria hygrometrica 1, 5, 8, 10 40 RU CE FI B1

Tab. 2. Continuiation

Species *villages **Fr (%) ***Habitats ****Ecological group Grimmia ovalis 4, 8, 10 30 RU CE A1 Grimmia pulvinata 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 100 RU CE A1 Homalothecium lutescens 1, 4, 5, 6, 7, 9, 10 70 ME CE B2 Homalothecium philippeanum 10 10 CE A1 A2 Homalothecium sericeum 1, 6, 9 30 CE RU ME A1 A2 Hygroamblystegium tenax 2, 8 20 CE RU A3 Hygroamblystegium varium 2 10 RU A1 A2 Hylocomium splendens 1 10 ME B2 Hypnum cupressiforme 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 100 CE RU ME A1 A2 B2 C1 C2 D Kindbergia praelonga 3, 4, 6, 8, 9, 10 60 RU CE FI ME B1 B2 Leskea polycarpa 5 10 CE C2 Leucodon sciuroides 1, 8, 9, 10 40 CE ME C2 Orthotrichum affine 3, 4, 7, 8, 9 50 CE RU ME C2 Orthotrichum anomalum 1, 2, 3, 6, 8, 9, 10 70 RU CE A1 Orthotrichum cupulatum 1, 10 20 CE A1 Orthotrichum diaphanum 3, 4, 5, 6, 7, 8, 9 70 RU CE ME A1 C2 Orthotrichum pumilum 2, 4, 8 30 RU CE ME C2 Oxyrrhynchium hians 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 100 RU CE FI ME B1 B2 Phascum cuspidatum 1, 2, 5, 8, 9, 10 60 RU FI ME B1 Plagiomnium cuspidatum 8, 10 20 CE B2 Plagiomnium rostratum 1, 2, 7, 8, 9, 10 60 RU CE ME B2 Plagiomnium undulatum 1, 2, 8, 10 40 CE ME B2 Platygyrium repens 10 10 CE C2 Platyhypnidium riparioides 2, 8 20 RU A3 Pleurozium schreberi 1, 10 20 CE ME B2 Pseudocrossidium hornschuchianum 8 10 RU B1 Pseudoscleropodium purum 6, 7, 8, 10 40 CE ME B2 Pylaisia polyantha 2 10 CE C2 Rhynchostegium murale 2, 4, 8, 10 40 CE RU A1 Rhytidiadelphus loreus 8 10 CE B2 Rhytidiadelphus squarrosus 8 10 ME B2 Rhytidiadelphus triquetrus 1, 7, 8 30 ME CE B2 Sciuro-hypnum populeum 1, 8, 9, 10 40 CE RU A1 A2 Schistidium apocarpum agg. 1, 2, 4, 7, 8, 9, 10 70 RU CE A1 Syntrichia montana 6, 8 20 CE A1 C2 Syntrichia ruralis 1, 2, 4, 5, 8, 9, 10 70 RU CE A1 A2 Syntrichia virescens 4, 6, 8 30 CE RU C2 Thuidium assimile 1, 8, 9, 10 40 CE ME B2 Tortella tortuosa 1 10 ME A2 Tortula muralis 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 100 RU CE A1 Tortula truncata 2, 10 20 FI RU B1 Weissia controversa 1, 9, 10 30 FI CE B1 *Villages: for description see Tab. 1 **Fr [%] = (n1/N) x 100, if n1 is number of sites, where has been the species recorded and N is the total sites number ***Habitats: RU – rural environment; CE – cemeteries, parks; FI – extensively managed fields; ME – meadows ****Ecological groups: A. Epilithic species: A1 – concrete and stone walls, fences; A2 – rocks and boulders; A3 – stream and stream banks; B. Epigeic species: B1 – bare damp soil; B2 – lawns, meadows; C. Epiphytic species: C1 – tree bases and trunks up to 50 cm above ground level; C2 – tree trunks at height 50 cm above ground level and higher; D. Epixylic species

50 57

40 40 30 36 31 20 24 24 20 20 18 Number ofspecies 10 17

0 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Villages

Fig. 2. Species number in the villages 1. Podbranč, 2. Borinka, 3. Horná Kráľová, 4. Močenok, 5. Kalná nad Hronom, 6. Horná Seč, 7. Drženice, 8. Párnica, 9. Príbelce, 10. Modrý Kameň

35 30

30 25 26 20 24 15 10 13 5 8 4 3 4 Number ofspecies 0 A1 A2 A3 B1 B2 C1 C2 D 31 49 12 4 A B C D

Ecological groups

Fig. 3. *Species number in the individual ecological groups * for abbreviations see Tab. 2

Rural environment (RU, 48 species, Fig. 4) On concrete and stone walls and fences basiphilous mosses were found. For several epilithic bryophytes, these are the only sites which allow them to survive in an environment with the absence of natural rocky substrates (e.g. Podunajská nížina Lowland). Representative species of these sites are Tortula muralis, Schistidium apocarpum agg., Grimmia pulvinata, Orthotrichum anomalum, Rhyn- chostegium murale, Sciuro-hypnum populeum. For paths and pavements epigeic and epilithic species are typical. These species with frequent vegetative reproduction are resistant to trampling, e.g. Bryum dichotomum, B. argenteum, Barbula convoluta, B. unguiculata and Pseudocrossidium hornschuchianum. Man-made habitats as gardens and courtyards are suitable localities for apophytic bryophytes such as Bryum argenteum, B. caespiticium, Funaria hygrometrica, Ceratodon purpureus. In lawns, especially if they are watered regularly, several pleurocarpous mosses occur (Cirriphyllum piliferum, Calliergonella cuspidata, Climacium dendroides, Pseudoscleropodium purum and others).

70 60 60 48 50

40 31 30 18 20 10

Number ofspecies 0 FI ME RU CE Habitats

Fig. 4. *Species number on the individual habitats * for abbreviations see Tab. 2

Meadows (ME, 31 species, Fig. 4) Pleurocarpous, tall mosses are a often part of the vegetation on meadows, especially on well drained sites, e.g. Rhytidiadelphus triquetrus, Calliergonella cuspidata and Pseudoscleropodium purum. On dry ground with low occurence of vascular plants also small acrocarpous and ephemeral bryophytes grow.

Fields (FI, 18 species, Fig. 4) Fields, mainly extensively cultivated, are colonized by annual and ephemeral epigeic bryophytes. The diversity of these species is especially high in colder and more humid areas of central and northern Slovakia (Kresáňová et al. 2005). On intensively managed and fertilizied fields, hemerophilous and nitrophilous species prevail (e.g. Marchantia polymorpha, Bryum argenteum, Ceratodon purpureus). Extensively exploited fields in close vicinity of settlements are destroyed by a housing construction, due to that several localities of agricolous bryophytes failed to confirm, e.g. Anthoceros agrestis.

Cemeteries and parks (CE, 60 species, Fig. 4) Comparing the various habitats and the sites in selected villages, the highest number of bryophytes was identified in cemeteries and parks. Several authors evaluated them as significant refuges for bryophytes in rural and urban environment, e.g. Fudali (2005), Mišíková, Jurčišinová (2013), Mišíková (2013). Degrees of hemeroby (HB) reflect the intensity of anthropogenic impacts on the landscape or habitats (Zechmeister, Moser 2001; Zechmeister et al. 2002). Polyhemerobic species (HB2–HB3) are mostly nitrophilous bryophytes, which grow on ruderal habitats. Euhemerobic bryophytes (HB4–HB5) occur in less eutrophic habitats, e.g. extensively managed fields and meadows inside the settlements. Mesohemerobic bryophytes (HB6–HB7) are particularly pioneer taxa growing on ruderal, as well as natural sites (e.g. non-fertilized meadows, abandoned orchards, quarries and old walls of the ruins) or other substrates and habitats close to natural. From oligohemerobic species (HB8, hemerophobic), only Homalothecium philippeanum and Rhytidiadelphus loreus were found on cemeteries. Overall, in observed villages hemerophilous bryophytes were prevalent (Fig. 5), while the polyhemerobic and euhemerobic species were more numerous (44 species) than the mesohemerobic ones (33 species). AP values (air purity / air pollution) (Düll 2010) indicate the level of sensitivity of different species to SO2 air pollution. Slightly to moderately sensitive species (AP1–AP4) can grow in an environment 3 with SO2 pollution from 0.11 to 0.16 mg/m . The so-called indicators of air purity (AP5–AP9) occur 3 mainly in areas where SO2 values are lower than 0.085 mg/m . In the study area, less sensitive species to SO2 pollution were over-represented (45 species, Fig. 6) than sensitive bryophytes (16 species, Fig. 6).

HB2‒HB5 6 11 14 13

HB6‒HB7 15 18

HB8 2

0 10 20 30 40 50 Number of species

Fig. 5. Degrees of hemeroby and species number

AP1‒AP4 11 10 17 7

AP5‒AP8 9 4 2 1

0 10 20 30 40 50 Number of species

Fig. 6. Air pollution indicators (AP1–AP4) and Air purity indicators (AP5–AP8)

Phytogeographical analysis The most of the observed species belongs to temperate ones (63%, Fig. 7). Sub-boreal species consti- tute 16% with several pleurocarpous mosses, which grow on meadows or lawns in parks and cemeteries. Within the sub-oceanic species (7%), bryophytes growing on bare soil of the fields and ruderal habitats were frequent. The group of sub-mediterranean species (7%) represents mainly thermophilous acrocarpous mosses, which often grow in anthropogenic habitats.

temperate species (63%) 4%3% 7% sub-boreal species (16%) 7% sub-mediterranean species (7%) 16% sub-oceanic species (7%) 63%

boreal montane species (4%) sub-continental sub- mediterranean species (3%)

Fig. 7. Phytogeographical analysis

Conclusions In rural environment, the strong anthropogenic influence resulted in a high presence of polyhemerobic and apophytic species. Hemerophobic species are rare inside of the villages. Bryophytes prefering habitats with low competition of vascular plants (bare soil, anthropogenic rocky substrates) established the prevailing group. The highest species diversity was observed on cemeteries and parks. These habitats are valuable refuges for several group of bryophytes, e.g. epiphytes, epilithic and epixylic species.

Acknowledgements This study was supported by the Grant Agency VEGA (Grant VEGA No. 1/0885/16).

References

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Abstrakt V desiatich skúmaných obciach Slovenska sme našli 81 druhov machorastov (Marchantiophyta, Anthocerotophyta, Bryophyta), pričom druhová diverzita sa pohybovala medzi 17 až 57 druhov. Naj- vyššia druhová diverzita bola zistená v obci Párnica, ktorá leží v severnej časti stredného Slovenska s vlhkejším a chladnejším podnebím, čo priaznivo ovplyvňuje výskyt viacerých machorastov. Najniž- šie počty druhov sme zistili v obciach južného Slovenska. Z hľadiska jednotlivých biotopov a stanovíšť je najvyššia druhová diverzita viazaná na cintoríny a parky, čo korešponduje s viacerými literárnymi údajmi. Z hľadiska ekologických skupín najviac druhov rastie na vlhkej holej pôde a na betónových a kamenných múroch, náhrobkoch. V rurálnom prostredí prevládajú poly- a euhemeróbne druhy oproti mezo- a oligohemeróbnym. Početnejšie sú druhy málo citlivé voči vyšším koncentráciám SO2 ako druhy senzitívne. Z fytogeografického hľadiska sú najčastejšie temperátne machorasty (63%).

Katarína Mišíková, Lucia Kokešová, Katarína Godovičová: Machorasty vybraných obcí Slovenska

Acta Botanica Universitatis Comenianae Vol. 50, 2015

ALIEN TAXA OF VASCULAR PLANTS OF THE URBAN ECOSYSTEM OF THE SELECTED AREA OF BRATISLAVA CITY, MUNICIPAL PART KARLOVA VES

Alena Rendeková, Michal Hrabovský, Jana Hanúsková, Ján Miškovic

Comenius University in Bratislava, Faculty of Natural Sciences, Department of Botany, Révová 39, 81102 Bratislava, Slovakia

Received 8 December 2015; Received in revised form 15 December 2015; Accepted 21 December 2015

Abstract The paper presents alien taxa of vascular plants recorded in the urban ecosystem of the selected area of Bratislava city, municipal part Karlova Ves. The area was divided into 18 sections, where 146 alien taxa of vascular plants were recorded during the vegetation season in 2014. It contains 82 archaeophytes and 64 neophytes, including 23 invasive taxa of vascular plants. The most common invasive neophytes in the monitored area were Conyza canadensis, Erigeron annuus and Robinia pseudoacacia.

Key words: archaeophytes, neophytes, ruderal flora, invasive plants

Introduction The study area is located in the southwest of Slovakia, on the border of two phytogeographic districts the Malé Karpaty Mts. and the Devínska Kobyla Hill, which causes a high diversity of the vegetation. The southern district Devínska Kobyla is a part of the unit Eupannonian xerothermic flora (Eupannoni- cum) within the region of the Pannonian flora (Pannonicum). The northen district Malé Karpaty belongs to the region of the West-Carpathian flora (Carpaticum occidentale), the region of the pre-Carpathian flora (Praecarpaticum) according to Futák (1984). The Malé Karpaty Mts. form a part of the temperate to warm area with average annual temperature of 7–9 °C. They are characteristic by the temperate and humid climate with cold winters in the higher altitudes and with the warm and moderately humid climate with mild winters in the lower altitudes and adjacent plains (Liška 1986). The contact of the Pannonian and Carpathian region is reflected in the composition of the local flora and the proportions of species growing in this territory. In addition to thermophilic species also some mountain species are found here (Bizubová et al. 1998). There was carried out a floristic research by Lumnitzer (1791) and Endlicher (1830) in the area. The most complete list of taxa present in the territory of Devínska Kobyla was elaborated by Kaleta in 1968. Záborský (1993) in the monograph of Karlova Ves edited by Bretová presented a survey of the most common plant species. Another publication Flora, Geology and Paleontology of Devinska Kobyla (Feráková et al. 1997) reviews the distribution of all non-vascular and vascular plants recorded in this phyto-geographical region and adjacent parts of the Podunajská nížina and Záhorská nížina lowlands. In this article we inform about the current distribution of the alien taxa of vascular plants in the selected part of Bratislava-Karlova Ves.

 Corresponding author: Alena Rendeková; e-mail: [email protected]

Material and Methods Flora in the territory of municipal part Bratislava-Karlova Ves – excluding Sihoť, Karloveské rameno, Slávičie údolie cemetery, Botanical garden, Zoo and SNP park in the Líščie údolie valley – and adjacent part of the municipal part Dúbravka was monitored during one vegetation season within the master's thesis study (Hanúsková 2015). The field research was carried out from March to October 2014. The study area was divided into 18 geographic sections (Fig. 1). Southern border of the study area is formed by the Botanická and Devínska cesta streets. Eastern border reaches the Vydrica watercourse valley.

Fig. 1. Monitored area divided into 18 sections

List of sections 1. Dúbravka, Dolné Krčace, Horné Krčace 11. Staré Grunty, South (M. Š. Trnavského street and surroundings) 12. Vydrica watercourse 2. Polianky 13. Dlhé diely, West (Majerníkova, Veternicová, Svíbová 3. Patrónka, North (Dúbravská cesta and Mokrohájska streets and surroundings) streets and surroundings) 14. Dlhé diely, North (Jána Stanislava street 4. Patrónka, South and surroundings) 5. Sitina hill 15. Dlhé diely, South-West (Jamnického, Iskerníková, 6. Líščie údolie street Stoklasová streets and surroundings) 7. Staré Grunty, North (Konvalinková, Na sitine, Vretenová, 16. Dlhé diely, South (Hany Meličkovej, Kresánkova, Slávičie údolie streets, Mlynská dolina street – North) Matejkova streets and surroundings) 8. Karlova Ves, North (Borská, Pernecká, Púpavová, 17. Dlhé diely, East (Adámiho, Baníkova, Janotova streets Zohorská streets and surroundings) and surroundings) 9. Karlova Ves, the central part (Čavojského, 18. The area of the Faculty of Natural Sciences Comenius Kempelenova, Tilgnerova streets and adjacent streets) University and adjacent streets (Botanická, Svrčia 10. Karlova Ves, South (Karloveská and Segnerova streets streets, Mlynská dolina street – South) and surroundings)

Furthermore, northen and north-western borders reach the cadastral areas of Bratislava-Lamač municipal part and Bratislava-Dúbravka municipal part. Western and south-western border of this area is the same as of the forest complex Devínska Kobyla, which belongs prevailingly to the cadastre of Devín municipal part (Bertová 1993). Alien taxa – excluding cultivated taxa not listed in Medvecká et al. (2012) – were recorded in these 18 sections of the area. The invasion status and residence time of the taxa was classified according to Medvecká et al. (2012). Herbarium specimens were deposited in herbarium of the Department of Botany, Faculty of Natural Sciences, Comenius University, Bratislava (SLO). Families were delimited according to APG III system. The nomenclature of taxa follows Marhold et al. (2007).

Results and discussion

We recorded together 146 alien taxa of vascular plants (Tab. 1), of which 82 belong to the archaeophytes and 64 to the neophytes. Frequent families were Asteraceae, Poaceae, Fabaceae, Brassicaceae and Rosaceae (Fig. 2). Although Slovak alien flora contains more neophytes than archaeophytes – 634 neophytes and 284 archaeophytes (Medvecká et al. 2012), there were found more archaeophytes than neophytes in the studied area. It includes 19 invasive neophytes – Acer negundo, Ailanthus altissima, Amaranthus retroflexus, Ambrosia artemisiifolia, Aster novi-belgii agg., Conyza canadensis, Erigeron annuus, Fallopia ×bohemica, F. japonica, Galinsoga parviflora, G. quadriradiata, Helianthus tuberosus, Impatiens glandulifera, I. parviflora, Lycium barbarum, Robinia pseudoacacia, Rumex patientia, Solidago canadensis and S. gigantea. The most common invasive neophytes were Conyza canadensis, Erigeron annuus a Robinia pseudoacacia, which occurred in almost all of the 18 sections. There are only four invasive archaeophytes in Slovakia (Medvecká et al. 2012) and all of them (Apera spica-venti, Atriplex tatarica, Cardaria draba and Echinochloa crus-galli) were present in the studied area in 2014. Currently, 34% of recorded neophytes are considered to be casual, 30% invasive, and 36% naturalized taxa. Many of invasive taxa (e.g. Robinia pseudoacacia, Solidago canadensis, S. gigantea, Aster novi- belgii agg., Fallopia japonica, Helianthus tuberosus, Impatiens glandulifera) were introduced as useful or decorative plants by travellers, beekeepers or gardeners. Therefore, introduction of new taxa should be controlled and regulated to avoid new invasions.

Tab. 1. Alien taxa found in the selected area of Bratislava-Karlova Ves in 2014 List of abbreviations used in text and Tab. 1 arch – archaeophyte nat – naturalized taxon neo – neophyte inv – invasive taxon cas – casual, temporarily introduced taxon

Invasion status Taxon Family Localities and residence time Acer negundo Sapindaceae inv/neo 1, 5, 7, 10, 11, 12, 16, 17, 18 Aesculus hippocastanum Sapindaceae nat/neo 3, 4, 7, 8, 9 Ailanthus altissima Simaroubaceae inv/neo 1, 2, 3, 4, 5, 7, 8, 9, 12, 15, 16, 17, 18 Amaranthus retroflexus Amaranthaceae inv/neo 1, 3, 6, 7, 11, 13, 15, 16, 17, 18 Ambrosia artemisiifolia Asteraceae inv/neo 2, 4, 5, 6, 11, 13, 14, 15, 18 Amorpha fruticosa Fabaceae nat/neo 18 Anchusa officinalis Boraginaceae nat/arch 5, 12, 13, 14, 16, 18 Anthemis arvensis Asteraceae nat/arch 15 Anthemis cotula Asteraceae nat/arch 12 Anthriscus cerefolium Apiaceae nat/arch 15,16,17,18 subsp. trichosperma Apera spica-venti Poaceae inv/arch 2 Arctium lappa Asteraceae nat/arch 2, 5, 6, 7, 13 Arctium tomentosum Asteraceae nat/arch 11 Artemisia absinthium Asteraceae nat/arch 2, 4, 7, 15, 16 Asperugo procumbens Boraginaceae nat/arch 18 Aster novi-belgii agg. Asteraceae inv/neo 1, 2, 3, 5, 6, 7, 11, 12, 15, 16, 18

Tab. 1. Continuation

Invasion status Taxon Family Localities and residence time Atriplex tatarica Amaranthaceae inv/arch 3, 7, 9, 10, 12 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, Ballota nigra Lamiaceae nat/arch 16, 17, 18 Berberis julianae Berberidaceae cas/neo 8, 15, 18 Berberis thunbergii Berberidaceae cas/neo 1, 3, 8 Berteroa incana Brassicaceae nat/arch 1, 2, 3, 4, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 18 Brassica napus Brassicaceae cas/neo 12 Bromus sterilis Poaceae nat/arch 1, 2, 4, 5, 6, 7, 8, 10, 10, 12, 13, 15, 18 Bromus tectorum Poaceae nat/arch 6, 7, 8, 12, 13 Bryonia alba Cucurbitaceae nat/arch 15, 18 Bunias orientalis Brassicaceae nat/neo 4, 12, 13 Buxus sempervirens Buxaceae cas/neo 5, 14, 17 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14,15, 16, Capsella bursa-pastoris Brassicaceae nat/arch 17,18 Cardaria draba Brassicaceae inv/arch 2, 4, 12, 13, 18 Carduus acanthoides Asteraceae nat/arch 1, 2, 4, 5, 6, 7, 11, 12, 13, 14, 15, 16, 18 Castanea sativa Fagaceae nat/arch 3, 8, 10, 18 Catalpa bignonioides Bignoniaceae cas/neo 1, 5, 8, 16, 17 Celtis occidentalis Cannabaceae nat/neo 7, 8 Cerastium tomentosum Caryophyllaceae cas/neo 13 Chelidonium majus Papaveraceae nat/arch 1, 2, 3, 5, 6, 7, 10, 12, 13, 14, 15, 17, 18 Chenopodium Amaranthaceae nat/arch 1, 8 polyspermum Chenopodium strictum Amaranthaceae nat/neo 1, 2, 4, 7, 8, 15 Cichorium intybus subsp. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, Asteraceae nat/arch intybus 16, 17, 18 Consolida regalis Ranunculaceae nat/arch 4, 16 1, 2, 3, 4, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, Convolvulus arvensis Convolvulaceae nat/arch 17, 18 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, Conyza canadensis Asteraceae inv/neo 17, 18 Cornus alba Cornaceae cas/neo 5, 7, 8 Cornus sericea Cornaceae nat/neo 8, 12, 18 Corylus colurna Betulaceae cas/neo 7 Crepis foetida Asteraceae nat/arch 6, 7, 8, 11, 13 Cynodon dactylon Poaceae nat/arch 10 Digitaria sanguinalis Poaceae nat/arch 1, 2, 5, 6, 7, 8, 10, 11, 13, 14, 15, 17, 18 Echinochloa crus-galli Poaceae inv/arch 1, 4, 6, 7, 11, 13, 14, 16 Elaeagnus angustifolia Elaeagnaceae nat/neo 1, 2, 8, 9, 12, 14, 15, 16, 18 Erigeron annuus Asteraceae inv/neo 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 Erysimum cheiranthoides Brassicaceae nat/arch 3, 18 Fallopia ×bohemica Polygonaceae inv/neo 12 Fallopia convolvulus Polygonaceae nat/arch 6, 7, 13, 18 Fallopia japonica Polygonaceae inv/neo 1, 2, 4, 7, 12, 14, 18 Fraxinus americana Oleaceae nat/neo 7 Fumaria officinalis Papaveraceae nat/arch 2, 13,15 Galeobdolon argentatum Lamiaceae cas/neo 1 Galinsoga parviflora Asteraceae inv/neo 6, 7, 8, 17 Galinsoga quadriradiata Asteraceae inv/neo 5, 8

Tab. 1. Continuation

Invasion status Taxon Family Localities and residence time Geranium pusillum Geraniaceae nat/arch 7, 8, 10 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, Geranium pyrenaicum Geraniaceae nat/neo 16, 17, 18 Helianthus tuberosus Asteraceae inv/neo 1, 7, 18 Hibiscus syriacus Malvaceae cas/neo 8, 11 Hippophaë rhamnoides Elaeagnaceae cas/neo 1, 3, 12 Hordeum murinum Poaceae nat/arch 2, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 18 Hordeum vulgare Poaceae cas/arch 10 Impatiens glandulifera Balsaminaceae inv/neo 12 Impatiens parviflora Balsaminaceae inv/neo 1, 2, 3, 5, 6, 12, 13, 15, 18 1, 2, 3, 4, 5, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, Juglans regia Juglandaceae nat/arch 18 Kerria japonica Rosaceae cas/neo 5, 15 Koelreuteria paniculata Sapindaeceae cas/neo 16 Laburnum anagyroides Fabaceae nat/neo 1, 7, 17 Lactuca serriola Asteraceae nat/arch 1, 2, 3, 6, 7, 8, 10, 11, 12, 13, 15, 18 Lamium amplexicaule Lamiaceae nat/arch 4, 18 Lamium purpureum Lamiaceae nat/arch 1, 2, 3, 4, 5, 6, 7, 12, 13, 15, 17, 18 Lathyrus tuberosus Fabaceae nat/arch 7, 12, 15 Leonurus cardiaca Lamiaceae nat/arch 18 Lepidium ruderale Brassicaceae nat/arch 2, 7, 8, 9, 10, 18 Lithospermum arvense Boraginaceae nat/arch 4 Lycium barbarum Solanaceae inv/neo 15, 18 Mahonia aquifolium Berberidaceae nat/neo 7, 12, 15, 17 Malva neglecta Malvaceae nat/arch 8 Malva sylvestris Malvaceae nat/arch 1, 2, 7, 8, 10, 11, 13, 14, 15, 16, 17, 18 Medicago sativa Fabaceae nat/neo 1, 2, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 Melilotus albus Fabaceae nat/arch 1, 2, 7, 15, 18 Melilotus officinalis Fabaceae nat/arch 1, 2, 3, 4, 7, 11, 15, 16, 18 Mercurialis annua Euphorbiaceae nat/arch 7, 13, 15, 18 Myosotis arvensis Boraginaceae nat/arch 12, 18 Onobrychis viciifolia Fabaceae nat/neo 3, 7, 12, 15, 18 Onopordum acanthium Asteraceae nat/arch 2 Oxalis fontana Oxalidaceae nat/neo 1, 7, 9, 16, 18 Panicum capillare Poaceae nat/neo 4, 10 Papaver rhoeas Papaveraceae nat/arch 2, 4, 7, 8, 10, 12, 13, 15 Papaver somniferum Papaveraceae cas/arch 2 Parietaria officinalis Urticaceae nat/arch 5 Parthenocissus Vitaceae nat/neo 2, 3, 5, 7, 12, 15, 18 quinquefolia Parthenocissus Vitaceae cas/neo 5, 7, 12, 16 tricuspidata Philadelphus Hydrangeaceae cas/neo 1, 8, 10, 15 coronarius Pinus nigra Pinaceae nat/neo 1, 2, 5, 7, 8, 9, 10, 11, 14, 17, 18 Pinus strobus Pinaceae nat/neo 5, 10 Pinus wallichiana Pinaceae cas/neo 7 Platycladus orientalis Cupressaceae cas/neo 7, 9, 10, 12, 13, 14 Portulaca oleracea Portulacaceae nat/arch 1, 6, 7, 8, 11, 12, 13, 17, 18

Tab. 1. Continuation

Invasion status Taxon Family Localities and residence time Prunus cerasifera Rosaceae nat/neo 1, 2, 4, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18 Prunus cerasus Rosaceae nat/arch 15,16 Prunus insititia Rosaceae cas/arch 1 Prunus laurocerasus Rosaceae cas/neo 1, 6 Prunus persica Rosaceae cas/arch 1, 11, 14, 15, 16, 18 Pseudotsuga menziesii Pinaceae cas/neo 2, 7, 17 Quercus rubra Fagaceae nat/neo 1, 7, 8, 11 1, 2, 4, 7, 8, 10, 11, 12, 13, 14, 15, 16, Reseda lutea Resedaceae nat/arch 18 Rhus typhina Anacardiaceae cas/neo 2, 5, 7, 8, 9, 15, 18 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, Robinia pseudoacacia Fabaceae inv/neo 16, 17, 18 Rumex patientia Polygonaceae inv/neo 11, 12, 13, 18 Saponaria officinalis Caryophyllaceae nat/arch 2, 7, 11, 15, 16 Senecio vernalis Asteraceae nat/neo 1, 8, 12, 18 Senecio vulgaris Asteraceae nat/arch 1, 2, 4, 6, 7, 8, 10, 12, 13, 16 Setaria pumila Poaceae nat/arch 1, 3, 4, 6, 8, 11, 14, 16, 18 Setaria verticillata Poaceae nat/arch 7, 13 1, 2, 3, 6, 7, 10, 11, 12, 13, 14, 15, 16, Setaria viridis Poaceae nat/arch 18 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, Silene latifolia subsp. alba Caryophyllaceae nat/arch 17, 18 Sisymbrium loeselii Brassicaceae nat/arch 2 Sisymbrium officinale Brassicaceae nat/arch 10, 15 Solanum nigrum Solanaceae nat/arch 5, 7, 8, 10, 13, 14, 18 Solidago canadensis Asteraceae inv/neo 1, 2, 7, 12, 16, 18 Solidago gigantea Asteraceae inv/neo 1, 2, 3, 5, 6, 7, 12, 13, 15, 18 Sonchus arvensis Asteraceae nat/arch 18 Sonchus asper Asteraceae nat/arch 5 Sonchus oleraceus Asteraceae nat/arch 2, 4, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18 Sophora japonica Fabaceae cas/neo 5, 8 Spiraea japonica Rosaceae cas/neo 3, 15, 18 Syringa vulgaris Oleaceae nat/neo 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17 Thlaspi arvense Brassicaceae nat/arch 1 Tripleurospermum Asteraceae nat/arch 1, 2, 4, 6, 7, 11, 13, 14, 16, 18 inodorum Triticum aestivum Poaceae cas/arch 12 Valerianella locusta Caprifoliaceae nat/arch 1, 3, 12, 13, 18 Verbena officinalis Verbenaceae nat/arch 1, 2, 7, 10, 11, 13, 15, 16, 18 Veronica arvensis Plantaginaceae nat/arch 2, 13 1, 2, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, Veronica persica Plantaginaceae nat/neo 17, 18 Veronica polita Plantaginaceae nat/arch 7, 8, 13, 18 Vicia hirsuta Fabaceae nat/arch 2, 4, 7, 12, 13 Vicia sativa Fabaceae nat/arch 2, 4, 5, 7, 12, 13, 18 Vicia villosa Fabaceae nat/arch 2, 4, 7, 13, 15, 18 Viola arvensis Violaceae nat/arch 2, 4, 7, 12, 13, 15, 18 Viola odorata Violaceae nat/arch 2, 13, 18 Vitis vinifera Vitaceae nat/arch 1, 6, 7, 8, 11, 14, 15, 16, 18

Fig. 2. Percentage of vascular plant families in the monitored area, followed by the code for the family: Ana – Anacardiaceae, Api – Apiaceae, Ast – Asteraceae, Bal – Balsaminaceae, Ber – Berberidaceae, Bet – Betulaceae, Big – Bignoniaceae, Bor – Boraginaceae, Bra – Brassicaceae, Bux – Buxaceae, Can – Cannabaceae, Cap – Caprifoliaceae, Car – Caryophyllaceae, Con – Convolvulaceae, Cor – Cornaceae, Cuc – Cucurbitaceae, Cup – Cupressaceae, Ela – Elaeagnaceae, Eup – Euphorbiaceae, Fab – Fabaceae, Fag – Fagaceae, Fag – Fagaceae, Ger – Geraniaceae, Hyd – Hydrangeaceae, Jug – Juglandaceae, Lam – Lamiaceae, Mal – Malvaceae, Ole – Oleaceae, Oxa – Oxalidaceae, Pap – Papaveraceae, Pin – Pinaceae, Pla – Platanaceae, Poa – Poaceae, Pol – Polygonaceae, Por – Portulacaceae, Ran – Ranunculaceae, Res – Resedaceae, Ros – Rosaceae, Sap – Sapindaceae, Sim – Simaroubaceae, Sol – Solanaceae, Urt – Urticaceae, Ver – Verbenaceae, Vio – Violaceae, Vit – Vitaceae.

Acknowledgements This study was supported by the Grant Agency VEGA, Grant No. 1/0380/13.

References

Bertová, L., 1993: Ohraničenie chotára a jeho vnútorné členenie. In: Bertová, L. (ed.), Karlova Ves, Vlastivedná monografia, p. 11-13, Alfa, Bratislava. Bizubová, M., Barančok, P., Kolény, M., Minár, J., Zaťko, M., 1998: Charakteristika prírodného prostredia. In: Trizna, M., Geografické spektrum, Diaľnica D2 Bratislava, Lamačská cesta – Staré Grunty, p. 21-37, Geografika, Bratislava. Endlicher, S., 1830: Flora Posoniensis, exhibens plantas circa Posonium sponte crescentes aut frequentius cultas, methodo naturali dispositas. Joseph Lander Posonii. Feráková, V., Kochjarová, J., Králik, E., Schwarzová, T., Záborský, J., 1997: Cievnaté rastliny. In: Feráková, V., Kocia- nová, E., (eds.), Flóra, geológia a paleontológia Devínskej Kobyly, p. 86-156, Litera, APOP, Bratislava. Futák, J., 1984: Fytogeografické členenie Slovenska. In: Bertová, L., (ed.), Flóra Slovenska IV/1, p. 418-419, Veda, Bratislava. Hanúsková, J., 2015: Floristické a vegetačné spektrum mestského ekosystému modelového územia Bratislava-Karlova Ves. Diploma thesis depon. in Comenius University, Bratislava. Kaleta, M., 1968: Príspevok k poznaniu Devínskej Kobyly. Zborn. Slov. Nár. Múz., Prír. Vedy, 14: 41-55. Liška, M., 1986: Horopis, vodopis a podnebie. In: Szomolányi, J., (ed.), Malé Karpaty Turistický sprievodca ČSSR, p. 12-21, Šport, Bratislava. Lumnitzer, S., 1791: Flora Posoniensis: Exhibens plantas circa Posonium sponte crescentes secundum systema sexuale Linneanum digestas. Siegfried Lebrecht Lipsiae. Marhold, K., Mártonfi, P., Mereďa, P. jun., Mráz, P., (eds.), 2007: Chromosome number survey of the ferns and flowering plants of Slovakia. Veda, Bratislava. Medvecká, J., Kliment, J., Májeková, J., Halada, Ľ., Zaliberová, M., Gojdičová, E., Feráková, V., Jarolímek, I., 2012: Inventory of the alien flora of Slovakia. Preslia, 84: 257-309. Záborský, J., 1993: Semenné rastliny. In: Bertová, L., (ed.), Karlova Ves, Vlastivedná monografia, p. 45-57, Alfa, Bratislava.

Abstrakt V článku prezentujeme nepôvodné taxóny cievnatých rastlín zaznamenané v urbánnom ekosystéme vybranej časti Bratislavy. Územie bolo rozdelené na 18 častí, na ktorých bolo zaznamenaných 146 ne- pôvodných taxónov cievnatých rastlín počas vegetačnej sezóny v roku 2014. Tento počet zahŕňa 82 archeofytov a 64 neofytov, vrátane 23 inváznych taxónov. Najrozšírenejšími inváznymi neofytmi na študovanom území boli Conyza canadensis, Erigeron annuus a Robinia psudoacacia.

Alena Rendeková, Michal Hrabovský, Jana Hanúsková, Ján Miškovic: Nepôvodné taxóny cievnatých rastlín urbánneho ekosystému vybranej časti Karlovej Vsi v meste Bratislava

Acta Botanica Universitatis Comenianae Vol. 50, 2015

ADDITION TO THE SURVEY OF ALIEN TAXA OF VASCULAR PLANTS OF THE URBAN ECOSYSTEM OF BRATISLAVA MUNICIPAL PART KARLOVA VES

Viera Feráková

Karloveská 23, 84104 Bratislava 4, Slovakia

Received 15 December 2015; Received in revised form 21 December 2015, Accepted 21 December 2015

Abstract The aim of the paper is to contribute to the floristic survey of the alien species of vascular plants of Bratislava, municipal part Karlova Ves as an addition to the list by Rendeková et al. (2015) compiled in the vegetation season of 2014. 11 naturalized archaeophytes, 24 naturalized neophytes (+ 1 invasive), 9 + 2 taxa with uncertain or revised residence status, 54 casual neophytes and 2 casual archaeophytes are included.

Key words: urban flora, Bratislava, municipal part Karlova Ves, vascular plants, alien taxa

Methodical notes

The additions concern mainly my own records from the vegetation seasons 2014 and 2015, rarely of previous years. For floristic data from the territory studied, which were already published, the corresponding citation is given. The nomenclature of taxa follows Marhold et al., eds. (2007) and it is in accordance with the publication of Medvecká et al. (2012 ) which served as a reference material for the classification of the alien vascular plants of the flora of Slovakia. Names, which deviate from the mentioned nomenclature and some infraspecific taxa (e.g. used in the latest volume of the publication Flóra Slovenska) are printed in bold. The names of taxa not included in the cited sources are marked with the symbol (!) and given with the author´s citation. Taxa listed in the Appendix follow the nomenclature of IPNI (The international plant names index). Herbarium specimens of selected species are kept in the collections of the Institute of Botany, Slovak Academy of Sciences (SAV). The delimitation of the territory and the numbering of localities is in accordance with the above cited paper (it does not fully correspond with the cadastre of Karlova Ves). In Karlova Ves are commonly occurring further taxa, classified in Medvecká et al. (2012) as „uncertain residence status“: Atriplex patula, Chenopodium album, Crepis setosa, Erodium cicutarium, Eragrostis minor, Polygo- num arenastrum, Polygonum aviculare, Tanacetum vulgare and sporadically Veronica hederifolia (all recorded but not listed in Rendeková et al. 2015). In the adjacent section of the cadastre of municipal part Karlova Ves (Karloveské rameno) not included in the study by the cited authors were recorded Bidens frondosa inv / neo and Rumex triangulivalvis nat / neo.

 Corresponding Author: Viera Feráková; [email protected]

43 Tab. 1. Additional list of alien taxa recorded in the selected area of Bratislava – Karlova Ves Abbreviations: arch – archaeophyte, neo – neophyte, cas – casual, temporarily introduced, nat – naturalized, inv – invasive, # invasive in the past, now with stable or decreasing populations, + Portulaca oleracea only on specific level has been already listed in Rendeková et al. (2015)

Invasion status Taxon and residence time References Localities Amaranthus albus nat / neo 18 Amaranthus blitum subsp. blitum nat / arch Feráková (2014) 8 Amaranthus powellii nat / neo 17 Arctium minus nat / arch 12 Artemisia annua nat / neo 17, 18 Atriplex oblongifolia nat / arch Zlinská, Janecová (2003) 8, 18 Bassia scoparia subsp. scoparia nat / neo Feráková (2014) 11 Chenopodium opulifolium nat / arch 12 Commelina communis nat / neo 8 Cymballaria muralis nat / neo 1, 6, 8, 10, 16 Descurainia sophia nat / arch 18 Diplotaxis muralis nat / arch 18 Erucastrum nasturtiifolium nat / neo 18 Euphorbia exigua nat /arch 5 Euphorbia helioscopia nat /arch 5, 18 Euphorbia peplus nat / arch 2, 5 Fraxinus pennsylvanica nat / neo 17 Fumaria vaillantii nat / arch 12 Geranium sibiricum nat / neo 6 Iva xanthiifolia nat # / neo 7, 10 Juncus tenuis inv / neo Feráková (2014) 8 sown in the city Lolium multiflorum nat / neo lawns Medicago x varia nat / neo 8, 10, 16 Morus alba nat / neo 10 Oenothera glazioviana nat / neo 7 Bíziková (1999) + Oxalis corniculata (Xanthoxalis corniculata) nat / neo many sites Feráková (2013) not. Oxalis dillenii (Xanthoxalis dillenii) nat / neo ibid. 18 Parthenocissus inserta nat / neo 6,17 Paulownia tomentosa nat / neo 8 Phytolacca americana nat / neo Letz (2012) 18 Polycarpon tetraphyllum subsp. tetraphyllum nat / neo Mereďa (2012) (nat / arch) 3, 8 Populus x canadensis nat / neo numerous sites +Portulaca oleracea subsp. granulatostellulata nat / arch Feráková et al. (2012) 8 (Poelln.) Danin et H. G. Baker P. oleracea subsp. papillatostellulata (Danin et nat / arch ibid. 3, 8, 9, 18 H. G. Baker) P. oleracea subsp. trituberculata (Danin et al.) nat / arch ibid. 7 J. Walter Ribes aureum nat / neo 18 Rudbeckia hirta nat / neo (cas) 8 Sagina apetala subsp. erecta (Hornem.) F. Herm. cas / neo? 10 Sedum rupestre subsp. erectum nat / neo 6, 8, 16 Sedum spurium nat / neo 6 Sempervivum tectorum nat / neo 8, 9, 10,13 Tanacetum parthenium nat / neo 18 Urtica urens nat / arch 5, 17, 18

44 Tab. 2. In the territory concerned cultivated and escaped species (in Medvecká et al. (2012) all except 2 taxa classified as cas / neo and additions) Species with evidence of escaping are marked with an asterisk (*)

Taxon References Localities Acer saccharinum 8 Ageratum houstonianum 8 Allium schoenoprasum var. schoenoprasum* 8 Amaranthus caudatus subsp. caudatus 8 Anagallis monelli L. ! * 8 Antirrhinum majus 1, 8 Buddleja davidii 7 Calendula officinalis* 8, 15 Callistephus chinensis* 2, 8 Caragana arborescens 7 Campsis radicans 3 Chaenomeles japonica (Thunb.) Spach ! 6, 9 Cheiranthus cheiri * 8, 9 Cotoneaster divaricatus* 13 Cotoneaster horizontalis 8, 16 Cydonia oblonga 17 Euphorbia maculata ( Chamaesyce maculata) Feráková (2014) 8 Forsythia suspensa 1, 2, 8, 9, 11 Gaillardia pulchella 18 Feráková (2011) SAV sec. Eliáš jun., Gypsophila scorzonerifolia 8 Dítě (2012) Heuchera americana 8 Hosta plantaginea 8 Hyacinthus orientalis* 9 Hylotelephium spectabile 2, 6, 8, 9 Ligustrum ovalifolium 2, 8, 9, 10 Lonicera maackii 3 Lonicera pileata 1, 8, 9, 10 Lonicera tatarica 3 Lunaria annua* 17 Malva mauritiana 3, 8 Narcissus poeticus* 2 Narcissus pseudonarcissus* 2, 3, 8, 16 Oenothera  fallax 7 Omphalodes verna 7 Panicum miliaceum subsp. miliaceum cas / arch 10 Papaver pseudoorientale 18 Persicaria orientalis* 8 Potentilla fruticosa 8 Phlox paniculata* 8 Portulaca grandiflora* 8, 10 Prunus dulcis cas / arch 8, 11 Pyracantha coccinea 1, 2, 6, 8, 13, 18 Rosa rugosa 8 Salvia sclarea* 8 Sedum pallidum M. Bieb. ! * 2, 8, 9,13 Sedum sarmentosum* Feráková (2014), Rendeková et al. (2014) 1, 2, 8, 13 Spiraea x vanhouttei 1, 6, 7, 8, 18 Stachys byzantina* 6, 7, 8

45 Tab. 2. Continuation

Taxon References Localities Symphoricarpos albus 5 Tagetes patula* 6, 8, 10 Tradescantia x andersoniana 8 Viburnum rhytidophyllum 6, 7 Vinca major 6, 9, 10 Viola x wittrockiana* 6, 9 Wisteria frutescens 1, 6, 9 Yucca filamentosa 8, 9, 10

Appendix: Other cultivated alien taxa Aucuba japonica Thunb. Magnolia stellata (Siebold et Zucc.) Maxim. Chamaecyparis sp. div. Picea pungens Engelm. group Glauca Caryopteris x clandonensis hort. Photinia x fraseri Dress Euonymus fortunei (Turcz.) Hand. - Mazz. Prunus subhirtella Miq. ´Jugatsu zakura´ Elaeagnus umbellata Thunb. Rosmarinus officinalis L. Jasminum nudiflorum Lindl. Salix x sepulcralis Simonk. Juniperus chinensis L. Santolina chamaecyparissus L. Lavandula angustifolia Mill. Thuja occidentalis L. Magnolia x soulangeana Soul.-Bod. Weigela florida (Bunge) A. DC.

References

Bíziková, L. 1999: Rod Xanthoxalis Small na Slovensku (s dôrazom na problematiku druhu Xanthoxalis corniculata (L.) Small v Bratislave). MSc. thesis, depon. in: Department of Botany, Faculty of Natural Sciences, Comenius University in Bratislava. 97 pp. Eliáš, P. jun., Dítě, D., 2012: Gypsophila L. In: Goliašová, K., Michalková, E. (eds.), Flóra Slovenska VI/3, p. 567-568. Feráková, V., 2014: Amaranthus blitum subsp. blitum, Bassia scoparia subsp. scoparia, Chamaesyce maculata, Juncus tenuis, Rumex triangulivalvis, Sedum sarmentosum. In: Eliáš, P., jun. (ed.), Zaujímavejšie floristické nálezy. Bull. Slov. Bot. Spoločn., Bratislava, 36, 2: 251-254. Feráková, V., Walter, J., Hodálová, I., 2012: Portulaca L. In: Goliašová, K., Michalková, E., (eds.), Flóra Slovenska VI/3, p. 50-64. Veda, Bratislava. IPNI (2004 onwards): The international plant names index. URL: http://www.ipni.org/index.html. Letz, R. D., 2012: Phytolacaceae R. Br. In: Goliašová, K., Michalková, E. (eds.), Flóra Slovenska VI/3, p. 28-37. Veda, Bratislava. Marhold, K., Mártonfi, P., Mereďa, P. jun., Mráz, P., 2007: Chromosome number survey of the ferns and flowering plans of Slovakia. Veda, Bratislava. Medvecká, J., Kliment, J., Májeková, J., Halada, Ľ., Zaliberová, M., Gojdičová, E., Feráková, V., Jarolímek, I. , 2012: Inventory of the alien flora of Slovakia. Preslia, 84: 257-309. Mereďa, P. jun., 2012: Polycarpon Loefl. ex L. In: Goliašová, K., Michalková, E. (eds.), Flóra Slovenska VI/3, p. 28-37. Veda, Bratislava. Rendeková, A., Kerekeš, E., Miškovic, J., 2014: Rare and interesting ruderal plant communities of Bratislava. Acta Bot. Univ. Comen., 49: 13-18. Rendeková, A., Hrabovský, M., Hanúsková, J., Miškovic, J., 2015: Alien taxa of vascular plants of the urban ecosystem in the selected area of Bratislava, municipal part Karlova Ves. Acta Bot. Univ. Comen., 50: 35-42 Zlinská, J., Janecová, J., 2003: Poznámky k rozšíreniu Atriplex oblongifolia Waldst. et Kit. v Bratislave. Biosozologia 1: 60-64.

Abstract Cieľom práce je prispieť k floristickému prehľadu nepôvodných druhov cievnatých rastlín zazname- naných v intraviláne bratislavskej mestskej časti Karlova Ves ako dodatok k zoznamu autorov Rendeková et al. (2015) z vegetačného obdobia roku 2014. Doplňujeme 11 naturalizovaných archeofytov, 24 natu- ralizovaných neofytov, z toho 1 invázny, 9 + 2 taxóny s neurčitým alebo revidovaným statusom, 55 ná- hodne zavlečených a pestovaných neofytov a 2 archeofyty.

Viera Feráková: Dodatok k prehľadu nepôvodných taxónov cievnatých rastlín urbánneho ekosystému Bratislavy, mestskej časti Karlova Ves

ACTA BOTANICA UNIVERSITATIS COMENIANAE

Volume 50

Vydala Univerzita Komenského v Bratislave vo Vydavateľstve UK Vyšlo v decembri 2015

Technická redaktorka: Darina Földešová

ISBN 978-80-223-4082-3 ISSN 0524-2371