ISSN 1408-3671 
UDK 57(497.4) 
izdajate! j/pu bi isher 
Društvo biologov Slovenije 
ISSN 1408-3671 
UDK 57(497.4) 
P-
BIOLOšKA KNllžNJCA 
VEčNA POT 111 
1Bno I J ,, 8lJANA 
b ~&'S-A,1 j (J[j 
ACTA 
BIOLOGICA 
SLOVENICA 
VOL. 47 ŠT. 1,2 LJUBLJANA 2004 
prej/formerly BIOLOŠKI VESTNIK 
izdajatelj/publisher 
Društvo biologov Slovenije 
Acta Biologica Slovenica 
ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2004 Vol. 47, Št. 1,2: 3-115 
Glasilo Društva biologov Slovenije - Journal of Biological Society of Slovenia 
Izdaja - Published by 
Društvo biologov Slovenije - Biological Society of Slovenia 
Glavni in odgovorni urednik - Editor in Chief 
Mihael Jožef Toman, e-mail: mihael.toman@uni-lj.si 
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Matija Gogala (SI), Nada Gogala (SI) Peter Maček (SI), Alenka Malej (SI), Andrej Martinčič (SI), 
Harald Niklfeld (A), Livio Poldini (1) , Boris Sket (SI), Robert Zorec (SI), Mitja Zupančič (SI), 
Thomas F. J. Martin (USA), Mark Tester (UK), Gerhard Thiel (D) 
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Acta Biologica Slovenica, Večna pot 111, SI-1001 Ljubljana, Slovenija 
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ISSN 1408-3671 UDK 57(497.4) 
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Acta Biologica Slovenica je indeksirana v - is indexed in: Biological Abstracts, Zoological records 
Introduction 
ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2004 Vol. 47, Št. 1: 3-11 
Sprejeto (accepted): 2003-11-21 
Coenological and synphysiological investigations on loess 
grassland vegetation (Salvio-Festucetum rupicolae) close to 
Godollo Hills (Hungary) 
Tibor SZERDAHELYJI, Szilvia FOTI1, Szilard CZOBEL', Janos BALOGH2, 
Zoltan NAGY1 & Zoltan TUBAJ,2 
'Department of Botany and Plant Physiology, Szent Istvan University, H-2103 
Godollo 
2Departmental Research Group of Hungarian Academy of Sciences, Szent Istvan 
University, H-2103 Godollo 
Abstract. Parallel coenological and synphysiological examinations were car-
ried out on three typical xerophilous loess grassland stands of Salvio-Festucetum 
rupicolae community in the Godollo Hills, near Isaszeg village. Three stand types, 
a Carex humilis, a Chamaecytisus austriacus and a Stipa dasiphylla dominated 
ones were investigated at the same spatial scale (mesoscale). In our sample area 
apparent dominance by three species (Festuca rupicola, Stipa dasiphylla, and 
Carex humilis) suggested that these stands of this subassociation have been 
emerged from three types of differing species composition. According to these 
analyses three groups dominated by Carex humilis, Chamaecytisus austriacus and 
Stipa dasiphylla oj festucetosum rupicolae subassociation of a stand of Salvio-
Festucetum rupicolae association were separated. Results of the synphysiological 
measurements show, that water shortage and senescence in autumn are responsible 
for the different physiological performances of the three types. In the well-watered 
summer period we found significant difference only between the physiological 
activity of the Carex- and Chamaecytisus- dominated types, however this differ-
ence is due to the significant LAI-difference and after relating photosynthesis to 1 
m2 leaf area, the difference is non-significant. 
Keywords: coefficient of variation, coenology, grassland, loess stands, multi-
variate analysis, Salvio-Festucetum rupicolae Z6lyomi 1958, synphysiology. 
Temperate grasslands cover large areas of the Earth's vegetation (COUPLAND 1992), and they 
are locate in one of the regions where the impact of globa! climate change is predicted to be high 
(MITCHELL et al. 1990). The temperate grassland surface has large and increasing areas with arid 
4 Acta Biologica Slovenica, 47 (1), 2004 
climate. Even in the middle ofEurope, Hungary has ·areas where the relatively low and unevenly dis-
tributed yearly precipitation results in semi-arid grasslands like loess grasslands. Loess grassland 
stands in the Carpathian Basin are among the richest in species considering the plant communities 
of the Pannonian vegetation. These stands are at the westem edge of the continental-Eurasian forest 
steppe-steppe zone. Unfortunately, intact stands exist only as small patches today. The Salvio-
Festucetum rupicolae association has severa! subassociations differing in their physiognomy. 
These stands were dominated by Festuca rupicola, but differed in physiognomy, texture, spatial 
pattem, vegetation dynamical traits and physiological activity from each other. (VIRAGH, BARTHA 
1998). This may partly be caused by the constantly changing long term dynamism of spots of species 
(therophytes, hemitherophytes, stoloniferous species). The long term processes are frequently mod-
ified in rates or in directions of s mali scale disturbances. These rapid changes are expressed by degra-
dation, decreased diversity of stands or invasion by advancing weeds or disturbance tolerant species. 
Coenological and synphysiological examinations were carried out on three typical xerophilous 
loess grassland stands ( Carex humilis, Chamaecytisus austriacus and Stipa dasiphylla dominated) of 
Salvio-Festucetum rupicolae Z6lyomi 1958 community in the Godoll6 Hills, near Godollo (Isaszeg 
village). The synphysiological measurements were started in three apparently different and arbitrar-
ily chosen types. 
We were interested whether these types could be detected also by multivariate analysis from 
many coenological samples of the area. We also tried to reveal what kind of synphysiological traits 
are characterising the physiognomically distinct, adjacent, mosaic-like types of the Jestucetosum sul-
catae subassociation. 
Materials and methods 
Study area, coenological survey 
Sample plots were chosen between villages Isaszeg and Kerepes (230 m a. s. l.) in typical and 
continuous stands ofthe Salvio-Festucetum rupicolae association (ZOLYOMI 1958). In this area the 
steppe grassland stands occupy marginal positions in the vicinity of Godollo Hills. The annual pre-
cipitation: 601 mm, the average temperature: 9,1 °C, the medium daily maximum: 14,1°C, the medi-
um daily minimum: 4, l 0 C. (KAKAS 1969). This flora spectrum is poor in characteristic species and 
contains several steppe species typical of the _Central Range. Hungarian loess vegetation strictly 
related to eastem European steppe vegetation. This flora composition of westem steppe fragments 
similar to e. g. Ukrainian steppe vegetation (ZOLYOMI & FEKETE 1994), so these sample plots of 
this area could represent adequately the East European loess vegetation. 
We tried to set up plots representative to this association, rich in species, diverse in physiogno-
my, far from plantations (Populus forest, hedges, etc.). 
The synphysiological study was carried out in June (summer aspect) and in September (autumn 
aspect) 2000. During June we made physiological measurements both under stressed and under non-
stressed conditions in the vegetation's fully developed phase, while in September measurements were 
made under wet conditions, but in the vegetation's senescent phase. The coenological survey was car-
ried out on 3rd July 2001. Seventy five quadrats of 2 x 2 m were chosen. The species list and cover 
values are detected. The species composition consists of 62 species. Dominant species with decreas-
ing cover values are the following: Festuca rupicola, Carex humilis, Dorycnium germanicum, 
Cytisus austriacus, Chrysopogon gryllus, Stipa dasiphylla. There are severa! subdominant species, 
T. Szerdahelvi, S. F6ti, S. Cz6bel, J. Balogh, Z. Nagy & Z. Tuba: Coenological and synphysiological... 5 
e. g.: Phleum phleoides, Seseli osseum, Teucrium chamaedrys, Galium verum,Helictotrichon pubes-
cens, Filipendula vulgaris, Euphorbia pannonica, Asperula cynanchica. Characteristic and accesso-
rial species the next: Salvia nemoralis, lsatis tinctoria and Agropyron repens, Agropyron caninum, 
Centaurea sadleriana, Hypericum perforatum, Falcaria vulgaris, Dactylis glomerata, Anthericum 
ramosum, Adonis vemalis. Por statistical evaluation several ecological characters of species were 
taken from Hungarian Database 1.2 (HORV ATH et al. 1995). The ecological indicator values of 
species (BORHIDI 1995) are given in percentage pro rata. These characters are: the relative temper-
ature requirement of species (TB), soil acidity (RB), hurnidity (WB), nitrate supply (NB), relative 
light intensity (LB), continentality (KB) and the types of social behaviour (SOC). The nature con-
servation ranks (NCR) (Simon 1988) are given in 10 categories separated in two groups. Proportion 
of the first group (U-unique, KV-strictly protected, V-protected, E-native species, K-accessorial 
species, TP-nature pioneers) reflects natura! conditions, proportion of the second group indicates the 
degree of degradation (TZ-disturbance tolerant native species, A-adventives, G-cultivated species, 
GY-weeds) compared to the Hungarian average. The categories of simplified flora element spectrum 
of Hungary (FL) contain the main groups completing with relationship of Pannonic viewpoint. This 
kind of simplification is suitable for the analysis. The coenosystematical groups (COENOS) are 
established for categories of the hierarchical system. Multivariate analysis on cover values of species 
was carried out by using the SYN-TAX program (PODANI 1993, 1997). Por the cluster analysis the 
Czekanowsi-index was used (PODANI 1993, 1997). 
Synphysiological methods 
COz-exchange, transpiration, air-temperature, relative hurnidity and vapour pressure, and stom-
atal conductance were measured by using a portable closed-loop IRGA gas exchange system (LI-
COR 6200) sampling the air in a plexi chamber of 60 cm diameter (ground area of the chamber is 
2826 cm2) and 70 cm height with three replicate measurements in each plot (BALOGH et al. 2002). 
PPFD values were recorded and LAI was estimated using sunfleck ceptometers (Decagon). Canopy-
surface temperature was measured with an infrared thermometer (MX4, Raytek). Soil water content 
was measured by a TDR reflectometer (ML2, Delta-T Devices) in three replications at 5, 10, 20 cm 
soil depths. Forty plots were measured in summer and 30 in autumn in each stand types. 
Results and discussion 
Coenological pattern 
The dendrogram of the sample plots can be seen on Figure l. Using the Czekanowski-index there 
are 7 well organized groups at 0.58 similarity value. The first group (1-37. sample plots) is charac-
terized by the dominance of Carex humilis, Chrysopogon gryllus, Chamaecytisus austriacus, 
Dorycnium germanicum, Teucrium chamaedrys and the presence of Phleum phleoides, Filipendula 
vulgaris (Carex dominated type). This fragment has three levels: the tall grasses (50-70 cm), the 
hemikryptophyte and chamaephyte species (30-50 cm) and th therophytes (5-15 cm). The levels have 
compact stand. 
The Chrysopogon gryllus is almost absent from the second group (2-50. samples), and there is 
less Chamaecytisus austriacus, Phleum phleoides, Filipendula vulgaris, Dorycnium germanicum, 
Teucrium chamaedrys too, however the dominance of Festuca rupicola is more pronounced, and the 
6 Acta Biolo!tica Slovenica, 47 (!), 2004 
O.B 
0.7 
0.6 
0.5 
0.4 
0.3 
0.2 
0.1 
Figure 1. Dendrogram of sample plots of Festucetum rupicolae stand. 
denominative species of this association, Salvia nemorosa is also present (Cytisus dominated type). 
This fragment is compact too, but smaller (50 cm) than the Carex type. 
The third group (3-68.) (Stipa dominated type) is characterized by lower cover values of Stipa 
dasiphylla, Chrysopogon gryllus, Chamaecytisus austriacus, Dactylis glomerata, Festuca rupicola, 
Filipendula vulgaris, Phleum phleoides, Teucrium chamaedrys, Helictotrichon pubescens. The four 
other groups are different from each other in species composition and cover values, but are coupled 
by the presence of Stipa dasiphylla. This fragment is not so compact, it comprised loose and tall 
grass tufts and spots (50-70 cm) and smaller other hemikryptophyte and charnaephyte species. There 
are sevaral gaps between the grass tufts (30-40 cm). 
Result of principal coordinates analysis (PCoa) shows 3 loose groups of sample plots (data are 
not shown), identical with the three types found by cluster analysis. The rest of sample plots contains 
rare species making them diverse and mosaic-like, not typical to festucetosum rupicolae subassosi-
ation. 
Analysing severa! standard coenological attributes the investigated area and the three grassland 
types can be characterized as follows. The distribution of temperature indicator values (TB) shows 
two peaks, similarly to the continentality diagrarn's two maximum values. The proportion of meso-
phyl and xerophyte species is more than 55 %. The high frequency of these species is characteristic 
for both open or closed grassland communities. 
The distribution of relative hurnidity indicator values (WB) shows a normal bell-shaped form 
(Table l.). Its maximum value is due to the high frequency of xerotolerant plants. The diagram is 
asymmetric in the case of xeromorph habitats. 
The distribution of soil acidity values (RB) sign the high frequency of basiphylous species in the 
area's three types. 10 % of them are lime indicator species and do not occur on acidic soil (Carex 
humilis, Chamaecytisus austriacus, Thalictrum pseudominus). 
T. Szerdahelyi, S. F6ti, S. Cz6bel, J. Balogh, Z. Nagy & Z. Tuba: Coenological and synphysiological... 7 
Table l. Percentage (%) distribution of relative humidity indicator values (WB) of the species in the 
investigated temperate loess grassland. 
categories 1 2 3 4 s 6 7 
WB values of Carex-type 0,15 3,75 20,6 5,86 1,2 1,65 0,15 
WB values of Cytisus-type 0,18 4,42 19,3 6,63 1,29 1,47 
WB values of Stipa-type 0,18 4,59 19,6 6,53 0,53 1,94 
Distribution of relative nitrogen indicator values (NB) can be seen in Table 2. Half of the species 
are characteristic of habitats very poor in nitrogen, 10 % of them live in places extremely pooF in 
nitrogen or in dry places (Seseli osseum, Minuartia _verna, Phleum phleoides). 
Table 2. Percentage (%) distribution ofrelative nitrogen indicator values (NB) of the species in the 
investigated temperate loess grassland. 
categories 1 2 3 4 s 6 7 
NB values of Carex-type 8,11 15,6 4,35 1,8 0,6 1,65 1,35 
NB values of Cytisus-type 8,29 15,3 3,13 1,47 1,47 1,66 2,03 
NB values of Stipa-type 8,11 18,2 3,17 1,06 0,18 1,94 0,71 
In terms of temperature (TB), water (WB) and soil (RB) requirements the distribution and the 
maximum of values are similar in the three types. The tendency of the distribution of nitrogen 
requirement (NB) is similar to the others, but the rate of Stipa type is higher at the maximum value 
(9% ), that at the Chamaecytisus type. 
Distribution of relative light indicator values (LB) shows high ratio of light plants (45%), full 
light plants of open habitats (20%) and shadow tolerant ones (32% ). There are only few typical shad-
ow plants (Table 3.). 
Table 3. Percentage (%) distribuiton of relative light indicator values (LB) of the species in the inves-
tigated temperate loess grassland. 
categories s 6 7 8 9 
LB values of Carex-type 0,3 0,45 11,6 15 5,86 
LB values of Cytisus-type 0,74 0,18 12,3 14 6,08 
LB values of Stipa-type 0,35 0,71 10,9 12,5 8,82 
The distribution of plants according to degree of continentality shows two peaks (Table 4.). The 
first maximum corresponds to the suboceanic species, mainly central European but expanding to 
East (Bromus sterilis, Teucrium chamaedrys, Galium verum, Agrimonia eupatoria), the second max-
Table 4. Percentage (%) distribuiton of species according to degree of continentality of the species 
in the investigated temperate loess grassland. 
categories 3 4 s 6 7 8 
KB values of Carex-type 1,35 9,46 5,56 4,05 9,61 3,15 
KB values of Cytisus-type 1,1 10,3 4,79 3,31 12 1,84 
KB values of Stipa-type 1,23 8,99 5,64 5,82 8,11 3,53 
8 Acta Biologica Slovenica, 47 (!), 2004 
imum sbows tbe continental-subcontinental species spread in East Europe (Festuca rupicola, Isatis 
tinctoria, Si/ene otites) . Tbe difference between the maximum values of ligbt requirement (LB) and 
nitrogen requirement (NB) of tbe Stipa and Chamaecytisus type is nearly tbe same (8%). The values 
of continentality (KB) are markedly different being 10 % bigber in tbe Chamaecytisus type, tban in 
tbe Stipa type. Tbis Stipa dominated group of plots is the most uniformly distributed out of tbe three 
types. 
Tbe distribution of categories of nature conservation values (SIMON 1988) sbows native species 
are tbe most frequent (60%), native accessorial species are less abundant (20%). The proportion of 
disturbance tolerant native species is low (10 %), as the proportion of weeds, cultivated plants and 
adventitious species too (less, tban 4%). Tbere is bowever considerable quantity of strictly protect-
ed and protected species of Hungary (8 % ), like Stipa dasiphylla, Centaurea sadleriana, Adonis ver-
nalis, Thalictrum pseudominus. 
According to distribution of floral elements (FL) the sbare by tbe Eurasian elements is tbe higb-
est (27%), the submediterranean and tbe Pontic-Pannonian elements (e. g. Chamaecytisus austria-
cus, /satis tinctoria) are present in considerable quantity (together 28 %) and there is a smaller quan-
tity (10%) oftbe Pannonian (Dianthus pontederae) and tbe Pannonian-Balkan elements (Euphorbia 
pannonica). Tbe proportion of cosmopolitan species is less than 5% (Koeleria cristata), and tbe rate 
of adventive species is low (1,5%, e. g. Erigeron canadensis, Onobrychis viciifolia). This sbows tbat 
tbis area is more or less intact. Tbe distribution of tbe flora! elements sbows tbat tbe Stipa type bas 
tbe bigbest quantity ofEurasian, continental and submediterranean elements, in contrast to tbe Carex 
type tbat almost bave tbe lowest levels of these. Tbe Chamaecytisus group bas tbe bigbest quantity 
of European and Pontic-Pannonian elements. 
Table 5 sbows the distribution of social bebaviour types (SOC). Generalists (G) and tbe competi-
tors (C) give balf of the species. Tbe cover by natura! pioneers (NP)(Myosotis stricta) and special-
ists (S)(Adonis vemalis, Thalictrum pseudominus) is remarkable, too (togetber 26 % ). Tbere are few 
(15 %) disturbance tolerant (DT) species (Achillea collina, Agrimonia eupatoria, Galium verum). 
Tbe quantity of weeds (W), alien competitors (AC) and ruderal competitors (RC) is insignificant 
(Viola arvensis, Descurainia sophia) , less than 4,5 %. 
Table 5. Percentage (%) distribution of social bebaviour types (SOC) of tbe species in tbe investigat-
ed temperat~ loess grassland. 
categories C s g np dt w rc ac 
soc. behav. typ. Carex-type 6,16 1,95 17,9 0,15 5,41 0,6 0,9 0,15 
soc. behav. typ. Cytisus-type 5,16 1,66 18,2 0,37 5,16 1,29 1,47 
soc. behav. typ. Stipa-type 7,41 1,06 18,7 5,29 0,18 0,53 0,18 
The clistribution according to tbe coenosystematic categories sbows the group of indifferent 
species is comprised of severa! xeropbilous species (Hypericum perforatum, Melandrium album) and 
species occurring due to anthropogenic disturbance (Erigeron canadensis), witb a sbare of 14 %. 
Presence of forest elements sbows tbe busby cbaracter of this area. The rest are the elements of grass-
land associations (80 % of ali) . Tbe distributions of tbe social bebaviour type (SOC) and of tbe nature 
conservation rank categories (NCR) are similar in the tbree types. 
The rate of monocotyledons/dicotyledons/Fabaceae sbows (Table 6.) that tbe quantity of 
Fabaceae species is double in the Stipa type as compared to tbe two other groups (Carex, 
T. Szerdahelyi, S. F6ti, S. Cz6bel, J. Balogh, z. Nagy & Z. Tuba: Coenological and synphysiological... 9 
Chamaecytisus). Effects of a warmer and drier climate may favor the spread of species from the 
Chamaecytisus and the Stipa types. 
Table 6. The percentage (%) rate of monocotyledons/dicotyledons/Fabaceae of the species in the 
investigated temperate loess grassland. 
categories monocotyl dicotyl fabaceae 
Carex-type 8,69 24,63 2,17 
Cytisus-type 10,52 22,8 1,75 
Stipa-type 10,71 22,61 4,76 
Synphysiological characteristics 
Synphysiological measurements were started in three apparently, physiognomically different 
types of the community. The above mentioned coenological considerations prove that there's many 
differences in the three types' composition, constraints by abiotic conditions, etc., but the physiolog-
ical perfonnance is not necessarily different. 
Considering the whole dataset of summer (120 plots) and autumn (91 plots) aspect physiologi-
cal measurements (temporal variability) it is obvious, that the average values and coefficient of vari-
ation of photosynthesis are higher in summer (Table 7.). Decreasing values of these variables till 
autumn is partly caused by senescence. The last two columns show that comparing summer water-
stressed (56 plots) and non-stressed (64 plots) periods separately, CV values are much smaller in the 
latter case. 
Table 7. Some data of the photosynthetical performance of a temperate loess grassland in summer 
and autumn 2000. 
Summer Autumn Non-stressed Water-stressed 
Carex-type, non- Carex-type, water-
aspect aspect summer period summer period 
stressed summer stressed summer 
period period 
Mean ofLAI (m2• m"2) 3,09 3,84 2,91 3,30 1,64 3,66 
SDofLAI 1,25 0,97 1,44 0,96 0,49 0,84 
CVofLAI (%) 40,35% 25,20% 49,34% 29,13% 30,05% 23% 
The estimated average value of LAI (Table 8.) was higher in autumn than in summer, but the 
method used for LAl-estimation does not distinguish photosynthetically active and fully senescent 
leaves. Nor did we pursue the interannual change of LAI, the measurements in summer may have 
not been at the highest LAias it can be inferred from increasing average LAI with decreasing CV in 
June (non-stressed period: 2000. 06.08., 13., 15., 16.; water-stressed period: 2000. 06. 21., 22.). 
Table 8. Some data of the estimated leaf area index. 
Summer Autumn Non-stressed Water-stressed 
Carex-type, non- Carex-type, water-
stressed summer stressed surnmer 
aspect aspect summer period summer period period period 
Mean of LAI (m'. m·') 3,09 3,84 2,91 3,30 1,64 3,66 
SDofLAI 1,25 0,97 1,44 0,96 0,49 0,84 
CVofLAI(%) 40,35% 25,20% 49,34% 29,13% 30,05% 23% 
10 Acta Biologica Slovenica, 47 (1), 2004 
Consideration of the three different types shows, that water shortage and senescence in autumn are 
responsible for the different physiological performances. In the well-watered summer period (soil water 
content: 12,38% in the Carex-type, 10 plots; 13,54% in the Chamaecytisus-type, 12 plots and 11,86% 
in the Stipa-type, 40 plots) we found significant difference only between the physiological activity of 
the Carex- and Chamaecytisus- dominated types, however this difference is due to the significant LAI-
difference and after relating photosynthesis to 1 m2 leaf area, the difference is non-significant. 
In the case of water-stressed summer period (soil water content: 6,55% in the Carex-type, 28 
plots; 7,54% in the Chamaecytisus-type, 28 plots) photosynthesis were significantly different between 
the types and the same was found for the autumn period (soil water content: 7,02 and 11,33% in the 
Carex-type on the two days of investigation, 10,89% in the Chamaecytisus-type) (Table 9.). 
Table 9. Some data of the photosynthetical performance of different types of the investigated grass-
land in different conditions. (, ** means significant difference when referred to 1 m2 leaf area) 
Non-stressed summer period Stressed /water deficient/ Autumn aspect 
summer period 
Carex-type Cytisus-type Stipa-type Carex-type Cytisus-type Carex-type Cytisus-type Stipa-type 
Mean of A (µmol•m·2•s·1) 17,89 29,15 22,55 2,53('*) 3,29(**) 3,24(*') 4,55(*,**) 2,87(') 
SDofA 8,00 13,72 10,35 1,53 1,32 1,83 1,60 1,38 
CVof A(%) 44,71% 47,07% 45,9% 60,45% 39,97% 56,39% 35,23% 47,9% 
These results contain both abiotic and biotic variability rates. At higher light intensities 
(PPFD>lO00 µmol.m-2.s-1) the average CO2 exchange rates were higher and CV s were lower (Table 
10.) except for the case of the stressed period measurements. 
There is a clear trend of increase of the value of photosynthesis and decrease of its CV (number 
of plots are: summer: 74, autumn: 44, water-stressed period: 24, non-stressed period: 50). 
Table 10. Data of photosynthetical performance at saturating light conditions. 
Conclusions 
Mean of A (µmol·m·2·s·1) 
SDofA 
CVofA(%) 
Summer aspect Autumn aspect s:::::s:::~ 
16,63 
12,69 
76,34% 
3,62 
1,66 
45,72% 
23,36 
9,80 
41,94% 
Stressed /water 
deficient/ summer 
period 
2,59 
1,60 
61,79% 
In a typical stand we tried to set up plots, which represents this Salvio-Festucetum rupicolae 
association. Statistical evaluation of several ecological characters of species was carried out togeth-
er with a hierarchical cluster analysis. According to these analyses three groups dominated by Carex 
humilis, Chamaecytisus austriacus and Stipa dasiphylla offestucetosum rupicolae subassociation of 
a stand of Salvio-Festucetum rupicolae association were separated. Results of the synphysiological 
measurements show that water shortage and senescence in autumn are responsible for the different 
physiological performances of the three types. In the well-watered summer period we found signif-
icant difference only between the physiological activity of the Carex- and Chamaecytisus- dominat-
ed types, however this difference is due to the significant LAI-difference and after relating photosyn-
thesis to 1 m2 leaf area, the difference is non-significant. 
T. Szerdahelyi, S. F6ti, S. Cz6bel, J. Balogh, Z. Nagv & Z. Tuba: CoenologicaJ and svnphysiologicaJ ... 11 
Acknowledgements 
The financial support of the Hungarian Scientific Research Foundation (OTKA-32586 project), 
the CARBOMONT (EVKZ-CT-2001-00125) and GREENGRASS (EVK2-CT-2001-00105) 5th EU 
Framework Research Projects are gratefully acknowledged. 
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8A, Natura] Grassland, Introduction and Western Hemisphere. Elsevier, New York, p. 1-6. 
HORVATH F., Z.K. DOBOLYI, T. MORSCHHAUSER, L. L6K6S, L. KARAS, T. SZERDAHELYI 1995: Fl6ra adatbazis 
1.2. Taxon-lista es attributum allomany. [Hungarian Flora Database 1.2. Taxon list and attribut-
um stands.]. Vacrat6t, pp. 267. 
KAKAS J. (ed.) 1969: Magyarorszag eghajlati atlasza. Adattar. [Climate atlas of Hungary. Data]. -
Akademiai kiad6, Budapest, pp. 263. 
MITCHELL J. F. B., s. MANABE v. MELESHKO, T. TOKIOKA 1990: Equilibrium climate change and its impli-
cations for the future. In: HOUGHTON J. T., G. J. JENKINS & J. J. EPHRAUMS (eds.): 
Climate change, The IPCC Scientific Assesment, Chapter 5. Cambridge University Press, New 
York, p. 131-172. 
PODANI J. 1993: SYN-TAX 5.0: Computer programs for multivariate dala analysis in ecology and sy-stem-
atics . - Abstr. Bot. 17: 289-309. 
PODANI J. 1994: Multivariate <lata analysis in ecology and systematics. SPB Publishing, The Hague. 
SIMON T. 1988: A hazai edenyes flora termeszetvedelmi-ertek besorolasa. [Natura! conservation ranks of 
Hungarian flowering plants.] . - Abstracta bot. 12: 1-23. 
V1RAGH K. , S. BARTHA 1998: Koalfci6s atrendez?desek a loszsztyeppretek kialakulasa fele tart6 
szukcesszi6 soran. [Coalition rearrangements of the succession in turn of origination of loess 
steppe.]. - Kitaibelia 337-339. 
Z6LYOMI B. 1958: Budapest es komyekenek tenneszetes novenytakar6ja. [Natura! vegetation of Budapest 
and its surroundings.].- In: PECS! M. (ed.): Budapest termeszeti kepe. [Natura! view of 
Budapest.]. - Akademiai kiad6, Budapest, 744 pp. 
Z6LYOMI B. & G. PEKETE 1994: The Pannonian loess steppe: Differentation in space and tirne. - Abst. Bot. 
18: 29-41. 
Introduction 
ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2004 Vol. 47, Št. 1: 13-20 
Sprejeto (accepted): 2003-11-21 
Seasonal and daily pattern, temporal and spatial variability of 
ecosystem CO2-exchange in a temperate Pannonian loess grassland 
Szilvia FOTI 1, Janos BALOGH2, Szilard CZOBEL1, Zoltan NAGY1, Sandor 
BARTHA3 & Zoltan TUBA1,2 
1Department of Botany and Plant Physiology, Faculty of Agricultural and 
Environmental Sciences, Szent Istvan University, H-2103 Gi:idi.ill6 
( e-mail: fotiszi@freemail.hu) 
2 Departmental Research Group of Hungarian Academy of Sciences, 
Szent Istvan University, H-2103 Gi:idi.i116 
3 Institute of Ecology and Botany, Hungarian Academy of Sciences, 
H-2163 Vacrat6t 
Abstract. In the photosynthetically most active spring, summer and autumn vege-
tation period the investigated grassland did maintain a relatively strong daytime carbon 
gain. During winter the grassland displayed a slight daytime carbon loss. These data 
suggest that the grassland was a weak sink for carbon in the investigated period. COz-
exchange variability during the day seemed to be independent from that of the daily 
photosynthetic radiation. Thus other factors like soil respiration, soil moisture content 
and temperature and their interactions could be responsible for the high daily variabil-
ity of grassland COz-exchange. The considerable temporal (daily and seasonal) vari-
ability of the grassland COz-exchange can be considered as a characteristic feature of 
the grassland COz-exchange. In the investigated loess grassland vegetation the variabil-
ity of COz-exchange showed clear dependence on measuring area, which is obvious in 
the CV of NEE. We hypothetised that the spatial scale with the lowest variability is the 
characteristic area of the grassland ecosystem's COz-exchange (COz-exchange physi-
ological unit). In general decreased variability indicates a more regulated state. 
Keywords: carbon-balance, temperate grassland, chamber technique, net 
ecosystem CO2-exchange, carbon gain 
Abbreviations: Net Ecosystem Exchange (NEE), Photosynthetic Photon Flux 
Density (PPFD), air temperature (Ta), COz-concentration (Ca), Coefficient of 
Variation (CV), Net COz-uptake (A), leaf area index (LAI) 
Detailed leaf-, individual- and macroscale CO2-exchange studies have long been conducted on 
grasslands (BREYMEYER ET AL. 1996). On the other hand there are relatively few information avail-
able on the micro-scale level, more specifically in temperate grasslands including loess grasslands. 
14 Acta Biologica Slovenica, 47 (1), 2004 
Out of the physiological processes, the carbon cycle based on photosynthesis-respiration bal-
ances of ecosystems is of primary importance. Grasslands are characterised by the fact that large part 
of their organic matter (C-content) is contained in the belowground living plant parts and in the soil 
(RICE ANO GARCIA 1994) and this feature is important also in terms of their C-cycle. The temporal 
pattem of the C-balance of a grassland is also influenced by soil respiration and microbial activity 
(DbRR ANO MDNNICH 1987). Investigation of the CO2-exchange rates of these grasslands is necessary 
to estimate their significance in the global carbon cycle and global climate change. 
Temporal dynamics of the photosynthetical activity and its relation to climate have been inten-
sively studied up to the present date. There are less information available on the relationships 
between the climate and phenology (leaf structure phenology) and the temporal variability of the 
COrexchange. Phenology should definitely be considered in a study conducted on a yearly tempo-
ral scale. Net COrexchange is primarily affected by senescence in the autumn period and not by the 
abrupt weather changes (HAM ANO KNAPP 1998). 
Moreover, patches of a grass stand are not alike. Considerable structural variability can be per-
ceived even in intensively managed grasslands with profound consequences on their primary func-
tioning. This also implies the necessity of carrying out scale-dependent stand level physiological 
studies when spatial variability is concemed. Closed chamber techniques are suitable tools for study-
ing small-scale spatial variability and dynamics of CO2 gas-exchange (ANGELL ET AL. 2001). 
The temperate Salvio-Festucetum rupicolae loess grassland (steppe) has also been investigated 
in structural, dynarnical and conservation terms for decades resulting in a considerable amount of 
information in these subject (Z6LY0MI ANO FEKETE 1994, V1RAGH ANO FEKETE 1984). At the same 
tirne, studies conducted on the spatial organization of these grasslands have revealed that the spatial 
scale of the most important processes (species exchanges, coexistence pattems, and diversity) is in 
the order of a few dm2 to m2 (MUCINA ANO BARTHA 1999). 
The aims of the present work are: i) to explore the daily and seasonal courses of CO2 gas-
exchange at a fixed spatial scale, ii) to describe the temporal (daily and seasonal) and spatial vari-
ability (heterogeneity) of the photosynthetical activity and iii) to study the spatial scale dependence 
of CO2 gas-exchange in a Pannonian loess grassland (steppe) ecosystem. 
Materials and Methods 
Study site 
The measurements were conducted in the years 2000-2001 on the loess grassland situated at the 
village Kerepes (G6d6116-Monor-Irsa Hills, 170 m a.s.l., 25 km south-east from Budapest). The cli-
mate is a temperate continental with hot dry summers and cold winters; mean annual precipitation 
550-600 mm or less; annual mean temperature of 11 °C; and large annual amplitude of temperature 
changes (22 °C). 
The investigated loess grassland 
The vegetation is a xeric temperate loess steppe (Salvio-Festucetum rupicolae pannonicum 
Z6lyomi). The community is dominated by Festuca rupicola, Chrysopogon gryllus, Stipa dasyphyl-
la, Cytisus austriacus, and Carex humilis. 
S. F6ti, J. Balogh, S. Cz6bel, Z. Nagy, S. Bartha & z. Tuba: Seasonal and daily pattern .. . 15 
The parent rock is sandy loess with thick humus- and nutrient-rich A layer. The original grassland 
is made up of more than 90 species. It is a perennial and overwintering, vertically well-structured (60-
80 cm height) grassland, with many broad-leaved dicotyledonous species. The loess grassland can be 
considered as the representative of the European temperate xeric grassland (steppe) vegetation. 
Measurements of the net ecosystem CO2-exchange rates 
Net ecosystem CO2-exchange rates (parallel with transpiration, air temperature, relative humid-
ity, vapour pressure and stomatal conductance) were measured by using a portable closed-loop IRGA 
(LI-COR 6200, operated in absolute mode) sampling the air in a cylinder-shaped plexi-chamber of 
60 cm diameter and 70 cm height, with three replicates in five plots. Mixing of the air in the cham-
ber was achieved by operation of mixing fans . Ambient conditions (Ta and Ca) at the beginning of 
each measurement have been re-established by lifting the chamber while the fans were running. The 
duration of a measurement was 10 to 25 seconds, therefore the changes in the Ta and Ca in the cham-
ber were small. PPFD values and canopy surface temperatures were recorded using ceptometers 
(Decagon) and an infra red thermometer (Raytek MX4). CO2 gas-exchange values from five plots 
were used to calculate average net photosynthesis values and daytime C-balances. Coefficient of 
variation was also calculated for each average. Daily courses of gas-exchange have been measured 
from sunrise to sunset (1.5-2 hours intervals) seasonally, (04/10/2000, 20/03/2001 , 23/05/2001, 
03/07/2001) to consider phenological effects, too. 
Set-up for measuring spatial scale-dependence of the ecosystem COz-exchange 
The set-up for estimating spatial dependence of CO2 gas-exchange on measuring area consisted 
of six chambers with different diameters. Ground areas of the six gas exchange chambers follow a 
logarithmic scale with the diameter of the chambers doubling from 7 .5 cm to 240 cm. The height of 
each chamber is 70 cm. The cylinder-jacket of the chambers has been arched from UV-B resistant 
water clean plexiglass. The air motion within the chambers is supplied by outer fan except for the 
two largest chambers, where the ventilation systems are within the chamber. The chambers are suit-
ed for measurements in closed system. The measurements have been carried out on 13th June 2001, 
in nine patches with three replications at each chamber size. 
Results and Discussion 
Daily courses of net CO2-exchange in the four seasons 
Daily courses of net ecosystem CO2-exchange in the four seasons (including soil and root respi-
ration in addition to photosynthesis), air temperature and PPFD average values from the five meas-
ured patches are presented (Fig. 1.). The aim was to describe the seasonal features of NEE, as based 
on these measurements. 
Autumn (04/10/2000) 
Air temperature was higher than expected at this date and ranged between 16.2-32.9 °C. 
Cloudiness caused 20-30 o/o CV considering PPFD. There was a strong correlation between NEE and 
PPFD until midday. After midday, stomata1 limitation of photosynthesis due to water shortage caused 
this correlation to become weaker at high Ta values. NEE reached its maximum (5 .37 µmol CO2m 2s·1) 
in the early moming hours, this value was far below those measured in spring or summer, which is 
16 Acta Biologica Slovenica, 47 (1), 2004 
the consequence of the decreased photosynthetically active LAI due to the autumn senescence of 
many species. 
-"! : 1 
; 10 
o 
E ' 
~ 
" o g, 
C\l ~ 
{i 
~ -10 
N 
Q -15 
C.) 
... ~ 1!!i 
" ~ 10 
o i ' 
:g, o 1 
5i ~ •/ 
8 -15 / 
4/10/2000 
•· ·•· 
23/05/2001 
_r-. 
(' ·"'-: · 
\ 
20/03/2001 
• . • ··· • .. ~ 
7/07/2001 
l :_: ""' ,,. 1500 ~ 25 ~ 
\._ 
\ · 
Q 20 -
1000 \ .i:'6 
15 l-
e 
u. 
o.. " o.. 
l:, 2000~ ·~ 30 
" 1500 ~ 25 -
o ~ 
[ 20 -~ 
10000 15 ..... 
u. 
o.. " soo o.. ~ "] ' I 
-20 ~--
_________ .,___ ___________ _, ' 
20 
Time (hours) Time (hours) 
Figure l.: Daily course of loess grassland C02 exchange (dots with error bars, solid line) measured on 60 
cm diameter stand plots, of the photosynthetic photon flux density (PPFD, triangles, dashed line) and of 
air temperature (Tair, squares, dotted line) on four seasonally different days. One symbol represents the 
average value of five stand plots. 
Late Winter - Early spring (20/03/2001) 
Soil respiration increased at Ta values above 15°C, balancing the C02-uptake. As a result NEE 
was negative throughout the day caused also by dawn frosts, small LAI values and below optimal Ta 
values for photosynthesis while PPFD was high. 
Spring (23/05/2001) 
Growth is most intensive in loess grasslands in this period of the year. NEE followed the rapid-
ly increasing Ta and PPFD values during the moming with the maximum of 9.51 µmol C02m 2s·1• 
From midday Ta was above 30°C and NEE declined with low soil water content (12% by volume) 
and high vapor pressure deficit of the air. 
Summer (03/07/2001) 
NEE followed the rapidly increasing Ta and PPFD values during the moming with the maximum 
of 10.68 µmol C02m 2s· 1• Ta range was narrower than that measured in May. Increasing cloud cover 
from early aftemoon caused decreasing PPFD and hence NEE values, with the break of the meas-
urements due to aftemoon rains. 
The observed large seasonal fluctuation of the daily C02-exchange rates also indicates that this 
pattem is subjected to large variation season by season and year by year due to the fluctuation of the 
climatic factors. Among others this inter-seasonal variability underlines the necessity of the contin-
uous long-term measurements. 
S. F6ti, J. Balogh, S. Cz6bel, Z. Nagv, S. Bartha & Z. Tuba: Seasonal and daily pattern. .. 17 
Temporal variability of grassland COz-exchange 
Daily course of variability of NEE in representative, stili rather variable, randomly selected 
patches of the loess grassland was investigated. PPFD <lata (average of the three replicates) and CV 
of NEE (of the three replicates) are presented for each patch (Fig. 2.). Large CV values in the early 
moming hours in May and July can be explained by the light intensity values around the compensa-
tion point (large part of the canopy is self-shaded), resulting in either positive or negative NEE val-
ues and therefore high standard deviation. CV values decreased later in the moming with the major-
ity of values between 10-30%. Increase of CV (NEE) in the aftemoon suggest water shortage 
induced, species and leaf specific, variable stomatal closure as the cause of high variation in NEE. 
roo l 
04/10/2000 20/03/2001 
2roo 
n=48 n=17 
C 
400 2000 
" ~: C, C: " >JO r 1soo ii . t o~ i 15 )( : ~. " • E ON • • 2, 
ZJO . ~ • 1000 (.) 
.r> •i· .., ~- Cl ~ lJ.. o o. 
> ~ ,t '! t • -~ o o. 
(.) 100 
" \l • o ~ - % roo .. ·, . . , . ' . ' . . : , • . . ~ 
23/05/2001 03/07/2001 
roo 2roo 
n=40 n=40 
~ o 
400 
$' i 2000 
~ t ., 
" 
o 
C, 
* t . ~: C: " >JO 10)() ii 15 )( • • " o E o ZJO t i 
• » • 2, (.) . o . " 1000 Cl lJ.. o ~ li" o. 
> . 1 • • t o. (.) 100 : "'° . . . e" . 
() } . 8 , • I • ' ' • ' ; 1 = ~ .. ' ] . '" 
05 09 13 17 05 09 13 17 
Time (hours) Time (hours) 
Figure 2.: Daily courses of the coefficient of variation of loess grassland C02 exchange measured on 60 
cm diameter single stand plots (CV, dots) and ofthe photosynthetic photon flux density (PPFD, diamonds) 
on four seasonally different various days. (One symbol represents the CV value of three independent meas-
urements on the same plots, n=number of measurements.) 
Spatial variability of the litter decomposition and soil respiration rates are the probable causes of 
the highly varying NEE at the tirne of the spring measurement. High CV s of NEE in October are 
most probably caused by highly varying PPFD during the day as opposed to the situation experi-
enced in May, when CV(NEE) was much smaller due to the steadily changing light conditions. 
Temporal variabilities of NEE in the patches and the average variability considering ali the patches 
are presented in Tab. 1. 
18 Acta Biologica Slovenica, 47 (1), 2004 
Concerning the daily temporal variability of grassland CO2-exchange, one of the most remark-
able observation was that after sunrise the COrexchange variability during the day seems to be 
rather independent from the considerable changes of the daily photosynthetic radiation. Thus other 
factors like soil respiration, soil moisture content and temperature and their interactions can be 
responsible for the high daily variability of grassland COrexchange. The considerable temporal 
( daily and seasonal) variability of the grassland COrexchange can be considered as the characteris-
tic feature of the grassland COrexchange. This reflects the necessity of the high number and contin-
uous measurements during the days and as much possible during the seasons. 
Tablel: Daily temporal and spatial variability of CO2 exchance rates on five different plots with 60 cm 
diameter (plot l- plot 5) of temperate loess grassland (04/10/2001). Variability is expressed as % value of 
variation coefficient (CV%). 
Periods of measurements Plot 1 Plot 2 Plot 3 Plot 4 Plot 5 Period's averaae 
08:07 - 08:24 54,0 229,0 219,0 49,0 98,0 98,1 
08:44 - 08:58 18,0 16,0 29,0 21,0 73,0 58,5 
09:17 - 09:30 991,0 330,0 66,0 84,0 54,0 92,5 
10:34 - 10:49 115,0 108,0 34,0 24,0 48,0 61,9 
11 :43 - 11 :55 34,2 74,6 21,3 18,5 14,2 41,0 
12:43 -12:56 245,8 187,9 283,9 39,5 185,4 246,9 
13:46 - 13:59 23,9 89,8 85,9 56,7 52,8 80,9 
14:43 - 14:56 48,7 52,5 114,6 164,0 167,3 212,5 
15:15 -15:36 135,0 39,7 15,4 513,3 180,8 568,9 
16:27 - 16:43 24,4 22,5 47,6 55,6 2'1-,9 46,0 
Spatial variability/spatial heterogeneity of' grassland CO2-exchange 
The spatial variability of grassland COrexchange rate can be seen as the sign of the spatial het-
erogeneity of the ecosystem COrexchange. CV s of NEE (Tab. l.) also demonstrate the spatially dif-
ferent behaviour of the five measured patches. 
Coenological studies have proved that a few dm2 to m2 sampling unit size is suitable for finding 
the highest variability in species composition and combinations in this grassland (MucINA AND 
BARTHA 1999, BARTHA ET AL.1997). Consequently, variability of LAI is also high at micro-scale (CV 
over 30% at 60cm) explaining in part the background of the spatial variability of COrexchange. 
From this one can conclude that parallel measurements of many unevenly distributed grassland 
plots with the same diameter are required. 
Daily maximum values of grassland CO2-exchange and daytime carbon gains 
Daily maximum values of NEE (Fig. 3.) are in good agreement with both the daytime carbon 
gain values (528, -864, 2624 and 2171 mgCm-2 on the four representative days, respectively) and 
with the phenological stages of the vegetation. The maximum of NEE and hence the carbon balance 
are negative in March, the auturnn NEE values are considerably lower than the ones in the summer. 
Consequently, in the photosynthetically most active spring, summer and auturnn vegetation period 
the investigated grassland did presumably maintain a relatively strong daytime carbon gain, while 
during winter the grassland displayed a slight daytime carbon loss. The above data indicate that in 
the year of 2000-2001 the investigated overwintering grassland vegetation was very probably acting 
as a carbon sink. 
S. F6ti, J. Balogh, S. Cz6bel, Z. Nagy, S. Bartha & Z. Tuba: Seasonal and daily pattem ... 19 
14 3000 
12 2500 
10 
2000 ,-; "' , ·
E 8 0 
15 1500 .[ i 6 
~ 
1000 ~ 
4 "' 
"' ]i -5 500 
X 2 ~ " ON o "' (.) o -r- (.)
-2 -500 
• -4 -1000 
04/1012000 20/03/2001 23/0512001 03/07/2001 
Figure 3.: Daily maximum values of C02 exchange rates (bars) and the carbon balance values of the inves-
tigated days (dots) in the temperate Joess grassland. 
Diameter of chambers (cm) 
•~ -• ~ __ l_s __ • • ;) 1~ i 
~ . i . \ I:: i 
/ · .X , __ ,/ 1· :: ! 
/ m ~ 
/ " 10 
-----------+ o 
0.1 
1e+1 1e+2 1e+3 1e+4 1e+5 
Ground area of chambers (cm2) 
Figure 4.: Spatial scale dependence of C02 exchange (dots) and its coefficient of variation (CV, diamonds) 
in temperate loess grassland. (One symbol refers to nine plots measured in three separate replicates on 
13/06/2001, the C02 exchange and the ground area values are plotted ona Jogarithmic scale.) 
Spatial scale-dependence of grassland CO2-exchange 
Spatial scale-dependence of NEE was investigated with different chamber sizes (different diam-
eters, 70cm height). Logarithrnic values are shown in Fig. 4. The regression COz-uptake values vs. 
chamber size shows good fit (p<<0.01). However CV of NEE shows scale-dependence with mini-
mum of variability at 60 cm patch diameter, suggesting this scale to be characteristic unit of this 
grassland, where the suparindividual regulation is the most pronounced. 
In the investigated loess grassland vegetation the variability of COz-exchange showed clear spa-
tial scale-dependence. The most probable factors which are candidates for causes of variability pat-
tem along the investigated space series are: the ratio of covered and uncovered soil surfaces, the spa-
20 Acta Biologica Slovenica, 47 (1), 2004 
tial heterogeneity of soil moisture, soil temperature and litter deposition, the changes of species com-
position (e.g. dicots/monocots ratio, plant density), the height and the physiognomical and microm-
eteorological structure of the canopy in the relation to the changes of the botanical composition. 
Presumably the spatial scale with the lowest variability can be considered as the characteristic scale 
of the COz-exchange (CO2-exchange physiological unit) of the grassland ecosystem. But this aspect 
and relationship between coenological (botanical composition) and physiological scale-dependence 
should be a matter of future detailed analysis. In general decreased variability indicates a more reg-
ulated state. Thus it is probably that the spatial scale with the lowest variability represents the 
supraindividually most regulated physiological - CO2-exchange- units of the grassland. 
Ackoowledgements 
The financial support ofthe Hungarian Scientific Research Foundation (OTKA-32586 project), the 
MEGARICH 4th and GREENGRASS 5th EU Framework Research Projects is gratefully acknowl-
edged. 
References 
ANGELL R. F. ET AL 2001: Bowen ratio and closed chamber carbon dioxide flux measurements over 
sagebrush steppe vegetation. Agricultural and Forest Meteorology 108: 153-161. 
BARTHA S.ET AL 1997: Spatiotemporal scales of non-equilibrium community dynamics: a methodological 
challenge. New Zealand Joumal of Ecology 21(2): 199-206. 
BREYMEYER A. l. ET AL (Eds.) 1996: Globa! Change: Effects on Coniferous Forests and Grasslands. 
Dč>RR H. & K. O. MiiNNJCH 1987: Annual variation in soil respiration in selected areas of the temperate 
zone. Tellus 39B: 114-121. 
HAM J. M. & A. K. KNAPP 1998: Fluxes of CO2, water vapor, and energy from prairie ecosystem during 
the seasonal transition from carbon sink to carbon source. Agricultural and Forest Meteorology 
89: 12-14. 
MucINA L. & S. BARTHA 1999: Variance in species richness and guild proportionality in two contrasting 
dry grassland communities. Biologica, Bratislava 54: 67-75. 
RICE C. V. & F. O. GARCIA 1994: Biologically active pools of soil C and Nin tallgrass prairie. In: 
DoRAND J. ET AL. (eds.): Defining soil quality for a sustainable environment. Spec. Pubi. No. 
35. MSoil Sci. Soc. Am., Madison, WI. 
VIRAGH K. & G. FEKETE 1984: Degradation stages in a xeroseries: composition, similarity, grouping, 
coordination. Acta Bot. Hung. 30: 427-459. 
Z6LYOMJ B. & G. FEKETE 1994: The Pannonian loess steppe: Differentiation in space and tirne. Abstracta 
Botanica 18: 29-41. 
Introduction 
ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2004 Vol. 47, Št. 1: 21-25 
Sprejeto (accepted): 2003-11-21 
Pollution of garden soils and vegetables in the Šalek valley 
Nives KUGONJČI, Boštjan POKORNY1, Melita STROPNIK1 
1 ERICo Velenje - Ecological Research & lndustrial Co-operation, Koroška 58, 
SI-3320 Velenje, Slovenia; e-mail: nives.kugonic@erico.si 
Abstract. Soil pollution and accumulation of heavy metals in the most com-
mon vegetables were investigated in the Šalek Valley, where the largest Slovenian 
thermal power plant of Šoštanj (ŠTPP) is located. Methods were chosen according 
to intemational and Slovenian standards. Heavy metal content in soils and plants 
was determined by ICP-MS, ETAAS and hydride technique after appropriate diges-
tion of samples. Results revealed that garden soils in the Šalek Valley are not pol-
luted in general with heavy metals. On the contrary, some plant species exceeded 
permitted Jevels of Cd. Our results showed that the most sensitive group of plants 
are stili affected, although emissions of heavy metals markedly decreased after the 
desulfurisation device was built at the ŠTPP. 
Key words: Vegetables, Pollution, Heavy metal accumulation, Cadmium, 
Lead, Mercury, Arsenic. 
High quantities of heavy metals are released into the environment by anthropogenic activities, 
such as mining, combustion of fuels and waste materials as well as other industrial processes. The 
fate and transformation of metals in a soil - plant system depends very much on soil type and pre-
vailing soil conditions (Ross 1996). In general availability of metal decreases as the pH leve! rises 
and the contents of clay and humus increase (MILLER & D0NAHUE 1990). Besides uptake from soils, 
plants can also derive significant amounts of some elements through foliar absorption. It depends on 
the plant species, its nutritional status, the thickness of its cuticle, the age of the leaf, the presence of 
stomatal guard cells, the humidity at the leaf surface and the nature of the solutes (ALL0WAY 1990). 
Heavy metals enter the biological cycle and are enriched in various plant organs (BERGMAN 1992). 
Plants in which roots or leaves are used for human or animal consumption are especially critical con-
cerning the accumulation of heavy metals. 
The main objective of the study was to assess the quality of soil and commonly grown vegeta-
bles which have been exposed to electricity production in the Šalek Valley. 
22 Acta Biologica Slovenica, 47 (1), 2004 
Material and methods 
The field experiment with lettuce (Lactuca sativa), endive (Cichorium endiviae) and carrot 
(Daucus carota - Nantes) as test plants was carried out. 28 locations in distances within 10 km from 
the ŠTPP where included in the experiment. Altitudes of experimental plots ranged between 312 and 
778 m above sea leve!. Seedlings oflettuce and endive were grown in the greenhouse; afterwards, 15 
plantlets were transplanted to each location. Carrot was sown directly on the experimental sites. Each 
species occuped approximately 3 m2 at each site. For each test species 12 - 15 plants were sampled. 
Individual parts of plants were separated as washed roots of carrot, washed and unwashed leaves of 
endive and lettuce. Plant samples were dried at 36°C and ground in an agate mortar. 250-500 mg DW 
plant samples were treated with 65 % HNO3 acid. Heavy metal concentrations were measured in 
three replicates. The electrothermal technique was used for the determination of Cd, As and Pb 
(Per!(in Elmer SIMAA 6000), flame atomic absorption spectrometry for the determination of Zn 
content (Analyst 100) and hydride technique for the determination of Hg content in plant after the 
acid dissolution technique with microwave heating (CEM MSP 1000 - Varian). Quality control was 
performed by comparison of results with the standard reference material NIST SRM 1515 Apple 
Leaves. 
Soil samples were taken from the topsoil (0 - 20 cm). An average soil sample from each sam-
pling site was prepared as a composite of 25 sub-samples taken from an area 250 m2 in size (ISO 
10381-1 1996). Soil samples were homogenized and ground in a ceramic grinder, then passed 
through a 2 mm plastic sieve before soil analysis (ISO 11464 1994). Pedologic soil parameters were 
determined as follows: pH in 0.1 M KCI solution, the content of organic matter by the Walkley-Black 
method, soil texture by pipette method, respectively (Janitzky 1986). For the analysis of metals con-
tent, the samples were ground further in an agate mili for 10 minutes then passed through 150 µm 
sieve. Heavy metal (Pb, Cd, As, Zn) content in soils was determined using flame and electrothermal 
atomic absorption spectrometric methods, while the hydride technique (Perkin Elmer SIMAA 6000) 
was used for the deterrnination of Hg. A standard reference material »Montana Soil« was used for 
analytical quality control. 
Results and disscusion 
A wide range of Pb, As, Cd, Zn and Hg content was determined in the garden soil of the Šalek 
Valley. Levels of heavy metal samples from the surface soil layer at the depth O - 20 cm are present-
ed (Table 1). 
Table 1: Heavy metals content of the surface soil (n = 28) in the Šalek Valley. 
min.-max. 
average 
stdev 
KV % 
n 
28 
28 
28 
28 
0.3 -1.3 
0.76 
0.25 
33.41 
23.6-77.9 
45.65 
12.18 
26.69 
92.0-348.0 
181.08 
59.24 
32.71 
4.8-138.0 
16.39 
24.17 
147.49 
0.07-0.48 
0.18 
0.09 
52.35 
N. Kugonič, B. Pokomy, M. Stropnik: Pollution of garden soils and vegetables ... 23 
The critical value for As (55 mg kg-1; Off. Gaz. Rep. Slov. 68/96) was exceeded twofold in the 
southem hilly margin of the Valley (Mali Vrh), thereofore this site has to be defined as polluted con-
sidering As. Levels of Cd, Pb, Hg and Zn do not exceed even waming values (Off. Gaz. Rep. Slov. 
68/96), which were 2 mg kg-1, 100 mg kg-!, 2 mg kg-1 and 300 mg kg-1, respectively, thus soils are 
not treated as polluted considering Cd, Pb, Hg and Zn. Additionally, the pedological results suggest 
that soil characteristics do not induce high heavy metal accumulation in plants. pH levels ranged 
between 4.8 and 7.1 with an arithmetic mean of 6.5, content of organic matter ranged between 3.6 
and 1.3 % with an arithmetic mean of 8.5, while the content of clay ranged between 5.4 and 18.4 % 
with an arithmetic mean of 8.2. 
Most of metals in plants originate from root uptake, but foliar uptake of some metals (especial-
ly Cd) may be relatively high as well (KABATA PENDIAS & PENDIAS 1984). Leafy vegetables are espe-
cially sensitive to foliar heavy metal absorption (ZUPAN & al. 1995). Results of our study revealed 
that the highest content of heavy metals was observed in edible green parts of lettuce (Table 2). 
Statistical analysis confirmed a significantly higher concentration of Cd in lettuce in comparison 
with other vegetables (MannWhitney U test: U = 3.5023, p < 0.001). 
Table 2: Ranges of heavy metals in vegetables from garden soils in the Šalek Valley. 
Vegetable n Cd Pb As Hg 
m!!kg-1DW mgkg-1DW mgkg-1DW ~ kir·1ow 
endive leaves unwashed min. -max. 28 0.14- 1.78 0.08-3.64 0.05 -1.46 0.01 -0.06 
(Cichorium endivae) average 0.40 0.83 0.31 0.01 
stdev 0.33 0.78 0.33 0.005 
KV% 84.34 93.90 107.88 30.79 
washed min.-max. 28 0.12-2.2 0.08-2.0 0.04-1.0 0.01 -0.05 
average 0.42 0.36 0.11 0.03 
stdev 0.44 0.45 0.10 0.04 
KV% 105.68 124.83 94.65 175.05 
lettuce leaves unwashed min. -max. 23 0.3-4.4 0.53-5.9 <1.0- 5.86 0.04-0.08 
(Lactuca sativa) average 0.88 1.61 / 0.06 
stdev 0.88 1.19 / 0.01 
KV% 99.94 73.60 / 23.78 
washed min.-max. 28 0.2-4.0 0.33-3.37 <1.0 - 1.53 0.05 -0.08 
average 0.96 0.97 / 0.07 
stdev 1.21 0.77 / 0.01 
KV% 125.60 79.57 / 14.85 
carrot roots washed min.-max. 28 0.19 - 1.4 0.41- 1.03 < 0.1 <0.05 
(Daucus carota) average 0.48 0.57 / / 
stdev 0.30 0.12 / / 
KV% 61.95 21.71 / / 
Over 95 % of washed lettuce samples exceeded the prescribed value for Cd in vegetables, which 
is 0.3 mgkg-1 DW (Off. Gaz. Soc. Fed. Rep. Yug. 59/83). Differences between Cd content in washed 
and unwashed edible green parts of vegetable were trivial, which suggest that a small fraction of Cd 
penetrates through the leaf cuticle (Figure lA). 
24 
., 
1 
1 
1 
1 
i 
- 1 _::. d .96•StdErr! .__ __ Cd __ ___ Cd __ __ ____. ~ ~=Stdj 
Acta Biologica Slovenica, 47 (1), 2004 
B: COl'l len1of Pb inw!Ohcd andunwashed lu.vn or ~ nuce 
-c:-r;;,tent of As m washed and unwashed kave, oflcttuce O: Contcnt ofHg in washed aml unwuhcd leevcs oflcttucc 
··•'-----
Io.1 116' Std&r 
C J i. l.OO"Sld.hr - ,.,-,,_---~ . ·-
0,072,---------- -
..... 
f 0,(16,1 
! j O,Ol!O 
-i-:-
[ • ! 
- , __J_ __ _]_ __ ,-=--
1 , 1 
S_~ 
•='------,---...__ 
I ,>196~td ~ .. 
• ~ l.flO"Sld En. 
Figure l(A - D): Differences between Cd, Pb, As and Hg content in washed and unwashed edible green 
parts of Jettuce (Lactuca sativa). 
On the contrary, Pb, As and Hg content significantly differed between washed and unwashed 
leaves and roots of carrot (Figure 1 B - D). Results also revealed that vegetable is not polluted in 
general with Pb, As and Hg, although on some plots (Topolšica, Mali Vrh) even washed lettuce 
leaves exceeded prescribed values, which are 3 mgkg-1 and 1 mgkg-1 DW for Pb and As, respective-
ly (Off. Gaz. Soc. Fed. Rep. Yug. 59/83). 
Conclusions 
It was estimated that ŠTPP had emitted 0,2 t of Cd, 22, 1 t of Pb, 4,5 t of As, 0,3 t of Hg and 298 t 
of Zn in the period 1981 - 2001 (POKORNY 2003). After the desulfurisation device was built in 1995 
the annual dust emission decreased from 8121 t in 1994 to 1077 t in 1999 (SEVŠEK 2000). Although 
exact <lata on heavy metal emissions are not known, our results revealed that atmospherical inputs, 
enriched with Cd, still affect the most sensitive group of plants. A high content of Cd in test plants is 
consistent with the data, which confirmed a significantly higher content of Cd in the surface layer of 
soil (0- 5 cm) in comparison with deeper layers (5 - 20 cm, 20- 30 cm) (KUGONIČ & STROPNIK 2001). 
References 
ALLOWAY B. J. 1990. Heavy metals in soils, 339 pp. - Blackie, John Wiley & Sons, Inc., London and 
Glasgow, New York. 
BERGMANN W. 1992. Nutritional disorders of plants, 386 pp. - Gustav Fischer Verlag, Jena, Stuttgart, New York. 
N. Kugonič, B. Pokomy, M. Stropnik: Pollution of !!arden soils and vegetables .. . 25 
ISO 10381-1, 1996. Soil quality - Sampling, Part 1: Guidance on the design of sampling programmes, 
45 pp. 
ISO 11466. 1995. Soil quality - Extraction of trace elements soluble in aqua regia, 6 pp. 
ISO/DIS 11047, 1995. Soil quality - Determination of cadmium, chromium, cobalt, copper, lead, man-
ganese, nickel and zine - Flame and electrothermal atomic absorption spectrometric methods, 
29 pp. 
JANITZKY P. 1986. Particle-size analysis. - In: SINGER M.J., JANITZKY P. (Eds.), Field and laboratory pro-
cedures in a soil chronosequence study, U.S . Geological Survey Bulletin, 1684, 11-16. 
ICABATA-PENDIAS A., H. PENDIAS 1984. Trace elements in Soils and Plants. CRC Press, lnc. Boca 
Raton, Florida, p. 315. 
KUGONIČ N., M. STROPNIK 2001. Heavy Metal levels on Soils and Plants on Agricultural Areas in the 
Šalek Valley, ERICo Velenje, ERICo Velenje, Ecological Research & Industrial Co-operation, 
Research Department, DP-24/02/01, Fina! report, October 2001, 173 p. 
MILLER R. W., R. L. DONAHUE 1990. Soils -An lntroduction to Soils and Plant Growth, Prentice Hall 
lnc., 768 pp. 
OFFICIAL JOURNAL OF THE REPUBLIC OF SLOVENJA 68/96. Degree on input of dangerous substances and 
plant nutrients into soil, 1996, 5769-5773. 
OFFICIAL JOURNAL OF THE SOCIALISTIC FEDERAL REPUBLIC OF YUGOSLAVIA 59/83. Ordinance of maxi-
mum allowed value of pesticides, another toxic substances, hormones, antibiotics and micotox-
ines in food stuff, 1983, 1635-1651. 
POKORNY B. 2003. Roe deer (Capreolus capreolus L.) organs and antlers as bioindicator of environ-
mental pollution with heavy metals, Doctoral thesis, ERICo Velenje, Ecological Research & 
lndustrial Co-operation, Research Department, 192 pp. 
Ross S . M . 1996. Toxic Metals in Soil-Plant Systems, 469 pp. - John Wiley & Sons, England. 
SEVŠEK I. 2000. Ekološke obremenitve okolja v obdobju 1980-1999. In: DEJANOVIČ B., C . RIBARIČ­
LASNJK (Eds), The Šoštanj Power Plant -Annual Report, Termoelektrarna Šoštanj, Šoštanj. 
ZUPAN M., V. HUDNIK, F. LOBNIK, H. GRČMAN 1995. Akumulacija kadmija, svinca in cinka v nekater-
ih kmetijskih rastlinah. Slovenski kongres o prehrani, april 21. - 25., Bled. 
lntroduction 
ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2004 Vol. 47, Št. 1: 27-30 
Sprejeto (accepted): 2003-11-21 
Harmful impact of exceptional cold air outbreak in april 1997 
on silver fir in Croatia 
Danko DIMINIC1, Boris HRAŠOVECI , Milan GLAVAŠI , Nenad POTOČIC2 
1 University of Zagreb, Faculty of Forestry, P.O. Box 422, 10002 Zagreb, 
Croatia; e-mail: danko.diminic@zg.htnet.hr 
2 Forest Research Institute, Cvjetno naselje 41, 10450 Jastrebarsko, Croatia 
Abstract. After a severe outbreak of cold air in mid and late April in 1997, 
locally occurring damages on twigs and needles of silver fir were recorded in some 
forests in Croatia. Field examinations in the first half of July 1997 revealed the 
damages on one-year and older needles while fully developed twigs of current year 
growth were left intact. Southem and western slopes with fir growing forests rep-
resented typically affected sites. Further laboratory analyses confirmed the field 
observation that no harmful insect or pathogens were the cause of these damages. 
Spread of symptoms, expressed more heavily at lower elevations, indicated a prob-
able connection with recent exceptionally cold air outbreak in mid-April. 
Physiological disturbances caused by potassium deficiency are discussed in the 
light of specific occurrences of damage symptoms on silver fir trees. 
Key words: Abies alba, cold air damages, potassium. 
Abiotic damages on forest trees are well documented and known in forest science and practice 
for a long tirne (EDLIN & NIMM0 1956). Among the commonest are injuries caused by cold air and 
late or early frost (REIF & PAPP 1995). Very often, such damages occur on tree species not fully adapt-
ed to their artificial habitat (such as plantations) or plants growing in the border zones of their natu-
ra! area of spread. In the case of silver fir (Abies alba Mill.) cold air damages are not common but 
are documented and known in the literature (HARTMANN & al. 1988). This paper deals with the rare 
event of atypical damages on sil ver fir in natural forest stands of central Croatia in the spring of 1997. 
Change of needle colour and dieback of twigs and branches was recorded in a relatively isolated area 
of roughly 40 ha with no visible cues to known biotic agents. At the same tirne, it was the year of 
extraordinary atmospheric events and cold air outbreak which occurred during mid and late April and 
hit the whole country, especially the maritime and mountainous region (BRzovrc 1999). As with the 
other tree species, the nature of cold hardiness of silver fir is strongly correlated with potassium 
physiology as potassium is known to increase frost resistance. Potassium deficient plants are more 
prone to winter damage (BAULE & FRICKER 1970, MENGEL & KrRKBY 1987, BERGMANN 1992). 
28 Acta Biologica Slovenica, 47 (1 ), 2004 
Based on the <lata of Diagnostic and Prognostic Service of Forest Research Institute J astrebarsko, 
on reported atypical symptoms on silver fir trees in May 1997, in the north Vinodol area (NW 
Croatia), this research was initiated to find out the possible causes. Impacts of phenological, climat-
ic and physiological factors were analysed in relation with spatially and temporally occurring dam-
ages in the research area. 
Materials and methods 
Samples were collected and trees were analysed on July 10, 1997, at two locations Treskavac and 
Kalic in the north Vinodol area. Both sites are located at altitudes between 900 and 960 m, with incli-
nation from O and 10 °. The site Treskavac covers an area of 17 .11 ha, with various expositions, and 
the site Kalic covers an area of 19.92 ha, with SW exposition. Sampled fir trees were randomly 
selected, felled and debarked to find out the possible presence of defoliators, suckers or xylophages 
(expectedly, bark beetles) and pathogenic fungi (wood decay and root rot fungi). 
Samples of needles, twigs and branches, with observed visible symptoms, were collected from 
sampled trees and randomly selected young firs. Standard laboratory analyses were carried out to 
examine the presence of possible harmful insect fauna or pathogenic fungi. 
In the year 2000, the second study was conducted to determine the possible influence of nutri-
ent dynamics on cold hardiness of silver fir, 5 plots (each 100 x 100 m) were chosen in the region of 
Gorski kotar: Belevine - at altitude of 800 m, SW exposition; Kupjački vrh - at altitude of 1000 m, 
SW exposition; Leska S - at altitude of 697 m, NW exposition; Leska D - at altitude of 708 m, NW 
exposition. Aerial distances between these plots and sites Treskavac and Kalic are approximately 20 
km. The 5th plot Sljeme was chosen on Medvednica (near Zagreb), at altitude of 954 m, S exposi-
tion. Current and 1-year old needles from a total of 141 trees were sampled monthly from February 
to November. Samples (24-36 trees per location, representing a 30 % size sample of all dominant and 
co-dominant trees) were pooled by location into a composite sample. Samples were dried at 105 °C 
and ground. After wet digestion (H2SO4 / HC1O4) potassium was deterrnined through flame pho-
tometer Eppendorf (AOAC 1996). 
Climatic <lata for 1996 and 1997 were obtained from the State Hydrometeorological Institute Zagreb. 
Results 
Field examinations of affected silver firs revealed irregularly distributed dead needles and twigs 
resulting with reddish tint of some parts of the crowns or whole trees. In adult trees some branches, 
randomly positioned in the crowns, showed the abovementioned symptoms. A definitive pattem of 
damage symptoms was observed on smaller trees and fir saplings: significantly highest proportion 
of one-year old needles was affected (those developed a year before). In most cases there was also 
an inexistence of current growth on these shoots. All of these symptoms were recorded with greater 
intensity at lower elevations in the research area. 
Examination of sampled trees in the field obtained no visible attack of sap suckers, defoliators, 
secondary xylophages nor wood decay or root rot fungi. Laboratory analyses of samples confirmed 
the field results. There were no pathogenic fungi, neither insect pests as possible causal agents of 
described symptoms in sites Treskavac and Kalic. 
D. Diminic, B. Hrašovec, M. Glavaš, N. Potočic: Harmful impact of exceptional... 
1,50 
1,30 
1,10 
i 
~ 0,90 
"C 
0,70 
0,30 
II III IV v VI VII VIII IX X 
Months 
XI 
--=i--1-yr.old 
needles 
-"l!t.)lll•Current 
needles 
• • Limit 
value 
29 
Figure 1: Seasonal dynamics of potassium coacentrations in silver tir needles in the year 2000 (averaged 
values from five locations). Limit value (0.50 %) after BERGMANN (1992). 
Dynamics of potassium concentrations in needles revealed a drop of K+ concentration in older 
needles due to its redistribution into younger tissues (Figure 1). Translocation of K+ from one-year 
old needles ( developed in 1999) towards buds is apparent just before the budburst in April, resulting 
in strong potassium deficiency and high K+ concentrations in current-year needles. 
Discussion 
The hypothesis on biotic complex as causal agents for the new case of silver fir dieback, based on the 
aetiology of already recorded damages in the area, was rejected. In spite of the large amount of wind-felled 
trees and woody debris, detailed field analysis of newly affected trees and laboratory check-up could not 
confirm the presence of any significant biotic agent. Spatial distribution of damaged trees, as well as their 
age-class structure, suggest that some abiotic factor must have had been involved in this event. 
A period of exceptionally low temperatures in mid and late April 1997 (both distinct minima, 
April 161h and 21 th , were lower than two standard deviations of the 30-year average) served asa basis 
for case study by which BRzovrc ( 1999) explained the mechanisms of local modifications of larger 
scale atmospheric processes in the area of Northern Mediterranean. 
What we consider highly relevant for this study is (I) the fact that the outbreak of cold air in April 
was one of the strongest ever recorded and (II) the affected area is considered a border zone of nat-
urally growing fir because of the milder climate due to the strong maritime influence from the west. 
This conforms to the conclusions by FoRSTER & al. (1988) and FoRDERER & al. (1990) in the case of 
similar large scale damages recorded in Switzerland after the winter 1986/87. 
30 Acta Biologica Slovenica, 47 (!), 2004 
According to BERGMANN (1992) potassium deficient plants are more prone to winter damage. 
Older leaves (needles) first reveal symptoms of potassium deficiency (ANIC 1973). The dynamics of 
potassium in the silver fir needles, as shown in the second study, reveals the translocation of potas-
sium from one-year old needles towards buds that are becoming physiologically active, leading to 
potassium deficiency in older tissues. This drop of K+ in older needles just before budburst in April 
coincided in 1997 with the outbreak of exceptionally cold air in the area. 
Synthesis of ali available <lata, gathered in the area of affected trees and from the separate study 
analyzing the nutrient status and cold hardines of silver tree in the region, lead to a conclusion that 
it must have been a 1997 exceptional cold air outbreak that initiated such damage symptoms. This is 
further backed up by the fact that at higher elevations, hit by the same climatic phenomenon, were 
no damages recorded. It is reasonable to deduct that the phenological differences of fir trees in dif-
ferent altitudes acted as key factors in the distribution of damage symptoms in the wider area. 
References 
ANIC, J 1973: Plant nutrition. Faculty of Agriculture, University of Zagreb, 180 pp. 
A0AC 1996: Official methods of analysis of AOAC Intemational, Association of Official Analytic 
Chemists Intemational, Arlington, VA. 
BAULE, H. & C. FrucKER 1970: The fertiliser treatment offorest trees. BLV-Verlagsges, Munchen, 223 pp. 
BERGMANN, W. 1992: Nutritional Disorders of Plants - Development, Visual and Analytical Diagnosis. 
Gustav Fischer Verlag Jena etc., 361 pp. 
BRZ0VIC N. 1999: A case study of a cold air outbreak on 20 April 1997 over Croatia. Croatian meteoro-
Jogical joumal, 33/34: 1-18. 
EDLIN H. L. & M. NIMM0 1956: Tree injuries. Thames & Hudson, London - New York, 167 pp. 
FORDERER L., J. THUDIUM, J. HERTZ, P. BUCHER, R. PANJ, R. HUBER & M. KNECHT 1990: 
Untersuchungen in Zusammenhang mit den Nadelverrotungen des Winters 1986/87 am 
Flascherberg (St. Luzisteig). Schweizerische Zeitschrift fi.ir Forstwesen, 141 (10): 837-849. 
FORSTER B., E. JANSEN, H. SCHERRER, M. LEIDIG, H. TURNER, R. HASLER, W. SCHONENBERGER, N. 
KuHN, W. KELLER, P. LUSCHER, J. BUCHER & w. LANDOLT 1988: Yerrotungen imrnergruner 
Nadelbaume im Winter 1986/87 in der Schweiz. Berichte Eidgenossische Anstalt fiir das 
Forstliche Versuchswesen, No. 307, 90 pp. 
HARTMANN G., F. NIENHAUS & H. BUTIN 1988: Farbatlas Waldschaden. Diagnose von 
Baumkrankheiten. Verlag Eugen Ulmer, Stuttgart, 256 pp. 
MENGEL, K. & E.A. KlRKBY 1987: Principles of Plani Nutrition. Intemational Potash Institute, Bern, 
686 pp. 
REIF A. & V. T. PAPP 1995: Spatfrostschaden in Sudwestdeutschland im Mai 1995. Allgemeine Forst 
Zeitschrift fi.ir Waldwirtschaft und Umweltvorsorge, 50 (23): 1282-1286. 
ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2004 Vol. 47, Št. 1: 31-43 
Sprejeto (accepted): 2003-11-21 
Algae of specific environments in Slovenia 
Alge posebnih okolij v Sloveniji 
Aleksandra KRIVOGRAD KLEMENČIČ1 , Danijel VRHOVŠEK2, 
Nataša SMOLAR-ŽVANUT2 and Gorazd KOSP 
1 Čušperk 51, 1290 Grosuplje, Slovenia 
2 Limnos d.o.o, Podlimbarskega 31, 1000 Ljubljana, Slovenia, 
Tel./Fax: 01 505 73 86 E-mail: info@Iimnos.si 
3 National Institute of Biology, Večna pot 111, 1000 Ljubljana, Slovenia 
Abstract. Algae studies were carried out in some specific biotopes of Slovenia: 
eutrophic lakes (Koseški bajer, Sotelsko jezero), peat bogs (Lovrenška jezera, 
Šijec), waterfalls (Savica, Krka falls, small waterfall on Pohorje), springs (springs 
at Medvedje Brdo and Pohorje, hot spring at Terme Čatež, mineral spring at Rimski 
vrelec), brackish waters (Fiesa lake, Dragonja estuary), hypereutrophic biotopes 
(manure water, constructed wetlands), aerial biotopes (concrete wall, stony wall, 
lime tree trunk (Tilia platyphyllos), limestone rock, Krška jama cave) and the 
Dragonja River. 
Samples were taken seasonally at 26 sampling sites in 1998, 1999, 2000 and 
200 l. Similarity in species structure and abundance of algae were determined with 
the Bray-Curtis coefficient of similarity. 
Altogether, 537 species and subspecies of algae (of nine classes) were deter-
mined and most of them belonged to Bacillariophyceae. 146 species and sub-
species were identified for the first tirne in Slovenia; of these 107 belonged to 
Bacillariophyceae, 28 to Cyanophyceae, six to Chlorophyceae, four to 
Zygnematophyceae and one to Xanthophyceae. 
The appearance of almost half of ali species and subspecies was limited to a 
single sampling site. Eight species (Achnanthes minutissima, Cymbella affinis, 
Cymbella silesiaca, Gomphonema angustum, Navicula veneta, Navicula sp., 
Phorrnidium sp. and Trentepohlia aurea) were present in more than half of ali spe-
cific biotopes. 
Key words: algae, eutrophic lakes, waterfalls, springs, peat bogs, aerial 
biotopes, constructed wetlands, caves, brackish waters, manure waters 
Izvleček. V prispevku je predstavljena raziskava alg v nekaterih posebnih 
biotopih v Sloveniji: eutrofnihjezerih (Koseški bajer, Sotelsko jezero), visokih bar-
32 Acta Biologica Slovenica, 47 (1), 2004 
jih (Lovrenška jezera, Šijec ), slapovih (Savica, slapovi na Krki, slapič na Pohorju), izvirih (izvir na 
Medvedjem Brdu in Pohorju, termalni izvir v Termah Čatež, mineralni izvir Rimski vrelec), 
brakičnih vodah Gezero v Fiesi, izliv Dragonje), hiperevtrofnih biotopih (gnojevka, rastlinskih čistil­
nih napravah), kopenskih biotopih (betonski zid, kamniti zid, deblo lipe (Tilia platyphyllos), 
apnenčasta skala, Krška jama) in reki Dragonji. 
Vzorci so bili odvzeti na 26 vzorčnih mestih v letih 1998, 1999, 2000 in 2001. Z Bray - Curtisovim 
koeficientom podobnosti je bila ugotovljena podobnost v vrstni strukturi in abundanci alg. 
Skupno je bilo določenih 537 vrst in podvrst alg iz devetih razredov, po številu določenih vrst so 
prevladovale Bacillariophyceae. 146 vrst in podvrst je bilo v Sloveniji prvič zabeleženih, od tega jih 
107 pripada razredu Bacillariophyceae, 28 razredu Cyanophyceae, šest razredu Chlorophyceae, štiri 
razredu Zygnematophyceae in ena razredu Xanthophyceae. 
Pojavljanje skoraj polovice vseh določenih vrst in podvrst je bilo omejeno na posamezna vzorčna 
mesta. Osem vrst (Achnanthes minutissima, Cymbella affinis, Cymbella silesiaca, Gomphonema 
angustum, Navicula veneta, Navicula sp., Phormidium sp. in Trentepohlia aurea) je bilo prisotnih v 
več kot polovici vseh posebnih biotopov. 
Ključne besede: alge, eutrofna jezera, slapovi, izviri, visoka barja, kopenski biotopi, rastlinske 
čistilne naprave, jame, brakične vode, gnojevka 
Introduction 
Algae are present in sea water, brackish water, freshwater and in aerial biotopes. Same of them 
exist also in specific environments like snow, hot springs, caves, peat bogs, etc. In Slovenia, the num-
ber of specific environments is very large because of its geographic diversity and a relatively low 
degree of pollution. Algae in such environments have been investigated only partly, despite the 
exceptional species diversity. 
Because most algal species are spread worldwide, they are not considered to be an endangered 
group of organisms. It is also difficult to consider a single algal species to be endangered. Changing or 
even disappearing of some ecosystems ( desiccation of swamps, regulation of rivers, building of artifi-
cial lakes, polluted rivers, etc.) causes quality and quantity changes in algal associations. In most cases, 
the species diversity lowers and consequently, the numerousness of species, which are tolerant to 
changes in ecosystems, increases. In the case of a permanent change of ecosystems ( desiccated swamps 
and peat bogs, etc.), the algal species in such areas could be !ost forever (Kosi & VRHOVŠEK 1996). 
We investigated algae in some specific biotopes of Slovenia, i.e. the biotopes with low pH, high 
temperature, high or low conductivity, low light, high velocity of water flow, low humidity, the envi-
ronments rich in food, etc. Our purpose was to establish the species structure and the abundance of 
species and subspecies in those biotopes. With regard to special ecological conditions, particular 
attention was given to the species and subspecies present only in specific biotopes and to those exist-
ing in various biotopes. The study was carried out in biotopes of Slovenia that have been investigat-
ed only partly or never before: eutrophic lakes (Koseški bajer, Sotelsko jezero), peat bogs (Šijec, 
Lovrenška jezera), springs (spring at Medvedje Brdo, spring on Pohorje, hot spring at Terme Čatež, 
mineral spring at Rimski vrelec), waterfalls (Savica, Krka falls, small waterfall on Pohorje), aerial 
biotopes (concrete wall, stony wall, !ime tree trunk (Tilia platyphyllos), limestone rock, Krška jama 
cave), the Dragonja River, brackish lake at Fiesa, manure water and two constructed wetlands (Barje, 
A. Krivograd Klemenčič. D. Vrhovšek. N. Smolar-Žvanut and G. Kosi: Algae of... 33 
Dragonja). Altogether, 26 sampling sites were investigated. For description of sampling sites, see 
KRIVOGRAD KLEMENČIČ (2001). 
Materials and Methods 
Samples were taken seasonally in 1998, 1999, 2000 and 2001. For the dates of sampling, see 
KRIVOGRAD KLEMENČIČ (2001). In lakes, peat bogs, springs, waterfalls and in the Dragonja River, the 
samples of periphyton for qualitative analysis were brushed from the surface of stones, rocks, wood, 
macrophytes and other sunk objects like bottles, plastic and iron objects, etc. In peat bogs, the Krka 
falls, the spring at Medvedje Brdo and the small waterfall on Pohorje, the samples of periphyton for 
qualitative analysis were also squeezed out of water mosses. In the mineral spring at Rimski vrelec 
and the hot spring at Terme Čatež, the samples of periphyton were brushed from the bottom and the 
walls of the fountains. 
Terrestrial algae were brushed from a limestone rock, a concrete wall, a stony wall and a lime 
tree trunk. In the Krška jama cave, algae were brushed from the rock bellow the light, which was 
most distant from the cave entrance. The samples of manure water were taken on the grassland above 
the manure pit. The samples of the Barje constructed wetland were taken from the collecting pool. 
The samples of phytoplankton were taken also in lakes (Koseški Bajer, Sotelsko jezero, Fiesa lake), 
peat bogs (Lovrenška jezera, Šijec) and in the Dragonja estuary. 
The samples were immediately bottled and preserved in a four per cent solution of formalde-
hyde, except the samples of manure water and the samples from both constructed wetlands, which 
were first examined and then preserved. All samples were treated by concentrated HN03 to deter-
mine the species from the Bacillariophyceae class. 
We determined the species and subspecies of algae using a light microscope and according to the 
following determination keys: LAZAR (1960), BOURRELLY (1966, 1968, 1970), STARMACH (1966, 
1968, 1972, 1974, 1977, 1980, 1983), GOLUBIC (1967), KRAMMER & LANGE-BERTALOT (1986, 1988, 
1991a, 1991b), POPOVSKY & PFIESTER (1990), HiNDAK (1978, 1996), CVIJAN & BLAŽENČIC (1996), 
LENZENWEGER (1996, 1997). Abundance was estimated by numbers from 1 to 5 (1-single, 2-rare, 3-
customary, 4-frequent, 5-dominant). Similarity in species structure and the abundance of algae were 
determined by the Bray-Curtis coefficient of similarity. 
Results and Discussion 
Altogether, 537 species and subspecies of algae ( of nine classes) were determined. Most of them 
(295) belonged to Bacillariophyceae, 116 belonged to Cyanophyceae, 58 to Chlorophyceae, 44 to 
Zygnematophyceae, eight to Xanthophyceae, six to Dinophyceae, five to Euglenophyceae, three to 
Chrysophyceae and two to Florideophyceae. For algal species lists with the estimation of abundance 
for individual sampling sites, see KRIVOGRAD KLEMENČIČ (2001). 
Figure 1 shows the algal structure by classes at all 26 sampling sites. In the eutrophic lakes of 
Koseški bajer and Sotelsko jezero, the Savica waterfall, the Krka falls, the small waterfall on 
Pohorje, the spring at Medvedje Brdo, the mineral spring at Rimski vrelec, the peat bog at Lovrenška 
jezera, the Krška jama cave, the Fiesa brackish lake and the Dragonja River, most species and sub-
species belonged to Bacillariophyceae, while in the Šijec peat bog those belonging to 
34 
"' Q) ·u 
Q) 
Q. 
"' .o
:, 
"' 
" C 
"' lil 
" ·u .. 
Q. 
"' o 
~ 
.o 
E :, 
C 
Figure 1: Alga! structure by classis at ali sampling sites 
Slika 1: Sestava alg po razredih na vseh vzorčnih mestih 
Acta Biologica Slovenica, 47 (!), 2004 
1 
•:: • Cyanophyceae 
• Florideophyceae 
!'il Dinophyceae 
lil! Xanthophyceae 
m Chrysophyceae 
• Bacillariophyceae 
l1l Euglenophyceae 
m Chlorophyceae 
cea 
Zygnematophyceae were predominant. In the spring on Pohorje, most species and subspecies 
belonged to Chlorophyceae and in the hot spring to Cyanophyceae. Cyanophyceae was predominant 
also in manure water, the Dragonja constructed wetland and in aerial biotopes (concrete wall, trunk 
of the lime tree). In the other two aerial biotopes (stony wall, limestone rock), most species and sub-
species belonged to Bacillariophyceae. 
146 new species and subspecies in Slovenia were determined, of these 107 belonged to 
Bacillariophyceae, 28 to Cyanophyceae, six to Chlorophyceae, four to Zygnematophyceae and one 
to Xanthophyceae (Table 1). 
Table 1: List of species and subspecies new to Slovenia 
Tabela 1: Seznam vrst in podvrst novih za Slovenijo 
A. Krivoerad Klemenčič . D. Vrhovšek. N. Smolar-Žvanut and G. Kosi: Algae of ... 
taxon 
CYANOPHYTA 
CYANOPHYCEAE 
Anabaena affinis Lemm. 
Borzia trilocularis Cohn 
Calothrix thermalis (Schwabe) Hansg. 
Gloeocapsa bituminosa (Bory) Kuetz. 
Lyngbya cryptovaginata Schkorbatoff 
Lyngbya hieronymusii Lemm. 
Lyngbya perelegans Lemm. 
Microcystis hansgirgiana (Hansgirg) Elenkin 
Microcystis viridis (Braun) Lemm. 
Nostoc spongiaeforme Agardh 
Oscillatoria laetevirens (Crouan) Gomont 
Phormidium angustissimum W. & G.S. West 
Phormidium dimorphum Lemm. 
Phormidium henningsii Lemm. 
Phormidium lignicola Fremy 
Phormidium rotheanum Itzig. 
Phormidium setchelianum Gomont 
Phormidium valderiae (Delp.) Geitler 
Plectonema terebrans Bomet & Flahault 
Pseudanabaena papillaterminata (Kisselev) Kukk 
Pseudospirulina amoena Pankow & Jahnke 
Schizothrixfriesii (Agardh) Gomont 
Spirulina jlavovirens Wislouch 
Spirulina gomontiana (Setchell) Geitler 
Spirulina meneghiniana Zanar. 
Spirulina tenuissima Kuetz. 
Synechocystis septentrionalis Skuja 
Tolypothrix cucullata Jaag 
HETEROKONTOPHYTA 
XANTHOPHYCEAE 
Characiopsis minima Pasch. 
BACILLARIOPHYCEAE 
Achnanthes amoena Hust. 
Achnanthes catenata Bily in Marvan 
Achnanthes lanceolata ssp. dubia (Grun.) Lan.-Bert. 
Achnanthes lanceolata ssp.frequentissima Lan.-Bert. 
Achnanthes oblongella Oestrup 
Achnanthes septata A. Cleve 
Amphora angusta (Greg.) Cleve 
Bacillaria paradoxa Gnalin 
Caloneis molaris (Grun.) Kramm. 
Cymatopleura solea var. apiculata (W.Smith) Ralfs 
Cymbella caespitosa (Kuetz.) Brun 
Cymbella cuspidata Kuetz. 
Cymbella descripta (Hust.) Kramm. & Lan.-B. 
Cymbella gaeumannii Meister 
Cymbella pusilla Grun. 
Cymbella tumidula var. lancettula Kramm. 
35 
36 
Denticula kuetzingii Grun. 
Denticula subtilis Grun. 
Diatoma ehrenbergii Kuetz. 
Diatoma moniliformis Kuetz. 
Epithemia turgida var. granulata (Ehr.) Brun 
Eunotia circumborealis Noerpel in Lan.-Bert. 
Eunotia denticulata (Breb.) Raben. 
Eunotia microcephala Krass. 
Eunotia paludosa Grun. 
Fragilaria biceps (Kuetz.) Lan.-Bert. 
Fragilaria capucina var.mesolepta (Rab.) Rab 
Fragilaria montana (Krass.) Lan.-Bert. 
Fragilaria parasitica var. subconstricta Grun. 
Frustulia spicula Amosse 
Gomphonema amoenum Lan.-Bert. 
Gomphonema clevei Hust. 
Gyrosigma nodiferum (Grun.) Reimer 
Gyrosigma tenuissimum (W.Smith) Cleve 
Gyrosigma wansbeckii (Dankin) Cleve 
Melosira moniliformis (Muell.) Agardh 
Melosira nummuloides (Dillwyn) Agardh 
Navicula accomoda Hust. 
Navicula aerophila Krass. 
Navicula angusta Grun. 
Navicula bryophila Pet. 
Navicula capitata var. capitata Ehren. 
Navicula cincta (Ehren.) Ralfs & Prit. 
Navicula duerrenbergiana Hust. 
Navicula erifuga Lan.-Bert. 
Navicula goeppertiana (Bleisch) H.L. Smith 
Navicula gregaria Donkin 
Navicula harderii Hustedt 
Navicula heufleriana (Grun.) Cleve 
Navicula incertata Lan.-Bert. 
Navicula integra (W.Smith) Ralfs 
Navicula libonensis Schoeman 
Navicula margalithii Lan.-Bert. 
Navicula menisculus var. upsaliensis Grun. 
Navicula mutica var. ventricosa Cleve & Grun. 
Navicula nivalis Ehren. 
Navicula oppugnata Hust. 
Navicula pseudokotschyi Lan.-Bert. 
Navicula recens Lan.-Bert. 
Navicula salinarum Grun. 
Navicula schroeterii Meister 
Navicula subhamulata Grun. & Van Heurck 
Navicula suecorum var. dismutica (Hust.) Lan.-Bert 
Navicula viridula var. linearis Hust. 
Navicula viridula var. rostellata (Kuetz.) Cleve 
Navicula vitiosa Schiman. 
Neidium bisulcatum (Lager.) C!eve 
Neidium ladogensis (Cleve) Foged 
Nitzschia angustatula Lan.-Bert. 
Nitzschia calida Grun. 
Acta Biologica Slovenica, 47 (1), 2004 
A. Krivograd Klemenčič, D. Vrhovšek, N. Smolar-Žvanut and G. Kosi: Algae of... 
Nitzschia capitellata Hust. 
Nitzschia commutatoides Lan.-Bert. 
Nitzschia compressa var. compressa (Bailey) Boyer 
Nitzschia constricta (Kuetz.) Ralfs 
Nitzschia dissipata var. media (Hant.) Grun. 
Nitzschiafiliformis var. conferta (Rich.) Lan.-Bert. 
Nitzschia flexa Schum. 
Nitzschia granulata Grun. 
Nitzschia levidensis var. salinarum Grun. 
Nitzschia linearis var. subtilis (Grun.) Hust. 
Nitzschia linearis var. tenuis (W.Smith) Grun. 
Nitzschia littoralis Grun. 
Nitzschia longissima var. genuina A.Cleve 
Nitzschia lorenziana Grun. 
Nitzschia navicularis (Breb.) Grun. 
Nitzschia perspicua Choln. 
Nitzschia recta var. robusta Hust. 
Nitzschia scalpelliformis Grun. 
Nitzschia sigma (Kuetz.) W.Smith 
Nitzschia sinuata var. delognei (Grun.) Lan.-Bert. 
Nitzschia sinuata var. tabellaria (Grun.) Grun. 
Nitzschia sociabilis Hust. 
Nitzschia vermicularis (Kuetz.) Hant. 
Nitzschia wuellerstorffii Lan.-Bert. 
Pinnularia divergens W. Smith 
Pinnularia microstauron var. brebissonii (Kuetz.) May. 
Pinnularia rupestris Ham. 
Pinnularia subcapitata var. hilseana ( Jan.) Muell. 
Pinnularia sudetica (Hilse) Peragallo 
Pleurosigma salinarum Grun. 
Pleurosigma strigosum W.Smith 
Rhizosolenia eriensis H.L.Smith 
Rhopalodia brebissonii Krammer 
Rhopalodia constricta (W.Smith) Kramm. 
Surirella brebissonii Kramm. & Lan.-Bert. 
Surirella constricta W.Smith 
Surirella striatula Turpin 
CHLOROPHYTA 
CHLOROPHYCEAE 
Elakatothrix biplex (Nyg.) Hindak 
Elakatothrix spirochroma (Rev.) Hindak 
Gloeobotrys monochloron Ettl. 
Koliella crassa Hindak 
Koliella variabilis (Nyg.) Hindak 
Scenedesmus velitaris Kom. 
ZYGNEMATOPHYCEAE 
Cosmarium pseudamoenum Wille 
Spondylosium pulchellum Arch. 
Spondylosium tetragonum W. West 
Staurastrum chaetoceras (Schr.) Smith 
37 
38 Acta Biologica Slovenica, 47 (1 ), 2004 
The appearance of almost half of ali determined species and subspecies (248) was limited to a 
single sampling point. Eight species were determined in more than a half of ali specific biotopes 
(Table 2). The Phormidium sp. species was most frequent, present in eutrophic lakes, waterfalls, 
springs, brackish waters, the Dragonja River, aerial biotopes and hypereutrophic biotopes. The 
Achnanthes minutissima species was present in eutrophic lakes, peat bogs, waterfalls, springs, hot 
spring, brackish waters, the Dragonja River and in aerial biotopes. The Navicula veneta species was 
present in eutrophic lakes, waterfalls, springs, brackish waters, the Dragonja River, aerial biotopes 
and hypereutrophic biotopes. The Trentepohlia aurea species was present in peat bogs, waterfalls, 
springs, a hot spring, the Dragonja River, aerial biotopes, Krška jama cave and hypereutrophic 
biotopes. The Cymbella silesiaca species was present in eutrophic lakes, peat bogs, waterfalls, 
springs, brackish waters, the Dragonja River and hypereutrophic biotopes. The Gomphonema angus-
tum species was present in eutrophic lakes, peat bogs, waterfalls, springs, brackish waters, the 
Dragonja River and aerial biotopes. The Navicula sp. species was present in eutrophic lakes, water-
falls, springs, brackish waters, the Dragonja River, aerial biotopes and hipereutrophic biotopes. The 
Cymbella a.ffinis species was present in eutrophic lakes, peat bogs, falls, springs, brackish waters, the 
Dragonja River and aerial biotopes. 
Table 2: List of alga! species determined in more than half of ali specific biotopes 
Tabela 2: Seznam vrst prisotnih v več kot polovici vseh posebnih biotopov 
taxon sampling sites 
'Phormidium sp. Koseški bajer, Sotelsko jezero, waterfall Savica, Krka falls, small waterfall 
on Pohorje, spring on Medvedje Brdo, spring on Pohorje, mineral spring 
Rimski vrelec, brackish lake Fiesa, river Dragonja, limestone rock, manure 
water, constructed wetland Dragonja 
Achnanthes minutissima Koseški bajer, Sotelsko jezero, peat bog Lovrenška jezera, waterfall Savica, 
Krka falls, small waterfall on Pohorje, spring on Medvedje Brdo, hot spring, 
mineral spring Rimski vrelec, brackish lake Fiesa, river Dragonja, limestone 
rock 
Navicula veneta Koseški bajer, Sotelsko jezero, waterfall Savica, Krka falls, small waterfall 
on Pohorje, spring on Medvedje Brdo, mineral spring Rimski vrelec, 
brackish lake Fiesa, river Dragonja, limestone rock, constructed wetland 
Dragonja 
Trentepohlia aurea peat bog Lovrenška jezera, waterfall Savica, small waterfall on Pohorje, 
spring on Medvedje Brdo, spring on Pohorje, hot spring, mineral spring 
Rimski vrelec, river Dragonja, stony wall, limestone rock, cave Krška jama, 
manure water, constructed wetland Dragonja 
Cymbella silesiaca Koseški bajer, Sotelsko jezero, peat bog Lovrenška jezera, waterfall Savica, 
Krka falls, small waterfall on Pohorje, spring on Medvedje Brdo, brackish 
lake Fiesa, river Dragonja, constructed wetland Barje 
Gomphonema angustum Koseški bajer, Sotelsko jezero, peat bog Lovrenška jezera, waterfall Savica, 
Krka falls, small waterfall on Pohorje, spring on Medvedje Brdo, brackish 
lake Fiesa, river Dragonja, limestone rock 
Navicula sp. Koseški bajer, Sotelsko jezero, waterfall Savica, Krka falls , small waterfall 
on Pohorje, spring on Medvedje Brdo, brackish lake Fiesa, river Dragonja, 
concrete wall, stony wall, manure water, constructed wetland Barje 
Cymbella affinis Koseški bajer, Lovrenška jezera, waterfall Savica, small waterfall on 
Pohorje, mineral spring Rimski vrelec, brackish lake Fiesa, river Dragonja, 
ston wall 
A. Krivograd Klemenčič, D. Vrhovšek, N. Smolar-Žvanut and G. Kosi: Algae of... 39 
Bray-Curtis Coefficient of Similarity 
The Bray-Curtis coefficient showed no significant similarity in sampling sites with high water 
velocity (Fig. 2). The Krka falls were more similar to the Sotelsko jezero and to the Koseški bajer 
than to the Savica waterfall. In the Dragonja River the differences depended more on the season than 
on the water velocity. Therefore, the velocity does not significantly influence the species structure 
and the numerousness of algal association. Regarding the eutrophic waters, there is a similarity 
between the Sotelsko jezero, the Koseški bajer and the Krka River, while there is a very low similar-
ity between them and the extreme eutrophic sampling sites (manure water and constructed wetlands), 
as only two common species were found there, i.e. Cymbella silesiaca and Navicula veneta. 
Similarity was determined between the brackish sampling points: the Fiesa lake and the Dragonja 
estuary. High influence of geological structure was also determined. All Dragonja River sampling 
sites (except the Dragonja estuary) were similar. Because of the limestone ground there was also a 
similarity between the Medvedje Brdo spring and the Savica waterfall. A higher similarity was 
expected between the spring and the small waterfall on Pohorje, but in the spring samples some ter-
restrial species (Navicula contenta, Klebsormidium flaccidum, Trentepholia aurea) were found. 
Thus, the results showed that the spring on Pohorje was more similar to the terrestrial sampling sites, 
like the limestone rock and the Krška jama cave. There was a high similarity between the terrestrial 
sampling sites: the lime tree trunk, concrete wall and stony wall where limitation factors were the 
absence of food and humidity. High water temperature was the major limiting factor for species 
structure and the numerousness of algal association in the Terme Čatež hot spring. 
0,9 0,8 0,7 0,6 0,5 0,4 
.n 
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Sotelsko jezero.10.8.1999 
Sotelsko iezero.20. 1 O. 1999 
Sotelsko Jezero 26.9.98 
Sotelsko jezero 9.5.99 
Sotelsko jezero 22.2.2000 
slapovi na Krki 13.2.2000 
slapovi na Krki 20.9.98 
slapovi na Krki 27.8.1999 
slapovi na Krki 23.10.1999 
slapovi na Krki 24.4.99 
Koseški bajer 1.8.1999 
Koseški bajer 4.5.99 
Koseški bajer 17.10.1999 
Koseški bajer 7.8.98 
Koseški baJer 19.2.2000 
Fiesa 29.7.1999 
Fiesa 7.4.99 
Fiesa 17.1.2000 
Fiesa 18. 1 o. 1999 
Fiesa 23.8.98 
Dragonja izliv 29.7.1999 
Dragon1a izliv 23.8.98 
Drago~a izliv 18.10.1999 
Dragonja izliv 17.1.2000 
Dragonja izliv 20.3.99 
Dragonja tolmun 29.7.1999 
DragonJa počasi t. 29.7.1999 
Dragonja Qrzica 29.7.1999 
Dragonja Skrline 18.7.98 
Dragonja tolmun 10.7.98 
Dragonja počasi t. 10.7.98 
Dragonja brzica 10.7.98 
Dragonja tolmun 20.3.99 
8~:~~~J: g~~!iJo~~gi99 
Drago11a Škrline 20.3.99 
Dragonja iočasi t. 17.1.2000 
Dragonia rzica 17.1.2000 
Dragonja krline 17.1.2000 
DragonJa tolmun 17.1.2000 
Dragonja tolmun 18.1 O. 1999 
Dragonja brzica 18.1 O. 1999 
g~;g~~J: ~:;~ 1d. ~: ~ g;g999 
Dragonja Skrline 18. 1 O. 1999 
Dragonja izvir 17.1.2000 
DragonJa izvir 18. 7.98 
Dragonja izvir 29.7.1999 
Dragonja izvir 20.3.99 
Dragonja izvir 18. 1 O. 1999 
sa,,,.;ca 22.8.1999 
Savica 25.3.2000 
Savica 8.8.98 
Savica 23.5.99 
sa,,,.;ca 2.11.1999 
izvir Medvedje B. 6.8.1999 
izvir Medvedje B. 2.4.99 
izvir Medvedje B. 17.10.1999 
izvir Medvedje B. 19.2.2000 
slap na Pohorju 7.8.1999 
slap na Pohorju 25.7.98 
slap na Pohorju 16. 1 O. 1999 
slap na Pohorju 3.6.00 
izvir Rimski v. 12.8.1999 
izvir Rimski v. 20. 1 O. 1999 
izvir Rimski v. 25.2.2000 
izvir Rimski v. 14.5.00 
!.Lovrenška j. 16.10.1999 
tl.Lovrenškaj.16.10.1999 
III.Lovrenškaj. 7.8.1999 
I.Lovrenškaj. 7.8.1999 
II.Lovrenškaj. 7.8.1999 
III.Lovrenškaj. 16.10.1999 
!.Lovrenška j. 3.6.2000 
III.Lovrenška j. 3.6.2000 
It.Lovrenška j. 3.6.2000 
!.Lovrenška j. 25.7.1998 
HI.Lovrenška j. 25.7.1998 
·. 25.7.1998 
22.8.1999 
2.11.1999 
15.5.99 
21.5.00 
11.10.98 
apnenčasta skala 23.10.99 
apnenčasta skala 22.2.00 
apnenčasta skata 9.5.00 
apnenčasta skala 24.4.99 
apnenčasta skala 15.8.99 
Krška jama 12.9.99 
Krška jama 13.2.00 
Krška jama 1.7.00 
Krška jama 20.9.98 
izvir na Pohorju 7.8.1999 
izvir na Pohorju 3.6.00 
izvir na Pohorju 16. 1 O. 1999 
deblo lipe 5.9.99 
deblo lipe 28.2.00 
deblo lipe 29.5.00 
deblo lipe 3.5.99 
betonski zid 20.5.00 
betonski zid 4.5.99 
betonski zid 5.9.99 
betonski zid 21.2.00 
kamniti zid 24.4.99 
kamniti zid 12.9.99 
kamniti zid 13.2.00 
kamniti zid 1.7.00 
termalni izvir 28.3.2000 
termalni izvir 16.9.00 
termalni izvir 15.1.2001 
termalni izvir 20.11.00 
gnojevka 3.11.00 
gnoJevka 22.1.01 
gnojevka 14.5.00 
gnojevka 10.8.00 
RCN Dragonja 25.11.00 
ACN Dragonja 28.1.01 
RCN Dragonja 25.7.00 
RCN Barje 12.4.00 
RCN BarJe 16.8.00 
RCN Barje 20.11.00 
RCN Barje 15.1.01 
A. Krivograd Klemenčič, D. Vrhovšek. N. Smolar-Žvanut and G. Kosi: Aleae of... 
Povzetek 
41 
V nalogi smo raziskovali alge nekaterih posebnih biotopov Slovenije v različnih letnih časih od 
leta 1998 do 2001 z namenom ugotoviti kvalitativno vrstno sestavo algnih združb in pogostost 
pojavljanja posameznih vrst in podvrst alg v določenih biotopih s posebnimi okoljskimi dejavniki 
(nizek pH, visoka temperatura, visoka ali nizka elektroprevodnost, pomanjkanje svetlobe, visoka 
hitrost vodnega toka, pomanjkanje vlage, visoka vsebnost hranil itd.). Zanimale so nas predvsem 
vrste, ki so prisotne samo v posameznih posebnih biotopih in vrste, ki glede na posebne ekološke 
pogoje lahko nastopajo v večih posebnih biotopih hkrati. Omejili smo se na sladkovodne, brakične 
in kopenske biotope, ki dosedaj še niso bili ali pa so bili le delno raziskani. Alge smo določevali v 
evtrofnih jezerih (Koseški bajer, Sotelsko jezero), visokih barjih (barje Šijec, Lovrenška jezera), 
izvirih (kraški izvir na Medvedjem Brdu, izvir na Pohorju, termalni izvir v Termah Čatež, mineral-
ni izvir Rimski vrelec), slapovih (lehnjakovi slapovi na reki Krki, slapič na Pohorju, slap Savica), 
raziskovali smo kopenske alge na apnenčasti skali, betonskem zidu, deblu lipe (Tilia platyphyllos) in 
v Krški jami ter alge v flišni reki Dragonji, brakičnem jezeru v Fiesi in hiperevtrofnih biotopih (gno-
jevki in rastlinskih čistilnih napravah na deponijah Barje in Dragonja). Z Bray - Curtisovim koefi-
cientom podobnosti smo ugotavljali podobnost v vrstni sestavi in pogostosti pojavljanja alg med 
posameznimi vzorčnimi mesti. 
Skupno smo na šestindvajsetih vzorčnih mestih določili 537 različnih vrst in podvrst alg iz 
devetih razredov. 295 vrst in podvrst pripada razredu Bacillariophyceae, 116 razredu Cyanophyceae, 
58 razredu Chlorophyceae, 44 razredu Zygnematophyceae, 8 razredu Xanthophyceae, 6 razredu 
Dinophyceae, 5 razredu Euglenophyceae, 3 razredu Chrysophyceae in 2 razredu Florideophyceae. 
146 določenih vrst in podvrst je novih za Slovenijo, od tega pripada 107 vrst in podvrst razredu 
kremenastih alg, 28 razredu Cyanophyceae, 6 razredu Chlorophyceae, 4 razredu Zygnematophyceae 
in 1 razredu Xanthophyceae. 
Pojavljanje skoraj polovice vseh določenih vrst in podvrst (248) je bilo omejeno na posamezna 
vzorčna mesta. Vrste Achnanthes minutissima, Cymbella affinis, Cymbella silesiaca, Gomphonema 
angustum, Navicula veneta, Navicula sp., Phormidium sp. in Trentepohlia aurea so splošno 
razširjene vrste, prisotne so bile v večini posebnih biotopov. 
Primerjava vrstnih sestavov algnih združb in pogostosti pojavljanja posameznih vrst in podvrst 
med vzorčnimi mesti z Bray - Curtisovim koeficientom podobnosti je pokazala, da si vzorčna mesta, 
kjer je bila hitrost vodnega toka velika, med seboj niso bila preveč podobna. Lehnjakovi slapovi na 
reki Krki so bili bolj podobni Sotelskemu jezeru in Kqseškemu bajerju kot slapu Savici in slapiču na 
Pohorju. Bray - Curtisov koeficient je pokazal med vzorčnimi mesti na reki Dragonji, ki se v 
glavnem razlikujejo le v hitrosti vodnega toka, večjo sezonsko kot krajevno odvisnost. Iz zgoraj 
naštetega lahko sklepamo, da hitrost vodnega toka ni bistveno vplivala na vrstno sestavo in 
številčnost algnih združb. Evtrofne vode: Sotelsko jezero, Koseški bajer in reka Krka so si bile med 
seboj podobne, medtem ko je bila njihova podobnost z ekstremno evtrofnimi vodami (gnojevko in 
RČN) izredno majhna, saj med njimi razen dveh (Cymbella silesiaca in Navicula veneta) ni bilo 
skupnih vrst. Med seboj sta si bili podobni tudi obe brakični vodi: priobalno jezero v Fiesi in izliv 
reke Dragonje. Na vrstno sestavo in številčnost algnih združb ima velik vpliv geološka podlaga. Vsa 
vzorčna mesta na flišni reki Dragonji (razen izliva) so si bila med seboj podobna. Podobna sta si bila 
tudi izvir na Medvedjem Brdu in slap Savica, oba ležita na apnenčasti podlagi. Pričakovali bi večjo 
podobnost med izvirom in slapičem na Pohorju, vendar smo v izviru na Pohorju določili nekaj 
kopenskih vrst alg (Navicula contenta, Klebsormidiumflaccidum, Trentepohlia aurea), zaradi česar 
42 Acta Bioiooica Slovenica, 47 (1), 2004 
je bil izvir na Pohorju bolj podoben kopenskima vzorčnima mestoma: apnenčasti skali in Krški jami. 
Precejšnja podobnost je bila med kopenskimi vzorčnimi mesti - deblo lipe, kamniti in betonski zid, 
kjer sta verjetno glavna omejujoča dejavnika za uspevanje alg pomanjkanje vlage in hranilnih snovi . 
V termalnem izviru v Termah Čatež pa na vrstni sestav in številčnost alg v prvi vrsti vpliva zelo viso-
ka temperatura izvirne vode. 
References 
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BOURRELLY P. 1968: Les algues d 'eau douce. Initiation a la systematique. Tome II. Les algues jaunes et 
brunes, Chrysophycees, Pheophycees, Xanthophycees et diatomees. Centre national de la 
recherche scientifique, Paris. 
BOURRELLY P. 1970: Les algues d'eau douce. Initiation a la systematique. Tome III. Les algues bleues et 
rouges les Eugleniens, Peridiniens et Cryptomonadines. Centre national de la recherche scien-
tifique, Paris. 
CVIJAN M., J. BLAŽENČic 1996: Flora algi Srbije. Cyanophyta. Naučna knjiga, Beograd, 290 pp. 
GOLUBIC S. 1967: Die Binnengewlisser. Algenvegetation der Felsen. Eine okologische Algenstudie im dinar-
ischen Karstgebiet. Band XXIII. E. Schweizerbart'sche Verlagsbuchandlung, Stuttgart, 183 pp. 
HINDAK F., P. MARVAN, J. KOMAREK, K. ROSA, J. POPOVSKY, o. LHOTSKY 1978: Sladkovodne riasy. 
Slovenske pedagogicke nakladatelstvo, Bratislava, 724 pp. 
HINDAK F. 1996: Kluč na určovanie nerozkonarenych vlaknitych zelenych rias (Ulotrichineae, 
Ulotrichales, Chlorophyceae). Slovenska botanicka spoločnost pri SAV, Bratislava, 73 pp. 
KOSI G. , D . VRHOVŠEK 1996: Sladkovodne alge. Narava Slovenije, stanje in perspektive. Zbornik 
prispevkov o naravni dediščini Slovenije. Društvo ekologov Slovenije, Ljubljana, pp. 143-146. 
KRAMMER K., H. LANGE-BERTALOT 1986: SiiBwasserflora von Mitteleuropa. Bacillariophyceae. 1 Tei! : 
Naviculaceae. Band 2/1. Fischer, Stuttgart, 876 pp. 
KRAMMER K., H. LANGE-BERTALOT 1988: SiiBwasserflora von Mitteleuropa. Bacillariophyceae. 2 Tei!: 
Bacillariaceae, Epithemiaceae, Surirellaceae. Band 2/2. Fischer, Stuttgart, 596 pp. 
KRAMMER K., H. LANGE-BERTALOT 1991A: SiiBwasserflora von Mitteleuropa. Bacillariophyceae. 3 Teil : 
Centrales, Fragilariaceae, Eunotiaceae. Band 2i3. Fischer, Stuttgart, 576 pp. 
KRAMMER K., H. LANGE-BERTALOT 1991B: SiiBwasserflora von Mitteleuropa. Bacillariophyceae. 4 Tei!: 
Achnanthaceae, Kritische Erglinzungen zu Navžcula (Lineolate) und Gomphonema. Band 2/4. 
Fischer, Stuttgart, 436 pp. 
LAZAR J. 1960: Alge Slovenije. Seznam sladkovodnih vrst in ključ za določanje. SAZU, Ljubljana, 117 pp. 
LENZENWEGER R. 1996: Bibliotheca phycologica. Band 101. Desmidiaceenflora von Osterreich. Tei! l., 
J. Cramer, Berlin-Stuttgart, 162 pp. 
LENZENWEGER R. 1997: Bibliotheca phycologica. Band 102. Desmidiaceenflora von Osterreich. Tei! 2., 
J. Cramer, Berlin-Stuttgart, 216 pp. 
PorovsKY J. , L. A. Pfiester 1990: SiiBwasserflora von Mitteleuropa. Dinophyceae. Band 6. Fischer, 
Stuttgart, 272 pp. 
STARMACH K. 1966: Flora slodkovodna palski . Cyanophyta-Sinice, Glauciphyta-Glaukofity. Tom 2. 
Panstwowe Wydawnictwo Naukowe, Warszawa, 808 pp. 
STARMACH K. 1968: Flora sladkovodna polski. Xanthophyceae - Roznowiciowe. Panstwowe 
Wydawnictwo Naukowe, Warszawa, 393 pp. 
STARMACH K. 1972: Flora sladkovodna polski. Chlorophyta III. Zielenice Nitkovate: Ulotrichales, Ulvales, 
Prasiolales, Sphaeropleales, Cladophorales, Chaetophorales, Trentepohliales, Siphonales, 
Dichotomosiphonales. Tom 2. Panstwowe Wydawnictwo Naukowe, Warszawa, 750 pp. 
A. Krivograd Klemenčič, D. Vrhovšek. N. Smolar-Žvanut and G . Kosi: Algae of ... 43 
STARMACH K. 1974: Flora slodkovodna polski. Cryptophyceae, Dinophyceae, Raphidophyceae. 
Panstwowe Wydawnictwo Naukowe, Warszawa, 517 pp. 
STARMACH K. 1977: F1ora slodkovodna palski. Phaeophyta-Brunatnice, Rhodophyta - Krasnorosty. 
Panstwowe Wydawnictwo Naukowe, Warszawa, 443 pp. 
STARMACH K. 1980: F1ora sladkovodna palski. Chrysophyceae - Zlotowiciowce (oraz zooflagellata wol-
nozyjace). Panstwowe Wydawnictwo Naukowe, Warszawa: 774 pp. 
STARMACH K. 1983: Flora sladkovodna polski. Euglenophyta - Eugleniny. Panstwowe Wydawnictwo 
Naukowe, Warszawa, 593 pp. 
ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2004 Vol. 47, Št. 2: 45-56 
Sprejeto (accepted): 2004-09-17 
Tissue culture of pyrethrum (Tanacetum cinerariifolium 
(Trevir.) Schultz Bip.) 
Tkivna kultura bolhača (Tanacetum cinerariifolium (Trevir.) 
Schultz Bip.) 
Mateja GSPANI, Margareta VRTAČNIKI , Jana AMBROŽIČ DOLINŠEK2*, 
Maja KOVAČ3, Marjana CAMLOH3, Jana ŽEL3 
I Faculty of Science and Engineering, University of Ljubljana, Vegova 4, P.O.B. 
18 / 1, 1000 Ljubljana, Slovenia; tel: +386(0)1 5613552, e-mail: 
mateja.gspan@lek.si, metka.vrtacnik@guest.ames.si 
2 Department ofBiology, University ofMaribor, Koroška 160, 2000 Maribor, 
Slovenia; fax:+386 62 28180, e-mail:jana.ambrozic@uni-mb.si 
('corresponding author) 
3 National Institute of Biology, Večna pot 111, 1000 Ljubljana, Slovenia; 
fax:+ 386(0) 14233388, e-mail: maja.kovac@nib.si, jana.zel@nib.si, 
marjana.camloh@nib.si 
Abstract. Pyrethrum (Tanacetum cinerariifolium), is plant species with the 
highest amount of natural insecticides - pyrethrins. An in-house information sys-
tem for the development of different plant tissue cultures and marketing of their 
products was designed. The application of the relational information system in set-
ting up a research hypothesis of high probability is discussed. By processing the 
information system, plant tissue cultures' parameters were identified, selected and 
modified. They were tested on the plant tissue culture of Tanacetum cinerariifoli-
um. The influence of jasmonic acid on axillary shoot differentiation was studied. 
An inhibitory effect of jasmonic acid on shoot tissue culture differentiation was 
proven (100 µM, 10 µM), and a prediction method for determination of the variable 
optimal concentration interval was presented (between 0,5 and 4,5 µM). In addition 
a HPLC method was introduced for pyrethrins determination. 
Key words: Tanacetum cinerariifolium, pyrethrum, plant tissue culture, shoot 
tissue culture, axillary shoot, natural insecticides, pyrethrins, jasmonic acid, infor-
mation system 
Izvleček. Bolhač (Tanacetum cinerariifolium) je rastlinska vrsta z največjo 
vsebnostjo naravnih insekticidov - piretrinov. Postavili smo informacijski sistem 
za razvoj različnih sistemov tkivnih kultur in trženje njihovih proizvodov. 
46 Acta Biologica S!ovenica, 47 (2), 2004 
Preučevali smo uporabo relacijskega informacijskega sistema za vpeljavo znanstvene hipoteze z 
visoko verjetnostjo. Z njegovo uporabo smo prepoznali, izbrali in priredili parametre sistema tkivnih 
kultur. Kot poskusni sistem smo uporabili tkivno kulturo bolhača in ugotavljali vpliv jasmonske 
kisline (JA) na tvorbo zalistnih poganjkov v tkivni kulturi. Dokazan je bil zaviralni učinek JA na 
diferencijo kulture poganjkov (100 µM, 10 µM) in predstavljena metoda za napoved intervala spre-
minjanja optimalne koncentracije JA (med 0,5 in 4,5 µM) . Uvedli smo metodo HPLC za ugotavljan-
je vsebnosti piretrinov. 
Ključne besede: Tanacetum cinerariifolium, bolhač, rastlinska tkivna kultura, tkivna kultura 
poganjkov, zalistni poganjki, naravni insekticidi, piretrini, jasmonska kislina, informacijski sistem 
Abbreviations: MS = Murashige and Skoog growth medium; JA = jasmonic acid; HPLC = high 
pressure liquid chromatography; IAA = indole-3-acetic acid; 2,4 D = 2,4-diclorophenoxy acetic acid; 
NAA = naphthalene acetic acid; BAP = 6-benzylamino purine; SD = standard deviation; R = corre-
lation index; EPA = Environmental Protection Agency 
Introduction 
About 10 000 million insect species cause foodstuff and wood damage. Some species are even 
fatal, especially for people (ELLIOT 1995). Butan extensive usage of synthetic persistent insecticides 
in the last fifty years has led to their dangerous accumulation in the environment and rapid insect 
resistance development (C0SHRAN 1995). One possible way to minimize the effect of insecticides on 
biological diversity, and at the same tirne to ensure food quality, is the application of biodegradable 
insecticides with low toxicity for Mammals. One of the most promising group of such insecticides 
are pyrethrins (CASIDA & QUISTAD 1995). 
Pyrethrins are natura! stereoisomer mixtures of six monoterpenes esters (Fig. l). The basic compo-
nents of pyrethrins are rethrolone (pyrethrolone, cinerolone and jasmolone) alcohols esterified with 
chrysanthemic monocarboxylic (pyrethrins I) or dicarboxylic/pyrethric (pyrethrins II) acid (TOMLIN 
1994, CROMBIE 1995). Pyrethrins precursor chrysanthemic acid is a volatile monoterpene formed from 
mevalonic acid of isoprenoid metabolism. Chrysanthemyl alcohol with its typical cyclopropane derives 
from two molecules of isopentenyl pyrophosphate (IPP). Oxidation of chrysanthemyl alcohol yields 
chrysanthemic acid. The both have been identified from explant derived callus of pyrethrum. 
Chrysanthemic acid is found not only as the precursor of chrysanthemic dicarboxylic/pyrethric acid and 
pyrethrins I/11 but also the important precursor in squalene synthesis (KEsKITALO 1999). 
R 
~ coo o 
Figure 1: Monoterpenes esters of pyrethrins. 
Slika 1: Monoterpenski estri piretrinov. 
R1 
pyrethrin I 
jasmolin I 
cinerin I 
pyrethrin II 
jasmolin n 
cinerin II 
R CH3, Rl CH=CH2 
R CH3, Rl CH2CH3 
R CH3, Rl CH3 
R C02CH3, Rl CH=CH2 
R C02CH3, Rl CH2CH3 
R C02CH3, Rl CH3 
M. Gspan, M. Vrtačnik, J. Ambrožič Dolinšek, M. Kovač, M. Camloh, J. Žel: Tissue culture of ... 47 
The detailed synthesis of rethrolones stili remains unclear. However the rethrolones, charecter-
ized by the cyclopentene, are derived most properly from fatty acid metabolism. The composition of 
the crude extracts of pyrethrum flowers is as follows: one third is the active constituents pyrethrins 
and two thirds are linoleic and linolenic acids (40 %), alkanes and terpenoides (CR0MBIE 1995, 
KESKITALO 1999). The origin of jasmolone presumably derives from linoleic acid via 12-oxophyto-
dienoic acid (12--oxo PDA). Experimentally is firmly established that 12 oxo-PDA undergoes hydro-
genations and 8--oxidations to form jasmonic acid (CR0MBIE 1995). 
A natura] means for controlling a wide range of insects and rapid insecticidal action by 
pyrethrins is found in the composition of esters mixture. It was also found that a moderate use might 
cause a slow insect resistance development (Coshran 1995). Pyrethrins were found in some species, 
e.g. T. coccineum, but more potent is pyrethrum (T cinerariifolium). From the middle of 19th cen-
tury to World War I, Dalmatia dominated in the pyrethrum production and trade. After the war the 
domination was replaced by Japan, and after World War II by Kenya, Tanzania and Australia 
(WAINAINA 1995, GULLICKSON 1995). There were always constraints on ensuring a sufficient supply 
because of the laborious work, demand for low labour costs, and weather dependency of plant pro-
duction. In recent years, the efficacy of pyrethrins and the unstable supply, which can no longer meet 
the world demand (JOVETIC & DE G0OIJER 1995, ROYAL SOCIETY OF CHEMISTRY 1988, 1996), stimu-
lated interest for their biotechnological production (HITMI & al. 2000). Analysis of patents concem-
ing pyrethrins in the last thirty-five years has shown a distinctive increasing research interest during 
the last decade (Fig. 2). 
30 -~ 
25 
""' 41 20 -.CI 
5 = = 15 "' .... 
= 41 .... 
~ 10 CI. 
5 
O+------------,-----------------< 
1960 1965 1970 1975 1980 1985 1990 1995 2000 2005 
Figure 2: Oscillating pyrethrins research trend in course of tirne and increasing interest in the last decade 
(CAplus, march 2000). 
Slika 2: Nihanje zanimanja za raziskovanje piretrinov v preteklih letih in naraščanje zanimanja za piretrine 
v zadnjem desetletju (CAplus, marec 2000). 
An alternative production of pyrethrins could be in vitro production by plant tissue cultures, the 
main advantage of which is flexible control of biotechnological processes. Therefore plant tissue cul-
48 Acta Biologica Slovenica, 47 (1), 2004 
tm:es are becoming a tool for producing many important products: e.g. quality food, wood, phy-
totherapy compounds, biopesticides etc (SASSON 1992, WALTON & al. 1999, RAVNIKAR & ŽEL 1992). 
The application of the tissue culture technique for the pyrethrum plant has developed also from the 
self-incompatibility of the plant (PAL & DHAR 1985). For the successful commercialisation of a 
biotechnological process the most important criterion must be fulfilled - less expensive in vitro in 
relation to in vivo pyrethrins production (JovETic & DE GooIJER 1995). 
The basic research problem which we tried to solve was, how to optimise tissue culture cultiva-
tion of a commercially potent plant to achieve quality products, e.g. pyrethrins. The problem was 
structured into severa! subproblems: (ELLIOT 1995) careful selection of a substance and its source -
plant, (COSHRAN 1995) selection of economic cultivating conditions, and (CASIDA & QUISTAD 1995) 
target-oriented development of cost-effective products. Fragmented information on the defined sub 
problems is dispersed among severa! bibliographic and factual sources, while the technological 
parameters are most often considered proprietary information and are therefore publicly available 
(HUMPRIES & al. 1991, GULLICKSON 1995). To increase the information content of the accessible and 
relevant data and information, a relational information system for plant tissue cultures development 
was designed. The system was tested on the experiment where the effect of JA on shoot production 
of pyrethrum was studied. 
Methods 
Tissue culture 
Plants of Tanacetum cinerariifolium were obtained from the island of Cres, Croatia. Their seeds 
germinated in vivo and two months old vegetative plantlets of 8 cm height were used in spring for 
establishment of the shoot tissue culture on MS medium (MURASHIGE & SKOOG 1962) supplement-
ed with 3 % sucrose, 1 % agar, 3 µM IAA and 2 µM BAP The pH was adjusted to 5.7 and the medi-
um was sterilized at 121 °C for 20 min. The induced axillary buds had been regulary subcultures, 
every 4 to 5 weeks, for a year. The tissue cultures were grown in a growth chamber at 23 +/- 1 °C 
under 100 - 110 µM m·2s-1 daylight illumination with a 16 h photoperiod. 
Axillary buds were separated and cultivated in 50 ml test-tubes on modified MS medium with 
different concentrations of JA and control medium without JA. In two experiments three JA concen-
trations were tested: 1 µM, 10 µM and 100 µM JA. 
Growth and differentiation measurement 
Two and four weeks after subculture (first experiment) the height of the central shoot and shoots 
number in fifteen test-tubes were determined, and the results were statistically evaluated by Student's 
t-test: * P < 0,05, ** P < 0,01, *** P < 0,001. In the second experiment the procedure was repeated 
after three and five weeks. 
Differentiation prediction 
To fulfil the remaining information gap a prediction method was introduced which wili have to 
be tested in further research. On the basis of ali and the selected average shoots number (data inside 
+/- 2 SD), measured after five weeks of cultivation, the curve with the highest correlation index (R) 
was determined: at concentration intervals between O to 1 O µM JA and between O to 1 µM JA. 
M. Gspan, M. Vrtačnik, J. Ambrožič Dolinšek, M. Kovač, M. Camloh, J. Žel: Tissue culture of... 49 
Isolation and quantification of pyrethrins 
Approximately 1 g of in vitro grown shoots was crushed in a mortar with a silicic sand and ¼ 
anhydrous sodium sulfate to obtain a homogenous powder. Pyrethrins were extracted two to three 
times with 5 ml/g FW petroleum ether (40-60°C). The clear separated extract was evaporated to dry-
ness with rotated evaporator, redissolved in 3 ml CH3CN, filtered through 0,22 µm mesh filter and 
analysed. 
Pyrethrins were analyzed by Waters HPLC system with a diode array (PDA) detector. 
Separations were performed on Nova Pack C18 column (Waters, 150 x 3.9 mm) using a gradi-
ent of CH3CN (solvent A) and Milli Q HzO (solvent B): 40 % solvent A gradually increased to 80 % 
during 25 min, followed by a further 10 min at 80 %. Flow rate was 1.4 ml/min. Absorbance was 
monitored at 225 nm. Pyrethrins in the sample were identified on the basis of retention times and 
characteristic absorption spectra. Standards used were cinerin I and II, pyrethrin I and II andjasmolin 
I and II (Pyrethrins technical mixture, PESTANAL, Riedel-de-Haen). 
Results and discussion 
Relational information system 
The in-house information system supports problem solving through establishing relationships 
among five main modules: (a) bibliography, (b) substances (properties, structures, producers, prod-
ucts), (c) world market, (d) plants and (e) photos (Fig. 3). Processing the relational information sys-
tem for identifying the most effective parameters for biotechnological production of the biosynthet-
ic high-yielding pyrethrum tissue culture done gave the following result (Table 1): 
Table 1: lnformation densities of breeding parameters' values of pyrethrum plant tissue cultures. 
Preglednica 1: Informacijska gostota parametrov vzgoje bolhača v rastlinskih tkivnih kulturah. 
Plant tissue culture 
cultivating 
parameters 
medium modification 
inoculum 
basic medium 
temperature 
irradiation 
mediumpH 
plant no. 
photoperiod 
light quality 
inoculum modification 
storage name 
storage temperature 
lnformation densities 
(number of defined values of 
specific parameter in documents) 
38 
14 
14 
14 
13 
10 
9 
9 
4 
1 
1 
1 
WUIW) 
MA:lKET 
PLANTS 
Figure 3: The structure of five main modules (oval boxes) of the relational information system on plant tissue culture development. 
Slika 3: Struktura petih glavnih modulov (ovalna polja) relacijskega informacijskega sistema za razvoj rastlinskih tkivnih kultur. 
GOVERNMENT 
PUBLICATIONS 
PA'll!NTS 
LABORATORY 
REPORTS 
RF..SEARCH 
ORGANISA TIONS 
OTIIER 
DOCUMENTS-. 
STA'll! 1 
STATE2 
STATE3. 
NATURAL 
SUBSTANCES 
SYNTHETIC 
ANALOGUES 
INPOR1"/EXPORT m 1997 
INPORT/EXPORT m 1998 
INPORT/EXPORT in 1999 
PYRETHRINS 
OTHERS .. 
AU.ETIIRINS 
RESMETHRINS 
PROPERTIES 
after 2 weeeks 
PROPERTJES 
after 3 weeh 
' PROPERTIES 
after 4 weeks 
CLASSIFICA TION 
PHYSICAL/ 
CHEMICAL 
PROPERTIES 
TOXICOLOGY/ 
ECOTOXICOLOGY 
PRODUCERS 
PRODUCER 
1 
ACTIV~ 
INGRADIENTS 
FORMULA TION 
USAGE 
SALE 
PRlCE 
M. Gspan, M. Vrtačnik, J. Ambrožič Dolinšek, M. Kovač, M. Camloh, J. Žel: Tissue culture of... 51 
Based on the information density (Table 1), the basic parameter which promotes plant produc-
tion is medium modification. This result led to setting up a high probability hypothesis which 
assumed that the target-oriented medium modification would have the strongest impact on the 
growth, differentiation and biosynthetic activity of the pyrethrum tissue culture. The information gap 
on medium modification possibilities was bridged by additional processing of the Bibliographic 
module of the Relational information system, which gave examples of medium modifications with 
pyrethrin biosynthesis stimulation effect (Table 2). 
In the literature the following pyrethrum tissue culture's stimulation medium modifications were 
recognised (Table 2): auxins 2,4 D, NAA; cytokinins BA, BAP, kinetin; vitamin ascorbic acid, 
sucrose, diluted basic medium MS and nutrient stress. It is important to stress that, regarding the bib-
liographic search in Chemical Abstracts, jasmonic acid (JA) was not reported as a pyrethrum plant 
growth regulator although it is quite widespread among plants. It is gradually synthesised from 
!inoleic acid, as is suggested for pyrethrins rethrolones (CROMBIE 1995). 
Table 2: Medium modifications with pyrethrins biosynthesis stimulative effect obtained from bibliograph-
ic module of the information system. 
Preglednica 2: Gojišča, ki pospešijo biosintezo piretrinov. Podatki iz bibliografskega modula informaci-
jskega sistema. 
Reference 
HITMI&al., 1998 
HITMI & al., 1997 
DHAR & PAL, 1993 
RAJASEKARAN & al., 1991 
RAVISHANKAR&al., 1989 
RAJASEKARAN & al., 1990 
Medium modification 
2 µM BAP; 21,5 µM NAA 
2 µM BAP; 21,5 µM NAA; ½ MS;l,8 g/1 sncrose 
2,3 µM 2,4 D; 2,2 µM BA 
0,9 µM 2,4 D; 23,2 µM kinetin; MS without agar and nitrates 
0,57 mM ascorbic acid; 0,9 µM 2,4 D; 2,3 µM kinetin 
0,9 µM 2,4 D; 23,2 µM kinetin 
Statistically proven inhibitory effect of jasmonic acid 
After two and four weeks an inhibitory effect of 100 µM JA on pyrethrum tissue culture growth 
and differentiation was observed and statistically proven (Figs. 4, 5). JA clearly inhibited number of 
30 ~--------------·--
25 
.. 20 .. 
..C: e 15 = = ** e 10 = .C: 
5 "' 
o 
OJA 1 JA 10 JA IOOJA 
JA concentrations ( µM) 
Figure 4: The effect of JA on number of shoots after two (light bar) and four (darker bar) weeks in tissue 
culture (first experiment). Average shoot number (n> 10), standard deviation (SD), and statistical significant 
differences (t-test) between control media without JA and with different concentrations of JA are shown. 
Slika 4: Vpliv JA na število poganjkov po dveh (svetel stolpec) in štirih (temen stolpec) tednih tkivne kul-
ture (prvi poskus). Prikazana so povprečna števila poganjkov (n>lO), standardne deviacije (SD) in sta-
tistično značilne razlike (t-test) med kontrolnim gojiščem brez JA in z različnimi koncentracijami JA. 
52 Acta Biolooica Slovenica, 47 (2), 2004 
shoots, especially on the highest JA concentration (Fig. 4), and the same trend was observed in sec-
ond experiment (data not shown). JA also inhibited the early lengthening of shoots. After two weeks 
of culture were shoots on the highest JA concentration significantly smaller then shoots on control 
media without JA (Fig. 5). Differences were not observed after four weeks of culture. 
I 
4,0 
3,5 
;; 3,0 
j 2,5 
g 2,0 
~ 1,5 
"E 1,0 
~ 
0,5 e 
o 
0JA 1 JA I0JA IOO JA 
JA concentrations ( pM) 
Figure 5: The effect of JA on central shoot length after two (light bar) and four (darker bar) weeks in tissue 
culture (first experiment). Average shoot number (n>lO), standard deviation (SD), and statistical significant 
differences (t-test) between control media without JA and with different concentrations of JA are shown. 
Slika 5: Vpliv JA na velikost poganjkov po dveh (svetel stolpec) in štirih (temen stolpec) tednih tkivne kul-
ture (prvi poskus). Prikazana so povprečna števila poganjkov (n>lO), standardne deviacije (SD) in 
statistično značilne razlike (t-test) med kontrolnim gojiščem brez JA in z različnimi koncentracijami JA. 
Prediction of Jasmonic acid stimulative effect 
Based on the results (Figs. 4, 5) it was assumed that a stimulatory JA concentration on pyrethrum tis-
sue culture differentiation may lie at the interval between O to 10 µM. At intervals between O to 10 µM JA 
and O to 1 µM JA the curve that mostly fit both the experimental raw data measured after five weeks of 
cultivation (second experiment) as well as selected data (+/- 2SD) was found (Figs. 6 and 7). According 
to the curves' peaks, which indicate the most copious pyrethrum tissue culture differentiation, the inter-
7 
30 ~-.--~-~--- -...---i--+----1---'--4----i 
o 9 JO 11 
JA concentrations (pM) 
Figure 6: Curves of average values from all ( •) and selected ( • ) <lata show at the interval between O and 
10 µM JA a high dependency (R2 = 0,9429; R2 = 0,818) between specific JA concentration and pyrethrum 
tissue culture differentiation. 
Slika 6: Krivulje povprečnih vrednosti vseh ( •) in izbranih ( •) podatkov kažejo v intervalu med O in 10 µM 
JA veliko odvisnost (R2 = 0,9429; R2 = 0,818) med koncentracijo JA in diferenciacijo bolhača v tkivni kulturi. 
M. Gspan, M. Vrtačnik, J. Ambrožič Dolinšek, M . Kovač, M. Camloh, J. Žel: Tissue culture of... 53 
val of optimal JA concentrations was determined. The same approach could be used in further research 
for prediction of other plant tissue cultures properties: e.g. biomass, growth index, pyrethrins content. 
60 
55 
} 50 
e 
Š 45 
1 40 
35 
30 
o 0,1 0,2 0,3 0,4 0,5 0.6 0.7 0,8 0,9 1, 1 
JA conccntrations ( µM) 
Figure 7: Average values from all ( •) and selected ( •) data show at the interval between O and 1 µM JA com-
plete dependency (R2 = 1) between specific JA concentration and pyrethrum tissue culture differentiation. 
Slika 7: Povprečne vrednosti vseh ( • )in izbranih ( • )podatkov kažejo v intervalu med O in l µM JA popol-
no odvisnost (R2 = 1) med koncentracijo JA in diferenciacijo bolhača v tkivni kulturi . 
Effect of jasmonic acid on pyrethrins content 
In all tested cultures six pyrethrins were detected: cinerin I and II, pyrethrin I and II and jasmolin 
I and II (Fig. 8). Among them pyrethrin I predominated. The total amounts of pyrethrins in the con-
j 
1,80; 
1.10J 
1.00-; 
,.so: 
1,<IO! 
1,301 
1,20j 
1,10-; 
1,orJi 
~ O,'"j 
o,eo;J 
i 
0,70 ] 
o.soj 
o.~j 
0.4 
0,30] 
O,~ 
:::i _,) 
i. i t: . 
ii _ ,.. ___ -- -- -~ --+ · 1 _j_ il ----;J :_ --· -~ 
' 30,oo -sa'.oo 
Figure 8: A representative chromatogram: Content of pyrethrins in pyrethrum shoots tissue culture grown 
four weeks on control medium without JA. 
Slika 8: Reprezentativni kromatogram: Vsebnost piretrinov v poganjkih bolhača po štirih tednih tkivne kul-
ture na kontrolnem gojišču brez JA. 
54 Acta Biologica Slovenica, 47 (2), 2004 
trol and JA treated cultures never exceeded 0.44 % of plant dry weight. These were much lower 
amounts then in flower heads, which contained 0.8 % to 2 % of pyrethrins per dry weight 
(RAJASEKARAN & al. 1990). The pyrethrins I\II ratio, especially important for insecticide activity, was 
0,1 or lower. However in flower heads this ratio is between 0.5 and 3.5 (RAJASEKARAN & al. 1990). 
In preliminary detections of pyrethrin content in shoots tissue cultures of pyrethrum, jasmoinc acid 
did not effect pyrethrin contents. 
The total amount was lower in tissue culture then in flower heads, grown in vivo. Further work 
by different inducers is needed to increase the synthesis of pyrethrins in pyrethrum tissue culture. 
Conclusions 
A systematic selection of economically important pyrethrins produced in tissue culture condi-
tions was outlined. Target-oriented research supported by the in-house information system is a time-
effective contribution to the less expensive in vitro pyrethrin production. 
The specific inhibitory effect of the chosen entry variable jasmonic acid on T. cinerariifolium tis-
sue culture's differentiation was praven. A method for variable optimal value prediction was present-
ed and applications for further research were indicated. 
Povzetek 
Bolhač (Tanacetum cinerariifolium) je rastlinska vrsta z največjo vsebnostjo naravnih insektici-
dov - piretrinov, ki imajo vrsto prednosti pred ostalimi insekticidi, kot sta hitra razgradljivost in 
majhna toksičnost za sesalce (JovETic & DE GooIJER 1995). Danes klasična proizvodnja zaostaja za 
svetovnimi potrebami (GULLICKSON 1995, JovEnc & DE GoorJER 1995), zato se je obnovilo zani-
manje za biotehnološko pridobivanje piretrinov. 
V našem delu smo postavili informacijski sistem za razvoj različnih sistemov tkivnih kultur in 
trženje njihovih proizvodov. Preučevali smo o uporabi relacijskega informacijskega sistema za vpel-
javo znanstvene hipoteze z visoko verjetnostjo. Z uporabo informacijskega sistema smo prepoznali, 
izbrali in priredili parametre sistema tkivnih kulturi. 
Ker do sedaj še ni bilo poročil o vplivu jasmonske kisline (JA) na tkivno kulturo bolhača, smo 
sistem preizkusili na preučevanju vpliva JA na tkivno kulturo poganjkov bolhača. Vzpostavili smo 
tkivno kulturo bolhača in ugotavljali vpliv JA na tvorbo zalistnih poganjkov v tkivni kulturi. Dokazan 
je bil zaviralni učinek JA na diferencijo kulture poganjkov (100 µM, 10 µM) in predstavljena meto-
da za napoved intervala spreminjanja optimalne koncentracije JA (med 0,5 in 4,5 µM). 
Uvedli smo tudi metodo HPLC za ugotavljanje vsebnosti piretrinov. V tkivni kulturi poganjkov 
smo določili vseh šest piretrinov: cinerin I in II, piretrin I in II in jasmolin I in II. Izmed piretrinov 
najbolj prevladuje piretrin I. Vsebnost piretrinov v poganjkih na kontrolnem gojišču in na gojišču z 
JA nikoli ne presega 0,44 % suhe mase, kar je veliko manj, kot je piretrinov v cvetnih glavicah, ki 
vsebujejo od 0,8 % do 2 % piretrinov v suhi masi. 
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ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2004 Vol. 47, Št. 2: 57-64 
Sprejeto ( accepted): 2004-09-17 
UV-B radiation screen and respiratory potential in 
phytoplankton in mountain lakes 
Zaščita pred UV-B sevanjem in dihalni potencial fitoplanktona 
iz visokogorskih jezer 
Mateja GERM 
National Institute of Biology, Večna pot 111, SI-1000 Ljubljana, Slovenia; 
tel: +386(0)14233388, e-mail: mateja.germ@nib.si 
Abstract. UV-B radiation screen and the respiratory potential (terminal elec-
tron transport system - ETS activity) were investigated in phytoplankton from five 
mountain lakes located on an elevation gradient from 1383 m to 2150 m a.s.l.. The 
amount of UV absorbing compounds in phytoplankton was generally higher on the 
lakes from higher elevation. The increased ETS activity of phytoplankton in high-
er lakes was suggested to reflect the energetic cost of generating the intemal mech-
anisms for photoprotection. 
Key words: mountain lakes, UV-B radiation, chlorophyll a, UV absorbing 
compounds, ETS activity, nutrient status 
Abbreviations: DOC - dissolved organic matter, ETS - electron transport sys-
tem, LK - Lake Krn, LKin. - Lake Kriško Inferior, LKSup. - Lake Kriško 
Superior, LL - Lake Ledvica, LP - Lake Planina, UV - ultraviolet, UV AC - UV 
absorbing compounds. 
Izvleček. Ugotavljali smo sposobnost za izgrajevanje UV zaščitnih snovi ter 
dihalni potencial (aktivnost terminalnega elektronskega transportnega sistema -
ETS) fitoplanktona na petih visokogorskih jezerih, ki ležijo na različnih nad-
morskih višinah, od 1383 do 2150 m. Vsebnost UV zaščitnih snovi je bila večja v 
fitoplanktonu iz višje ležečih jezer. Večja aktivnost ETS fitoplanktona iz višje 
ležečih jezer odraža potrebo po energiji, ki jo organizmi rabijo za mehanizme, s 
pomočjo katerih se zaščitijo pred sevanjem. 
Ključne besede: visokogorska jezera, UV-B sevanje, klorofila, UV absorbira-
joče snovi, aktivnost ETS, obremenjenost s hranili 
Okrajšave: DOC - raztopljena organska snov, ETS - elektronski transportni 
sistem, LK - Krnsko jezero, LKln. - Spodnje Kriško jezero, LKSup. - Zgornje 
Kriško jezero, LL - Jezero v Ledvicah, LP - Jezero na Planini pri Jezeru, UV -
ultravijolično, UV AC - UV absorbirajoče snovi. 
58 Acta Biolo2ica Slovenica, 47 (2), 2004 
Introduction 
The increase of UV radiation at the Earth's surface, due to the gradual depletion of ozone in the 
atmosphere (WILLIAMS0N 1995), has caused great concern about the consequent effects on terrestri-
al (BJORN 1999, GABERŠČIK & al. 2001, GABERŠČIK & al. 2002a) and aquatic ecosystems (HADER & 
al. 1998, GERM & al. 2002a,b) . Mountain lakes are potentially vulnerable to climate changes due to 
their high elevation and, usually, their small size. Data on the dependence of intensity of UV-B on 
elevation differ. Increases of UV-B radiation (280-320 nm) are reported to range from 6-8% 
(CALDWELL & al. 1980) to 20% (BLUMTHALER & al. 1993) per 1,000 m of elevation. Higher lakes 
usually contain lower concentrations of DOC which is considered to be the important factor control-
ling the penetration of UV into water (HuovrNEN & al. 2003). Organisms in higher and cleaner lakes 
are therefore exposed to higher UV-B doses, usually without any refuge from damaging solar radia-
tion (WILLIAMSON 1995). UV-B radiation affects many biological and physiological processes in pri-
mary producers (HADER & al. 1998, RozEMA & al. 1997). Experimental studies show that species 
and populations originating from naturally high UV-B locations i.e. from high elevations or low lat-
itudes are less sensitive to UV-B radiation than those from low UV-B locations (SULLIVAN & al. 1992, 
VrLLAFANE & AL. 1999). Protection against direct and indirect influences of UV-B is most important 
in photosynthetic organisms that depend on solar radiation as the primary source of energy in their 
natura! environment (HESSEN & al. 1995). One of the important responses of phytoplankton to UV 
is the synthesis ofUV absorbing compounds, such as mycosporine-like amino acids (MAAs), which 
protect the cells by preventing UV radiation from reaching and damaging vita) molecules such as 
nucleic acids, especially DNA (HADER & al. 1998, BJ6RN 1999, GABERŠČIK & al. 2002a, GERM & al: 
2002a) . There is an increased need for energy during stress (AMTH0R 1995, GERM & GABERŠČIK 
2003) since the establishment of protective mechanisms i.e. synthesis of component materials 
demand additional supplies of energy from the respiratory process (GULM0N & M00NEY 1986). 
In this study we have estimated the respiratory potential, and the ability of phytoplankton from 
mountain lakes Jocated on an elevation gradient, to produce UV screening substances. 
Materials and methods 
The study was carried out during summer 2001 and 2002 in the Julian Alps in NW Slovenia. The 
lakes differ in elevation, trophic status, dimensions and type of activities in the watershed. Their 
characteristics are listed in Table l. 
Chlorophyll (Chl.) a: samples of water were taken with a Van Dorn sampling device (Wildco 
Ltd., USA) from the boat. 0.5 to 3 litres of water at 2.5 m intervals from the lake surface to the bot-
tom at the deepest point of the lakes were collected around noon and immediately filtered through 
Whatman GF/F filters and frozen. The samples were homogenised in 2 ml of extraction solution 
(90% (v/v) acetone) and centrifuged at 8500 g, 4°C, for 4 min in a top refrigerated ultracentrifuge 
(2K15, Sigma, Osterode, Germany) and the absorbance of the supernatant measured. The equation 
of JEFFREY & HuMPHREY (1975) was used to calculate the concentration of eh!. a. 
UV absorbing compounds: the basic procedure used for extracting UV screening substances 
follows the method of CALDWELL (1968). The frozen samples (see above) were homogenised in 5 ml 
extraction solution containing methanol : distilled water : HCl (37% v/v) (79:20: 1 v/v/v), incubated 
for 20 minutes and centrifuged (1600 g, 10°C, 10 min) in a top refrigerated ultracentrifuge. The 
M. Germ: UV-B radiation screen and reseiratorl:'. eotential in .. . 59 
absorbance of the supematants was measured from 280 to 400 nm at intervals of 1 nm. The relative 
amounts of UV absorbing compounds were determined by integrating the values of absorbance and 
normalised to the concentration of chl. a. 
Table 1. Key characteristics of studied lakes. Geographical characteristics are listed according to DOBRAVEC 
AND ŠIŠKO (2002). 
Tabela 1. Osnovne značilnosti preučevanih jezer. Geografske značilnosti so povezete po DOBRAVEC IN ŠIŠKO 
(2002). 
Lake Kriško Sup. Kriško In. Ledvica Planina Krn 
Geogr. position N 46°24'32" N 46°23'59" N 46°20'25" N 46°18'40" N 46°17'09" 
E 13 °48'34" E 13 °48'24" E 13 °47'12" E 13 °49'56" E 13 °41 '08" 
Altitude 2150 1880 1830 1430 1383 
Surface 0.662 0.862 2.187 1.562 4.534 
Depth 9 9 15 II 18 
Tota! P 14.07 11.38 21.24 111.66 19.4 
Total N 1.75 1.83 2.03 2.65 1.86 
chl. a 0.85 0.57 0.37 14.31 6.36 
Temperature 7.5 9.8 6.6 9.0 10.8 
Secchi disc bottom bottom bottom bottom bottom 
Max penet.UV-B 9 8 7 1.5 5 
Trop. state oligotrophic oligotrophic oligotrophic hypereutrophic eutrophic 
Legend: Geogr. position (geographical position), altitude (m), surface (ha), depth (m), total P (total phosphorus, 
µg/1) , total N (lota! nitrogen, mg/1), eh!. a (chlorophyll a, µg/1) , temperature (0 C), Secchi disc (m), max. penet. 
UV-B (maximum penetration of UV-B , m), trop. state (trophic state). 
Legenda: Geogr. position - geografska lega, altitude - nadmorska višina (m), surface - površina (ha), depth -
globina (m), total P - celoten fosfor (µg/1), total N - celoten dušik (mg/!), eh!. a - klorofil a (µg/1), temperature -
temperatura (°C), Secchi disc - Sekijeva plošča (m), max. penet. UV-B - maksimalna globina prodiranja UV-B 
(m), trop. state - trofično stanje. 
Terminal electron transport system activity: the terminal electron transport system (ETS) 
activity of mitochondria was measured as described by PACKARD (1971). Samples of water taken 
from the vertical profile, were filtered through 100 µm mesh size to remove zooplankton, and then 
through Whatman GF/F filters. Samples were homogenised in 4 ml of ice-cold buffer in a mortar fol-
lowed by an ultrasound homogenizer (4710; Cole-Parmer, Vemon Hills, IL, USA) at 40W and cen-
trifuged (8500 g, 2°C, 4 min) in a top refrigerated ultracentiifuge. The 0 .5 ml of supematant was 
mixed with 1.5 ml of substrate solution and 0.5 ml of iodo-nitro-tetrazolium-chloride (INT) solution 
and incubated for 40 minutes at room temperature. During incubation tbe INT was reduced to for-
60 Acta Biologica Slovenica, 47 (2), 2004 
mazan. The absorption of formazan was measured at 490 nm. ETS activity was calculated from the 
rate of INT reduction, which was converted to the equivalent amount of oxygen (KENNER & AHMED 
1975), and normalised to the concentration of eh!. a. 
Chemical analyses: samples of water were taken as stated above. The contents of total phospho-
rus (Valderrama 1981) and total nitrogen (A.P.H.A. 1998) were measured spectrophotometrically. 
Results and discussion 
The trophic levels of the lakes varied with the elevation; the lakes above 1800 m were oligotroph-
ic while those at lower altitudes were eutrophic to hypereutrophic. Average oftotal phosphorus, total 
nitrogen and concentration of eh!. a, as well as temperature in water colurnn were measured in year 
2001 and 2002 (Tab. 1 ). The correlation between ETS activity and concentration of phosphorus was 
r=0.98 (p<0.05), in agreement with del Giorgio (1992). Factors like latitude, altitude, land use, veg-
etation cover and geological composition of watershed can strongly influence the leve! of incident 
radiation and the optical properties of the water column (KARENTZ & al. 1994 ). Clean water ecosys-
tems in high elevation mountains, may receive high doses of UV-B light at significant depths 
(HESSEN & al. 1995). The maximum penetration depth of UV-B was significantly negatively corre-
lated to the content of eh!. a (r=-0.94, p<0.05). The maximum penetration depth of UV-B radiation 
was highest in Lake Kriško Sup., and lowest in Lake Planina. Depth of maximum penetration reflects 
the DOC concentrations and the presence of planktonic organisms (NIELSEN 1996). Hodoki & 
Watanabe (1998) also proved, that attenuation coefficient for UV-A (320-400 nm) and UV-B (280-
320 nm) was highly correlated with the concentration of eh!. a. The amount of eh!. a in lake water 
was highest in the hypereutrophic Lake Planina (Tab. 1). The highest concentration of eh!. a was 
observed to be close to the bottom in most ofthe lakes. This has been reported for other high-moun-
tain lakes (SoMMARUGA & GARCIA-PICHEL 1999). T6TH & al. (1995) suggested that the photosynthet-
ically most active zone had been forced deeper in the lake due to UV-B. 
SOMMARUGA & GARCIA-PICHEL (1999) reported about the maximum amount of uv absorbing 
compounds (normalised to the concentration of eh!. a) close to the bottom. That was not the case in 
all studied lakes (Fig. 1). The bioaccumulation of UV absorbing compounds in copepods has been 
suggested to be the reason for the observed gradient (SOMMARUGA & GARCIA-PICHEL 1999). 
VrLLAFANE & al. (1999) also observed a high rate of bioaccumulation of these compounds in the food 
web. The possible reason for the concentration of UV absorbing compounds reaching a maximum 
near the bottom was probably the sedimentation of phytoplankton, coupled with the fact that UV 
absorbing compounds were chemically more stable than eh!. a. Phytoplankton from transparent, high 
elevation and oligotrophic lakes generally contained more UV absorbing compounds than specimens 
from lower elevation lakes (Fig. 1 ). These results showed that the production of UV absorbing com-
pounds in primary producers in the high elevation lakes was likely a response to the higher flux of 
UV-B. The frequently observed correlation between UV-B radiation and the concentration of UV 
absorbing compounds in many primary producers shows that these substances protect vulnerable tar-
gets in organisms (SOMMARUGA & GARCIA-PICHEL 1999, GABERŠČIK & AL. 2002A, GERM & AL. 
2002A, WINKEL-SHIRLEY 2002). 
M. Germ: UV-8 radiation screen and respiratory potential in .. . 
8 
„ 7 
13 
C) 
:i 
8 5 
j 4 
·1: 
~ 3 
ai 
6 2 
u 
<( 
5 
LK LP LL 
Lake 
61 
LKln. LKSup. 
Fig. l. Amount of UV absorbing compounds (normalised) in phytoplankton on the lake surface (white bars), 
5 m deep (light grey bars), and bottom (dark grey bars) (n=3, mean±SD). 
Sl. l. Vsebnost UV absorbirajočih snovi (normirano) v fitoplanktonu na površini (beli stolpci), na 5 metrih 
(svetlo sivi stolpci) in na dnu (temno sivi stolpci) (n=3, povprečje±SD). 
The synthesis of seeondary substanees is energetically eostly and they are produeed if the dam-
age due to UV-B is bigger than metabolie eosts for produetion (GABERŠČIK & al. 2002b). ETS aetiv-
ity is a measure of the metabolie potential of organisms. ETS aetivity per volume of water was high-
er in Jower, eutrophie lakes (Fig. 2), which had high eoneentration of eh!. a (Tab. 1). Severa! inves-
tigations (CHo & AzAM 1990, DEL GIORGJO 1992) showed a signifieant eorrelation between ETS and 
eh!. a in a wide range of lakes. On the other hand, ETS aetivity (normalised to the eoneentration of 
eh!. a) was higher in the pytoplankton from more oligotrophie lakes reeeiving higher UV-B doses 
(Fig. 3). It has already been reported, that ETS aetivity of pytoplankton increased under enhaneed 
UV-B radiation (FERREYRA & al. 1997, GABERŠČIK & al. 2002a, GERM & al. 2002a). This ean be 
explained by the inereased need for energy, for meehanisms involved in photoproteetion, i.e. produe-
tion of UV absorbing eompounds. 
>-~ :1 ..... :c: 
0 ..... : .... 
"' o {/) 
t- 2, LLJ 
25 
20 
15 
10 
5 
o 
KL PL LL 
Lake 
Klin. KLSup. 
Fig. 2. ETS activity (µIOzlllh) in phytoplankton in vertical profile (n=6-9, mean±SD). 
Sl. 2. Aktivnost ETS (µ]Ozll/h) v fitoplanktonu na vertikalnem profilu (n=6-9, povprečje±SD). 
62 
:.C »-
i~ 
"' u C) CI) :l. 
f- o w 
2: 
5 
4 
3 
2 
o 
KL PL LL 
Lake 
Acta Biologica Slovenica, 47 (2), 2004 
Klin. KLSup. 
Fig. 3. ETS activity (normalised) in phytoplankton in vertical profile (n=6-9, mean±SD). 
Sl. 3. Aktivnost ETS (normirano) v fitoplanktonu na vertikalnem profilu (n=6-9, povprečje ±SD). 
In conclusions, the present study reveals, that phytoplankton from high mountain and oly-
gotrophic lakes protected their vulnerable targets in the cells by synthesis of UV screening sub-
stances. This process required additional energy, derived from respiratory potential. 
Povzetek 
Naraščanje UV sevanja na Zemeljski površini, ki je posledica tanjšanja ozonske plasti v atmos-
feri, je predmet mnogih raziskav. Čeprav je delež UV-B sevanja v sončnem sevanju majhen, je nje-
gov vpliv na primarne proizvajalce velik. Visokogorska jezera so še posebej občutljivi sistemi na kli-
matske spremembe. Po nekaterih podatkih se UV-B sevanje (280-320 nm) na vsakih 1000 m nad-
morske višine poveča za 6-8%, oz. za 20%. 
Visokogorska jezera vsebujejo navadno nizke koncentracije raztopljenih organskih snovi, ki v 
veliki meri vplivajo na prodiranje UV sevanja v vodo. Organizmi v visokogorskih in čistih jezerih so 
tako izpostavljeni višjim odmerkom UV-B sevanja brez možnega zatočišča pred škodljivimi žarki. 
UV-B sevanje vpliva na mnoge biološke in fiziološke procese pri primarnih proizvajalcih. 
Ugotavljali smo sposobnost fitoplanktona za izgrajevanje UV zaščitnih snovi ter dihalni poten-
cial (aktivnost terminalnega elektronskega transportnega sistema - ETS) na Jezeru na Planini pri 
Jezeru, Krnskem jezeru, Jezeru v Ledvicah ter Spodnjem in Zgornjem Kriškemu jezeru, ki ležijo na 
različnih nadmorskih višinah, od 1383 do 2150 m. Vsebnost UV zaščitnih snovi je bila večja v fito-
planktonu iz višje ležečih jezer. Aktivnost ETS (normalizirana) je bila večja v Jezeru v Ledvicah ter 
obeh Kriških jezerih. Večja ETS aktivnost fitoplanktona iz višje ležečih jezer odraža potrebo po 
energiji, ki jo organizmi rabijo za mehanizme, s pomočjo katerih se zaščitijo pred sevanjem. 
Acknowledgements 
This research was a part of SLO Alpe2 (3311-01-2183388) financed by Ministry of Education, 
Science and Sport of Republic Slovenia. The financial support is gratefully acknowledged. Author is 
grateful to Dr. Roger H. Pain for correction of the paper. 
M. Germ: UV-B radiation screen and respiratory potential in ... 63 
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ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2004 Vol. 47, Št. 2: 65-73 
Sprejeto (accepted): 2004-09-17 
The phenotypic plasticity of Glyceria fluitans growing over the 
water/land gradient 
Fenotipska plastičnost vrste Glyceria fluitans na prehodu 
voda/kopno 
Nina KRŽIČ*, Alenka GABERŠČIK* & Mateja GERM** 
Department of Biology, Biotechnical Faculty, University of Ljubljana* & 
National Institute of Biology**, Večna pot 111, SI-1000 Ljubljana, Slovenia; 
tel: +386(0)14233388, e-mail: nina.krzic@bf.uni-lj.si (Corresponding author), 
mateja.germ@nib.si 
Abstract. The amphibious species Glyceria fluitans successfully thrives in an 
intermittent ecosystem Lake Cerknica, where floods altemate with dry periods. 
The species grows over the environmental gradient from open water to dry land. 
The plant's phenotypic plasticity was studied analysing anatomical, morphological, 
biochemical and physiological characteristics in specimens from different loca-
tions. Floating leaves were thicker, having abundant aerenchyma, lower chloro-
phyll a+b contents and higher chlorophyll alb ratio. The relative amounts of total 
UV-B and UV-A screening compounds per leaf area were high and similar in all 
forms that indicated possibly saturated amounts. The measurement of fluorescence 
parameters revealed no disturbance in energy harvesting since the values of poten-
tial and effective photochemical efficiencies, photochemical and non-photochemi-
cal quenching were similar over the gradient. 
Key words: Glyceria fluitans, land/water gradient, amphibious character 
Izvleček. Amfibijska vrsta Glyceriafluitans se pojavlja v presihajočem ekosis-
temu Cerkniškega jezera, kjer se poplave izmenjujejo s sušnimi obdobji. Vrsta 
porašča širok pas na prehodu iz vode na kopno. Na podlagi nekaterih anatomskih, 
morfoloških, biokemijskih in fizioloških analiz rastlin na prehodu smo ugotavljali 
fenotipsko plastičnost vrste. Plavajoči listi so bili debelejši, z obsežnejšim aerenhi-
mom, nižjo vsebnostjo klorofila a+b in višjim razmerjem med klorofiloma a in b. 
Relativna vsebnost UV-B in UV-A zaščitnih snovi na listno površino se med 
proučevanimi oblikami ni bistveno razlikovala kar kaže na saturirane vrednosti. 
Izmerjeni parametri fluorescence so odražali nemoten privzem energije, saj so bile 
vrednosti potencialne in dejanske fotokemične učinkovitosti ter fotokemičnega in 
nefotokemičnega dušenja podobne na celotnem prehodu iz vode na kopno. 
Ključne besede: Glyceria fluitans, prehod med kopnim in vodo, amfibijske 
značilnosti 
66 Acta Biologica Slovenica, 47 (2), 2004 
Introduction 
Macrophytes are limited to the macroscopic flora including aquatic spermatophytes, pterido-
phytes and bryophytes. Their growth form usually classifies them in four-group system, which is 
widely accepted: emergent macrophytes (i.e. Phragmites australis), floating-leaved macrophytes 
(i.e. Nuphar luteum), free-floating macrophytes (i.e. Eichhornia crassipes) and submerged macro-
phytes (i.e. Myriophyllum spicatum). Some plants are not restricted to one category only due to their 
amphibious character (Fox 1992). Plants/species with an amphibious character plants are found 
among all major groups of plants, including mosses, fems and angiosperms (HUTCHINSON 1975). 
In the intermittent water bodies, such as Lake Cerknica, water depth and water retention play a 
major role in the occurrence of plant species (FERNANDEZ-ALAEz & al. 1999). The water leve! rais-
ing drowns emergent plants, while lowering exposes submerged ones to drying, freezing or heat 
(CRONK & FENNESSY 2001). Plants/species with an amphibious character plants possess either dis-
tinct aerial and aquatic leaves (heterophylly) either different shoots or growth forms (HUTCHINSON 
1975, Fox 1992). The phenotypic plasticity enables the species to colonize a range of habitats. It is 
an expression of flexibility in evolutionary strategy of the species (MENADUE & CR0WDEN 1990). 
Arnphibious character gives them the ability to photosynthesize under contrasting conditions in 
aquatic and terrestrial environment (GERM 2002). Beside morphological adaptations, amphibious 
plant species may also respond to the fluctuations of water by changing the reproduction strategy 
(B0ULTON & BROC 2001). 
Scarce information on Glyceriafluitans is available in the literature (JOGAN 1996). We hypothe-
sised tbat the species possessing temporal or spatial segregated submerged, floating and emergent 
shoots might have a competitive advantage over less tolerant submerged or emergent macrophytes in 
habitats characterised by water fluctuations, such as Lake Cerknica. To get an insight into survival 
strategy of G. fluitans, growing over environmental gradient in an intermittent Lake Cerknica, we 
studied some anatomical, morphological, biochemical and physiological characteristics. The latter 
included the measurements offluorescence parameters that give us useful information about the abil-
ity of plants to tolerate the water leve! fluctuations. 
Materials and methods 
Area description 
The study was carried out in Lake Cerknica (45°45'N, 14°20'E) during extremely dry vegetation sea-
son in June 2003. The closed depression of Cerkniško polje extends on an area of 38 km2 and usual!y the 
area of 25 km2 is flooded when the polje changes into a lake. The Lake Cerknica is locus typicus for inter-
mittent lakes and karst poljes. The water reaches its normal leve! of 550 m a.s.l. in spring and late auturnn 
to early winter (KRANJC 2002a, KRANJC 2002b). Usually in summers the lake becomes dry. 
Material studied 
Glyceriafluitans L. belongs to Poaceae family (MARTINČIČ & al. 1999). It colonizes more or less 
permanent, shal!ow still or slowly running water. It is widespread to scattered in lowland and lower 
montane area rarely exceeding 600 m a.s.l. (JOGAN 1996). We examined different growth forms of 
G. fluitans growing over environmental gradient from open water to dry land (1 - open water; 2 -
water-land interface, 3 - 2 rn from water body, 4 - dry land). 
N. Kržič, A. Gaberščik & Germ: The phenotypic of G/1,ceria fl utains growing ... 67 
Morphological and anatomical analyses 
In samples collected at different locations we measured the total stem length and counted the 
number of leaves per stem. We determined the thickness of leaves, spongy tissue and palisade tissue 
and calculated the palisade/spongy tissue ratio. Dry weights were obtained after drying the plant 
material at 105°C for 24 hours. 
Biochemical analyses 
For determination of chlorophylls (chl a+b and chl alb ratio) we followed the equations devel-
oped by JEFFREY & HUMPHREY (1975). The chlorophyll extracts were made using 90% (v/v) acetone 
solution. The chlorophyll contents were calculated considering absorption values at 647, 664 and 
750 nm measured with UVNIS Spectrometer System (Lambda 12, Perkin-Elmer, Norwalk, CT, 
USA). The extracts of UV-A and UV-B screening compounds we made using the methanol solution 
Šmethanol : distilled water : HCI (37% (v/v)) = 79:20: lC. We followed the method proposed by 
CALDWELL (1968). The extinctions of the samples were measured in the UV-B spectral range 280-
320 nm and UV-A spectral range 320-400 nm (Lambda 12, Perkin-Elmer, Norwalk, CT, USA). The 
extinctions were integrated for UV-A and UV-B range and expressed in relative units per dry mass 
and per area. 
Measurements of fluorescence parameters 
Measurements of chlorophyll a fluorescence were carried out on clear days during dry period in 
summer 2003. Photosynthetic photon flux density (PPFD) was more than 1100 mmol/m2s, relative 
humidity around 40% and temperature (air and water) was in range between 20°C and 25°C. The 
measurements of chlorophyll fluorescence were made using a modulated fluorometer (OS-500 fluo-
rometer; OPTI-SCIENCES, Tyngsboro, MA, USA). The actual (yield) and potential (Fv/Fm) photo-
chemical efficiencies were measured to estimate the disturbance to light harvesting in different plant 
forms from different locations. Values of Fv/Fm were obtained after dark-adaptation by using dark-
leaf elips for 10 minutes. Fv stands for variable fluorescence. This is the difference between maxi-
mal (Fm) and minimal (Fo) fluorescence in dark-adapted leaves excited with a saturating beam of 
white light. The yield of illuminated sample was defined as (F'm-F'o)/F' m (F'm is maximal and F' o 
is minimal fluorescence of light-adapted leaves) (CAMPBELL & al. 2003). The photochemical (qP) 
and non-photochemical (qN) quenching was traced using a series of saturating pulses of white light 
(PPFD=8000 mmol/m2s; 0.8s). Sample leaves for kinetics determination were dark adapted for 30 
minutes. 
Statistical analysis 
The measurements were carried out on 5 parallel samples. Standard deviations (SD) were calcu-
lated. The analysis of variance (one factor ANOVA) was performed to estimate the differences 
among locations over environmental gradient. 
Results and discussion 
The intermittent Lake Cerknica is characterised by extreme water leve! fluctuations . The flora of 
Cerkniško polje is very diverse, due to great diversity of biotopes and specific water regime 
(MARTINČIČ 2002). The life histories of plants in the lake are intimately coupled to the periodicity of 
the water regime (GABERŠČIK & al. 2004). The water level fluctuations are a limiting factor for the 
68 Acta Biologica Slovenica, 47 (2), 2004 
plant growth, development and reproduction (GABERŠČIK & URBANC-BERČIČ 2002); therefore the 
growth of plants/species with an amphibious character is favoured (GABERŠČIK & MARTINČIČ 1992, 
MARTINČIČ 2002, MARTINČIČ & LESKOVAR 2002). 
G. fluitans thrives at the locations were the water is present also in dry period. At that tirne this 
species occupies the locations from open water to dry land, exhibiting different growth forms in con-
trasting environment. The analysis of the habitus of plants revealed that plants in open water devel-
oped more leaves than terrestrial ones, but the length of the plants was the highest in both extreme 
Iocations. The Ieaves became thinner with the increasing distance from the water body, due to less 
developed spongy tissue (Tab. 1). Looser tissue, well developed in floating specimens, might 
improve the floating ability of leaves. Abundant aerenchyma contributes to lower specific leaf weight 
of floating leaves, even though they are much thicker. Thicker floating leaves were also found in a 
case of Polygonum amphibium (GABERŠČIK & MARTINČIČ 1992). P. amphibium exhibited only slight 
differences in palisade/spongy tissue ratio. It seems that thicker floating leaves are an attribute of 
many plants/species with an amphibious character (HurcHINSON 1975, NIELSEN 1993). Ona con-
trary, the floating leaves in Batrachium peltatum were thinner than aerial ones (NIELSEN & SAND-
JENSEN 1993). 
Tab. 1. Morphological, anatomical and biochemical characteristics of Glyceria jluitans growing over the 
environmental gradient (1 - open water; 2 - water-land interface, 3 - 2 m from water body, 4 - dry land) in 
Lake Cerknica. 
Tab. 1. Morfološke, anatomske in biokemijske značilnosti vrste Glyceria jluitans rastoče v različni oddal-
jenosti od prqste vodne površine (1 - odprta vodna površina, 2 - prehod med vodo in kopnim, 3 - 2 m od 
vodnega telesa, 4 - kopno) na Cerkniškem jezeru. 
Characteristic Location 
1 2 3 4 
Tota! steam length [cm] 89±7 48 ±3 66±4 87 ± 10 
No. leaves per steam 7.8 ± 0.4 6. 7 ± O.S 6.6 ± 0.9 4.9 ±0.9 
Leaf thickness [µm] 539 ± 120 372 ± 74 256 ±38 180 ± 22 
Palisade/spongy tissue 0.28 ±0.08 0.44 ±0.06 0.80± 0.22 2.15 ± 0.47 
Spec. leaf weight [g/dm2] 33.8 ± 6.9 35.2 ± 9.0 39.1 ± 2.8 40.7 ± 1.6 
Spec. leaf area [dm2/g] 0.031 ± 0.008 0.028 ± 0.001 0.025 ± 0.002 0.024 ± 0.001 
Chl a+b [mg/gDMJ 7.19 ± 1.34 14.87 ± 2.45 10.05±2.09 8.49 ± 2.17 
Chl alb 7.19 ± 2.95 3.95 ± 0.31 3.90 ± 0.48 5.35 ± 3.17 
Amphibious species exhibit great variability in chlorophyll content (GERM & GABERŠČIK 2003). 
The floating leaves of G. fluitans had lower chlorophyll a+b content (mg g-1 dry rnass) than the emer-
gent leaves (Tab. 1). The investigations of Callitriche copocharpa and Batrachium peltatum are in line 
with our results (NIELSEN 1993, NIELSEN & SAND-JENSEN 1993), but this was not the case in Batrachium 
aquatile (NIELSEN 1993). We deterrnined the highest values of chlorophyll alb ratio in floating leaves. 
This might be a consequence of the fact that a lot floating leaves were more exposed to irradiation, 
while aerial Ieaves were erect and therefore they avoided high irradiance flux during midday. 
The ability of plants to protect themselves from UV radiation plays the important role in open habi-
tats. The majority of primary producers is able to produce UV screening compounds that present an UV-
B selective filter (BJORN 1999). This appears to be an important protective mechanism because they could 
effectively reduce detrimental effect of UV-B radiation in leaves (MIDDELTON & l'ERAMuRA 1993). In G. 
fluitans the relative amount of total UV-B and UV-A screening compounds per unit of dry mass (Fig. 1) 
N. Kržič, A. Gaberščik & Germ: The phenotypic of Gli'ceria f1 utains growing ... 69 
was the highest in floating leaves. The investigations reveal that UV absorbing compounds are mainly 
synthesised in epidermis of the leaves that prevent UV penetration into the mesophyll (DAY & al. 1996). 
Por that reason we also compared the amounts of total UV-B and UV-A screening compounds per unit 
of leaf area. The values showed no significant difference among different forms. These results are in 
agreement with other researches of plants/species with an amphibious character (GERM & al. 2002). The 
comparison of the amounts determined in G. fluitans with other amphibious and terrestrial plants 
revealed relatively high values in the former. That indicated possibly saturated amounts of UV-B and UV-
A screening compounds. The production is probably genetically fixed and less dependent on environ-
mental factors. A sirnilar phenomenon was found in Potamogeton nodosus (LES & SHERIDAN 1990). 
.. ~ 20 • uv-A QUV-B 6000 • uv-A oUV-B 
E 15 l~JJ 
~ 
~ 
~-h 
Cl 
.§. ~ 4000 10 c 
l1l1li u:i = .o 5 u:i 2000 Ol .o > Ol ::, o > o ::, 
2 3 4 2 3 4 
Transect Transect 
Fig. 1. The amount of UV absorbing compounds of Glyceria fluitans growing over the environmental gra-
dient (1 - open water; 2 - water-land interface, 3 - 2 m from water body, 4 - dry land) in Lake Cerknica. 
Sl. 1: Količina UV absorbirajočih snovi pri vrsti Glyceria fluitans rastoče v različni oddaljenosti od proste 
vodne površine (I - odprta vodna površina, 2 - prehod med vodo in kopnim, 3 - 2 mod vodnega telesa, 4 -
kopno) na_ Cerkniškem jezeru. 
The water-logging should not cause a physiological stress in macrophytes, although photochem-
ical efficiency might be disturbed due to other stresses which are the consequence of water leve! fluc-
tuation; i.e. drought (COLOM & VAZ.ANA 2003), photoinhibition (GABERŠČIK & MAZEJ 1995, RALPH 
& BURCHETI 1995, BEER & BJ0RK 2000, MoUGET & TREMBLIN 2002, CAMPBELL & al. 2003) and UV-
B radiation (CONDE-ALVAREZ 2002, GABERŠČIK & al. 2002, GERM ET al. 2002). Our results showed 
no significant differences in potential (Fv/Fm) and actual (yield) photochemical efficiencies of PS II 
in G. fluitans growing over the environmental gradient (Fig. 2, Tab. 2). Fv/Fm was close to the opti-
Q) • Fv/Fm oyield (.) 
C 
0,8 tl1 Q) (.) Jlll 
(/) 
~ 
o 0,6 1 
:@ 
>, 0,4 
.C 
0.2 l C. e 
o 
:C o (.) 
2 3 4 
Transect 
Fig. 2. Photochemical efficiency of Glyceria fluitans growing over the environmental gradient (1 - open 
water; 2 - water-land interface, 3 - 2 m from water body, 4 - dry land) in Lake Cerknica. 
Sl. 2: Fotokemična učinkovitost vrste Glyceria fluitans rastoče v različni oddaljenosti od proste vodne 
površine (1 - odprta vodna površina, 2 - prehod med vodo in kopnim, 3 - 2 mod vodnega telesa, 4 - kopno) 
na Cerkniškem jezeru. 
70 Acta Biologica Slovenica, 47 (2), 2004 
mal values (0.83) at all locations as an indication of normal function of PS II. Lower values of yield 
indicated the presence of stress with no respect to the location and form. WALDHOFF & al. (2002) also 
found no correlation between the changing water levels and the Fv/Fm ratios of the tree species 
Symmeria paniculata. The leaves fully retained the potential to photosynthesize during the aerial and 
aquatic phases. Fv/Fm ratio in Genipe americana also remains constant despite flooding conditions 
(MIELKE & al. 2003). Our study revealed no differences in photochemical (qP) and nonphotochemi-
cal (qN) quenching in G. fluitans appearing in different growth forms over the gradient water-land 
(Fig. 3). Photochemical quenching had greater values and affected the actual photochemical efficien-
cy more than non-photochemical quenching. Values of qN in Genipe americana increase by flood-
ing, while qP remains constant (MIELKE & al. 2003). 
Fig. 3. Photochemical and non-photochemical fluorescence quenching in Glyceria fluitans growing over the 
environmental gradient (1 - open water; 2 - water-land interface, 3 - 2 m from water body, 4 - dry land) in 
Lake Cerknica. 
Sl. 3. Fotokemično in nefotokemično dušenje fluorescence pri vrsti Glyceriafluitans rastoče v različni odd-
aljenosti od proste vodne površine (1 - odprta vodna površina, 2 - prehod med vodo in kopnim, 3 - 2 m od 
vodnega telesa, 4 - kopno) na Cerkniškem jezeru. 
C) 1,5 
C • qP oqN :C 
" C Q) 
1 ' 
~~ 
::, 
er 
Q) 
" C Q) 0,5 
" v, ~ 
o 
::, o .L u:: 
2 3 4 
Transect 
Tab. 2. The significance of differences of investigated plant characteristics among 4 locations over environ-
mental gradient water/land (ANOVA) (1 - open water; 2 - water-land interface, 3 - 2 m from water body, 4 
- dry land) in Lake Cerknica. 
Tab. 2. Satistična značilnost razlik pri proučevanih značilnostih rastline med 4 lokacijami na okoljskem gra-
dientu voda/kopno (ANOVA) (1 - vodno telo, 2 - prehod med vodo in kopnim, 3 - 2 mod vodnega telesa, 
4 - kopno) na Cerkniškem jezeru. 
Characteristic Significance Characteristic 
Tota! steam length [cm] *** UV-A [int./gDM] 
No. leaves per steam *** UV-B [int./gDM] 
Leaf thickness [µm] *** UV-A [int./cm2] 
Palisade/spongy tissue *** UV-B [int./cm2] 
Spec. leaf weight [g/dm2] * Fv/Fm 
Spec. leaf area [dm2/g] NS Yield 
Chl a+b [mg/gDM] *** Photchemical quenching 
Chl alb NS Non-photchemical quenching 
*** p < 0.001; ** p < 0.01; * p < 0.05 ; non significant (NS) p > 0.05. 
Significance 
NS 
* 
NS 
** 
NS 
* 
*** 
*** 
N. Kržič, A. Gaberščik & Germ: The phenotypic of Gl) ceria flutains growing ... 71 
Conclusions 
The floating shoots of G. fluitans develop thicker leaves than emergent ones. They have abun-
dant aerenchyma that contributes to lower specific leaf weight and their floating ability. 
The floating leaves had higher chlorophyll alb ratio than the emergent ones, which might be due 
to higher irradiance of direct sunrays. The relative amount of total UV-B and UV-A screening com-
pounds showed no significant difference among diverse forms of G. fluitans . 
No significant differences in potential and effective photochemical efficiencies as well as in pho-
tochemical and non-photochemical quenching were foµnd over the environmental gradient. These 
fluorescence characteristics indicate normal functioning of PS II. 
Acknowledgements 
This research was financed by Ministry of Education, Science and Sport, Republic of Slovenia 
through the programs »Biology of plants (Pl-0212) « and » Young researchers«. 
Povzetek 
Glyceria fluitans je vrsta z amfibijskim značajem in raste v presihajočem ekosistemu 
Cerkniškega jezera, ki ga označujejo izmenjave poplav in sušnih obdobij. V sušnem delu leta, ko 
nivo vodne gladine upade, porašča širok pas na prehodu iz vode na kopno. G. fluitans v odvisnosti 
od vodnih razmer, ki so pogojene prostorsko ali časovno, tvori morfološko različe poganjke. V vodi 
razvije poganjke, ki so potopljeni ali plavajoči, na kopnem pa zračne poganjke. V raziskavi smo ugo-
tavljali fenotipsko plastičnost vrste na prehodu iz vode na kopno. Spremljali smo nekatere 
anatomske, morfološke, biokemijske in fiziološke parametre posameznega tipa poganjkov. V nar-
avnem okolju smo s pomočjo parametrov fluorescence ugotavljali uspešnost prestrezanja energije. 
Rezultati so pokazali, da so plavajoči listi G. fluitans debelejši, z obsežnejšim aerenhimom, ki 
zmanjšuje specifično listno težo in prispeva k večji plovnosti. Nižja vsebnost klorofila a+b in višje 
razmerje med klorofiloma a in b pri plavajočih listih pa sta odraz večje jakosti sevanja zaradi direk-
tnih sončnih žarkov. Realtivna vsebnost UV-B in UV-A zaščitnih snovi na listno površino je bila v 
vseh proučevanih oblikah podobna in je nakazovala saturirane vrednosti. Izmerjeni parametri fluo-
rescence klorofila a v FS II so odražali normalno fotosintezno aktivnost vrste na celotnem prehodu 
iz vode na kopno. Vrednosti potencialne fotokemične učinkovitosti (Fv/Fm) so bile na celotnem pre-
hodu blizu optimalni vrednosti 0,83 . Dejanska fotokemična učinkovitost (yield) je bila nižja od 
potencialne, kar je rezultat rahlega stresa, vendar pa razlik na gradientu nismo opazili. Tudi vrednos-
ti fotokemičnega in nefotokemičnega dušenja so bile podobne na celotnem prehodu iz vode na 
kopno. 
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Introduction 
ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2004 Vol. 47, Št. 2: 75-81 
Sprejeto (accepted): 2004-09-17 
Pollen grain bioassay for environmental contamination 
biomonitoring 
Biomonitoring polucije okolja z analizo poškodovanosti pelod-
nih zrn 
Jasna PARADIŽ and Milan LOVKA 
National Institute of Biology Ljubljana, Večna pot 111, SI-1000 Ljubljana, 
Slovenija; tel: +386(0)14233388, e-mail: jasna.paradiz@guest.arnes.si 
Abstract. Pollen grain bioassay near some highways was performed in 45 
plant species for identification of environmental genotoxic impact on naturally 
growing plants due to environmental traffic contaminants. A relationship between 
the duration of heavy traffic on particular road sections and the degree of develop-
mental and morphological changes of pollen grains was indicated. Increased fre-
quency of pollen deformation after more than 20 years of traffic influence was 
established, indicating the applicability of pollen grain bioassay for genotoxic risk 
assessment of chronic low leve! contamination impact in natura! habitats. 
Key Words: naturally growing bioindicator plants, traffic contaminants, pollen 
grain deformation 
Izvleček. V biomonitoringu onesnaženosti okolja zaradi prometa smo anal-
izirali stopnjo poškodovanosti pelodnih zrn pri 45 vrstah rastlin z naravnih rastišč 
ob naših avtocestah. Ugotovili smo, da je stopnja poškodovanosti peloda na 
poskusnih lokalitetah povezana s trajanjem prometa na posameznih avtocestnih 
odsekih, povečan obseg poškodovanosti peloda je bil pri biondikatorskih rastlinah 
na rastiščih, obremenjenih z vplivi prometa več kot 20 let. Ti rezultati kažejo, da je 
analiza poškodovanosti pelodnih zrn uporabna za sledenje kroničnih vplivov nizk-
ih nivojev zračne polucije z genotoksičnimi agensi in oceno ogroženosti rastlin na 
naravnih rastiščih. 
Ključne besede: samonikle bioindikatorske rastline, onesnaževanje z 
izpušnimi plini, poškodovanost pelodnih zrn. 
Environmental contamination caused by highway traffic has been identified as a serious ecolog-
ical problem. Air contaminants produced by motor traffic and released along the highways and main 
76 Acta Biologica Slovenica, 47 (2), 2004 
road connections are particularly hazardous to puhlic health and the environment. Low concentra-
tions of many environmental pollutants are considered as harmless, but for the genotoxic contami-
nants there is no safe <lose or concentration, and environmental hazard identification is required. 
Naturally growing plants are exposed to a changing mixture of environmental contaminants, and 
when used as bioindicators of long lasting exposure to complex pollution impact they have a key role 
in genotoxic risk evaluation of low contamination levels in natura! habitats. 
Our previous observation in plant natura! habitats with elevated contamination load (PARADIŽ & 
LOVKA 1998, 1999a, 1999b, 2000) and literature <lata (KORMUTAK 1996, MIČIETA & MURIN 1996, 
MULCAHY 1981, MURIN 1995) suggest that with many different species, plant bioassay results could 
give a more complex information about the pollution threats in the environment. Using 45 species of 
naturally growing plants in pollen grain bioassay near some highways in Slovenia, we have per-
formed screening of genotoxicity for risk identification of environmental impact of heavy traffic in 
natura! habitats. 
Material and Methods 
Experimental sites near some highways in Slovenia, conceming the duration of traffic on the spe-
cific road sections were chosen for biomonitoring with pollen grain bioassay. At 8 localities 
(Bizovik, Arja vas, Vransko, Čebulovica, Divača, Dolenja vas, Zajčica, and Sela) plants in their habi-
tats had been exposed to traffic influence for the period up to 10 years, at 2 localities (Povodje and 
Brdo) for 10 to 20 years, and at 5 localities (Gabrje, Ivanje selo, Ravnik, Trebnje and Žiče) for 20 to 
30 years. Localities at Komen, Predmeja, Pugled, and Škofja Loka with no obvious traffic pollution 
sources were used for the comparison. 
For pollen grain bioassay flower buds were sampled in 45 species of plants growing naturally at 
experimental and control sites (Tab. 1) on 18th, 241h, and 31 st of March, 7th of April, 19th and 26th of 
May, 2nd, 9th, 17th and 23rd of June 1999. The samples were fixed in ethanol acetic acid mixture (3:1), 
then kept at -200C in a refrigerator. 
Microscopic slides of fully developed anthers were made by staining in aceto--carmine. 
Generally, five slides from five different flowers per bioindicator species at the site were examined. 
In at least 300 pollen grains per slide, stained (viable) and empty (sterile) grains, as well as pollen 
size and shape deformations were observed under an optical microscope. Deformation frequencies 
of pollen grains, as well as portions of morphological changes in relation to the duration of highway 
traffic for up to 10 years, for 10-20 years and for 20-30 years are presented. 
Results 
In naturally growing bioindicator plants at the control sites, over 95% of stained (fertile) pollen 
grains were observed. In plants at experimental sites near highways, deformation frequency of pollen 
grains increased with the duration of traffic influence. The frequency of empty (sterile) grains 
increased up to 10% after 30 years of traffic influence (Fig. 1). Smaller size (for about 1/3 of the nor-
mal one) as well as shape deformations were less frequent than empty not stained grains, however, 
their portion increased with the duration of highway traffic as well (Tab. 2). 
J. Paradiž and M. Lovka: Pollen grain bioassay for environmental... 77 
Table 1. List of bioindicator species for pollen grain bioassay within sampling periods from March to June 
at experimental sites near highways and the control sites. Duration of traffic influence for particular road 
sections in brackets is stated. 
Tabela l. Seznam rastlinskih vrst vključenih v analizo poškodovanosti pelodnih zrn od marca do junija na 
poskusnih lokalitetah ob odsekih avtocest in kontrolnih lokalitetah. V oklepajih je navedeno trajanje 
prometa na posameznih odsekih avtocest. 
Sampling Bioindicator plants 
March Anemone nemorosa L. 
Capsella bursa-pastoris (L.) Med. 
CornusmasL. 
Corydalis cava (L.) Schweigger & Koerte 
Corylus avellana L. 
Crocus albiflorus Kit. ex Schult. 
Crocus napolitanus Mord. & Loisel. 
Crocus variegatus Hoppe & Hornsch. 
Daphne mezereum L. 
Erythronium dens-canis L. 
Galanthus nivalis L. 
Hacquetia ep ipactis (Scop.) DC. 
Helleborus niger L. 
Helleborus odorus W. & K. ex Willd. 
Lamium purpureum L. 
Leucojum vernum L. 
Omphalodes verna Moench 
Oxalis acetosella L. 
Polygala chamaebuxus L. 
Potentilla erecta (L.) Riiuschel 
Pulmonaria officinalis L. 
Symphytum officinale L. 
Tussilago farfara L. 
Vinca minor L. 
April/May Campanu/a rapunculus L. 
Crocus napolitanus Mord. & Loisel. 
Erythronium dens-canis L. 
Lamium orvala L. 
Lamium purpureum L. 
Melittis melissophyllum L. 
Myosotis sylvatica Ehrh. ex Hoffm. 
Oxalis acetosella L. 
Polygala chamaebuxus L. 
Potemilla erecta (L.) Riiuschel 
Ranunculus acris L. 
Ranunculus repens L. 
Symphytum officinale L. 
Trifolium pratense L. 
Trifolium repens L. 
Vinca minor L. 
June Calamintha grandiflora (L.) Moench 
Campanula rapunculus L. 
Coronilla emeroides Boiss. & Sprun. 
Coronilla varia L. 
Dianthus san uineus Vis. 
Erigeron annuus (L.) Pers. 
Experimental and control sites 
Bizovik(!), Arja vas (2), Trebnje (30) 
Arja vas (2) 
Komen, Žiče (23) 
Komen 
Povodje ( 14) 
Dolenja vas (4), Gabrje (25) 
Bizovik(!), Povodje (14), Brdo (18), Trebnje (30) 
Komen 
Trebnje (30) 
Brdo (18) 
Komen 
Ravnik (27) 
Pugled 
Pugled, Dolenja vas (4) 
Arja vas (2) 
Povodje (14) 
Ravnik (27) 
Arja vas (2) 
Pugled 
Čebulovica (4) 
Povodje (14), Ziče (23), Ivanje selo (27) 
Žiče (23) 
Vransko (2), Divača (4) 
Vransko (2) 
Sela (7) 
Trebnje (30) 
Sela (7), Povodje (14), Brdo (18) 
Povodje (14) 
Bizovik (1), Povodje (14) 
Pugled 
Povodje (14), Brdo (18) 
Trebnje (30) 
Pugled 
Pugled 
Bizovik (1) 
Sela (7), Povodje (14) 
Bizovik (1) 
Sela (7) 
Bizovik (1) 
Sela (7) 
Ravnik (27) 
Skofja Loka 
Vransko (2) 
Komen 
Komen 
kofja Loka 
78 
Coronilla emeroides Boiss. & Sprun. 
Coronilla varia L. 
Dianthus sanguineus Vis. 
Erigeron annuus (L.) Pers. 
Filipendula vulgaris Moench 
Geranium sylvaticum L. 
Lilium martagon L. 
Lysimachia nummularia L. 
Muscari racemosum (L.)Mill. em. Lam. & DC. 
Potentilla erecta (L.) Rauschel 
Ranunculus acris L. 
Ranunculus bulbosus L. 
Ranunculus repens L. 
Sanicula europaea L. 
Symphytum officinale L. 
Valeriana officinalis L. 
12 
",fl. 10 
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Q, 
t 
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8 
6 
4 
2 
o 
Acta Biologica Slovenica, 47 (2), 2004 
Vransko (2) 
Komen 
Komen 
SkofjaLoka 
Komen 
Ravnik (27) 
Gabrje (25) 
Škofja Loka 
Komen 
Škofja Loka 
Predmeja, Škofja Loka, Arja vas (2) 
Dolenja vas (4), Zajčica (4) 
Gabrje (25) 
Predmeja 
Arja vas (2) 
Divača (4) 
O Control 
[J 1 0Years 
• 20 Years 
III 30 Years 
March April/May 
Sampling period 
June 
Figure l. Pollen grain bioassay in naturally growing plants at experimental sites near highways and the con-
trol sites. 
Slika 1. Poškodovanost peodnih zrn pri naravno rastočih rastlinah na poskusnih lokalitetah ob avtocestah in 
kontrolnih lokalitetah. 
J. Paradiž and M. Lovka: Pollen grain bioassay for environmental ... 79 
Table 2. Portion (%) of size (small) and shape deformations of pollen grains in naturally growing plants near 
highways and the control. 
Tabela 2. Delež (%) malih in deformiranih pelodnih zrn pri naravno rastočih rastlinah ob avtocestah in kon-
troli. 
Traffic March April/May June 
influence Size Shape Nr. Size Shaee Nr. Size Shaee Nr. 
Control 7,2 0,1 7 4,2 0,0 3 3,4 0,1 11 
10 Years 5,6 4,1 10 5,0 3,2 9 13,3 1,8 5 
20 Years 9,5 2,0 5 4,5 11,7 5 
30 Years 8,7 11,8 9 1,5 1,7 2 4,6 6,3 4 
Nr. - Number of bioindicator species used in the pollen grain bioassay 
Discussion 
The genotoxic risk of environmental contarnination due to increasing traffic density and enlarged 
highway network in our country is of concem to both puhlic health and the environment. P!ants in 
their habitats are exposed to a broad spectrum of ecological and pollution risks, and they as bioindi-
cators of long lasting exposure to genotoxic effects in the environment are used in biomonitoring pol-
lution in view of environmental risk assessment (BESSONOVA & al. 1996, GICHNER & VELEMINSKY 
1999, JABEEN & ABRAHAM 1996, KORDYUM & SID0RENK0 1997, M0NARCA & AL. 1999, MULLER & 
AL. 1991, PARADIŽ & LOVKA 1998, 1999a, 1999b, 2000). 
In genotoxic risk identification, reduced pollen viability is one of the serious pollution impacts 
on reproductive capacity of plants that might link to ecological consequences. When the amount of 
stained pollen grains decrease Jess than 10%, pollen fertility of warm season species is not serious-
ly affected (TEDESCO & AL. 2002), but under normal climatic conditions pollen abortion in plants is 
Jess than 5% (MURIN 1995). This is in agreement with our results in bioindicator plants; the average 
frequency of 3% of empty (sterile) pollen grains at the control sites was established. 
At experimental sites near highways, the frequency of empty pollen grains (generally due to 
irregularities during meiosis) in bioindicator plants increased with the duration of traffic to 10% after 
30 years of traffic influence (Fig. 1), as well as the portion of size and shape deformations (Tab. 2). 
These results indicate that the genotoxic impact of long lasting environmental pollution might be 
related with traffic contaminants. 
When naturally growing plants for biomonitoring genotoxicity of pollutants are used, synergy 
with other environmental factors, such as meteorological conditions could not be excluded. With suc-
cessive sampling times at experimental sites near highways, the influence of extreme temperature or 
drought in sumrner on the results (Fig. 1) could be verified. But generally, prolonged sampling peri-
od for pollen grain bioassay is needed because of blooming season of various plant bioindicators. 
On the basis of the present results, pollen grain bioassay for continuous biomonitoring could be 
suggested, in addition to screening of detailed cytogenetical damage in plants, as well as the routine 
measurements of atmospheric contaminants for risk assessment of traffic pollution impact in the 
environment. 
80 Acta Biologica Slovenica, 47 (!), 2004 
Acknowledgements 
This work was part of a project supported by the Ministry of Science and Technology, Slovenia. 
Povzetek 
Ugotavljali smo vpliv škodljivih onesnaževalcev prometa na stopnjo poškodovanosti pelodnih 
zrn pri naravno rastočih rastlinah ob avtocestah. V raziskavo smo vključili 45 rastlinskih vrst in 
jemali cvetne popke od marca do julija za opazovanje sprememb v razvoju in obliki pelodnih zrn. 
Vzorčili smo na lokalitetah: Bizovik, Arja vas, Vransko, Čebulovica, Divača, Dolenja vas, Zajčica in 
Sela z vplivi prometa do 10 let; Povodje in Brdo z vplivi prometa 10 do 20 let; Gabrje, Ivanje selo, 
Ravnik, Žiče in Trebnje z vplivi prometa 20 do 30 let. Za primerjavo rezultatov smo uporabili 
lokalitete, oddaljene od glavnih prometnih cest (Komen, Predmeja, Pugled in Škofja Loka). 
Na poskusnih lokalitetah ob posameznih odsekih avtocest, kjer se odvija promet več kot 20 let smo 
ugotovili povečano stopnjo poškodovanosti pelodnih zrn pri naravno rastočih rastlinah. To kaže na 
uporabnost analize poškodovanosti pelodnih zrn za ugotavljanje dolgotrajnega vpliva zračne polucije 
na rastline v naravnem okolju. Z nadaljevanjem tovrstnih analiz bi doprinesli rezultate o škodljivih 
vplivih nizkih nivojev kontaminacije za oceno stopnje ogroženosti rastlin obremenjenih rastišč. 
Refereces 
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ment of industrial pollutant mutagenicity. Tsitologiya i Genetika 30 (5): 70-76. 
G!CHNER T. & J. VELEMINSKY 1999: Monitoring the genotoxicity of soil extracts from two heavily pol-
luted sites in Prague using the Tradescantia stamen hair and micronucleus (MNC) assays. Mutat. 
Res. 426: 163-166. 
lABEEN C. & S. ABRAHAM 1996: Impact of gaseous pollutants from the Kerala Minerals and Metals Ltd. 
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KORDYUM E. L. & P. G. SIDORENKO 1997: The results of cytogenetic monitoring in angiosperm species 
of plants in the zone of radionuclide contamination after the Chernobyl NPP accident. 
Tsitologiya i Genetika 31(3): 39-46. 
KORMUTAK A. 1996: Development and viability of silver fir pollen in air-polluted and non-polluted habi-
tats in Slovakia. Forest Genetics 3 (3): 147-151. 
MIČIETA K. & G. MURIN 1996: Microspore analysis for genotoxicity of a polluted environment. Environ. 
Exp. Bot., 36 (1): 21-27. 
MONARCA S., D. FERETTI, A. ZANARDINI, E. FALISTOCCO & G. NARDI 1999: Monitoring of mutagens in 
urban air samples. Mutat. Res. 426: 189-192. 
MULCAHY D. L. 1981: Pollen tetrads in the detection of environmental mutagenesis. Environ. Health 
Persp. 37: 91-94. 
MURIN A. 1995: Basic criteria for selection of plant bioindicators from the regional flora for monitoring 
of an environmental pollution. Biologia, Bratislava, 50 (1): 37-40. 
MOLLER M., H. GUTrENBERGER, D. GRILL, B. DRUŠKOVIČ & J. PARADIŽ 1991: A cytogenetic method 
for examining the vitality of spruces. Phyton, 31 (1): 143-155: 
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PARADIŽ J. & M. LOVKA 1998: Plant bioassays of possible environmental contamination. In: Abstract 
boo k European Environmental Mutagen Society (EEMS) 28th Annual Meeting, September 7-1 O, 
1998, Salzburg, P 128. 
PARADIŽ J. & M. LOVKA 1999A: Pollen grain bioassay for environmental pollution screening. Phyton 
(Austria) Special issue: »Plant Physiology« 39 (3): 175-180. 
PARADIŽ J. & M. LOVKA 1999B: Bioindicator plants for the detection of genotoxicity in the environment. 
Pharmacology and Toxicology, Vol. 85, Supplement I, Abstracts from EEMS-99 29th Annual 
Meeting of the European Environmental Mutagen Society, 1999, Copenhagen, Denmark, P-IVa-
9: 35. 
PARADIŽ J. & M. LovKA 2000: Chromosome aberration assay in naturally growing plants for environ-
mental biomonitoring. In: Proceedings of the 2 nd Congress of the Genetic Society of Slovenia 
with international participation, Bled, September 13 - 17, 2000, Genetic Toxicology: 365-366. 
TEDESCO S. B., M. T. SCHIFINO-WITIMANN & M. D . AGNOL 2002: Meiotic behaviour and pollen fertil-
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ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2004 Vol. 47, Št. 2: 83-94 
Sprejeto (accepted): 2004-09-17 
Respiration rate and respiratory electron transport system 
(ETS) activity of chironomid larvae from mountain lakes 
(NW Slovenia) 
Stopnja dihanja in aktivnost dihalnega elektronskega 
transportnega sistema (ETS) pri ličinkah trzač iz gorskih jezer 
(SZ Slovenija) 
Tatjana SIMČIČ 
National Institute ofBiology, Večna pot lll, Sl-1000 Ljubljana, Slovenia; 
tel: +386(0)14233388, e-mail: tatjana.simcic@nib.si 
Abstract. Respiration rate (R) and electron transport system (ETS) activity 
were measured in larvae offour chironomid species, Chironomus thummi (Kieffer), 
Paratanytarsus austriacus (Kieffer), Procladius (Holotanypus) spp., and 
Zavrelimyia barbatipes (Kieffer) from three mountain lakes. ETS activity and res-
piration rate, measured at a standard temperature of 20 °C, differed significantly 
between species (ANOVA: p<0.001). ETS activity ranged from 10.3 µL 0 2 mg- 1 
DW h- 1 for Zavrelimyia to 27.6 µL 0 2 mg-1DW h-1 for Paratanytarsus. The lowest 
respiration rate was observed for Procladius (4.0 µL 0 2 mg- 1DW h- 1) and the high-
est for Paratanytarsus (12.4 µLO2 mg-1 DW h- 1). ETS activity and respiration rate 
of chironomids decreased with increasing size of the animals (p<0.001); b values 
for ETS activity and respiration rate were 0.82 and 0.66, respectively. Respiration 
rate correlated significantly with ETS activity for ali four species investigated. This 
study revealed that the ETS/R ratio differed between species (ANOVA: p<0.001). 
A low ratio (0.98) was determined for Zavrelimyia, while Procladius showed the 
highest value (3 .88). The differences can be explained by body size, and the eco-
logical preference and tolerance of each chironomid species. 
Key words: Chironomidae, Diptera, benthos, ETS/R ratio, ETS activity, respi-
ration, metabolism 
Izvleček. Stopnjo dihanja in aktivnost elektronskega transportnega sistema 
(ETS) smo določali pri ličinkah štirih različnih vrst trzač , Chironomus thummi 
(Kieffer), Paratanytarsus austriacus (Kieffer), Procladius (Holotanypus) spp., in 
Zavrelimyia barbatipes (Kieffer), ki smo jih nalovili v treh gorskih jezerih. Aktivnost 
ETS in stopnja dihanja, ki smo ju merili pri standardni temperaturi 20 °C, sta se 
značilno razlikovali med vrstami (ANOVA: p<0.001). Aktivnost ETS se je gibala 
84 Acta Biologica Slovenica, 47 (2), 2004 
med 10.3 µL 0 2 mg·1DW h·1 pri ličinkah Zavrelimyia in 27.6 µL 0 2 mg-t DW h·1 pri ličinkah 
Paratanytarsus. Najnižjo stopnjo dihanja smo določili pri ličinkah Procladius (4.0 µL 0 2 mg-1DW h·1), 
najvišjo pa pri ličinkah Paratanytarsus (12.4 µL 0 2 mg· 1DW h· 1). Aktivnost ETS in stopnja dihanja 
ličink sta se zmanjševali z naraščajočo velikostjo živali (p<0.001); vrednosti b sta znašali 0.82 za 
aktivnost ETS in 0.66 za dihanje. Opazili smo pozitivno korelacijo med stopnjo dihanja in aktivnos-
tjo ETS pri vseh štirih vrstah. Raziskava je pokazala značilne razlike v razmerju ETS/R med vrsta-
mi (ANOVA: p<0.001). Nizko razmerje (0.98) je bilo določeno pri ličinkah Zavrelimyia, medtem ko 
je bilo pri ličinkah Procladius določeno najvišje razmerje (3.88). Različna razmerja so posledica raz-
lik v vrstno-specifični telesni velikosti ter različne ekološke preference in tolerance pri posamezni 
vrsti trzač. 
Ključne besede: Chironomidae, Diptera, bentos, razmerje ETS/R, aktivnost ETS, dihanje, 
metabolizem 
Introduction 
The family Chironomidae is widely distributed in ali types of aquatic environments, where they 
form the major part of nearly all freshwater communities, and they occupy a variety of niches. The 
relatively short life cycle and large total biomass of the numerous larvae confer ecological energetic 
significance on this taxon, as both consumers and prey, and the partitioning of ecological resources 
by a large number of species presumably enhances the biologic stability of aquatic ecosystems 
(MERRITI & CUMMINS 1984). 
As chironomid larvae play an important role in aquatic food webs, an estimation of their respi-
ratory energy loss is essential in order to estimate the energy flow through the ecosystems. Direct 
determination of respiration usually involves incubating animals in a controlled environment, and 
determining the time-dependent change in oxygen concentration. These methods are tirne consum-
ing and impractical. The development of enzymatic techniques, however, has allowed metabolic 
activity to be estimated from the electron transport system activity (OWENS & KING 1975, PACKARD 
1985). The electron transport system (ETS)-assay, based on the reduction of tetrazolium salt (INT) 
in the presence of a cell-free homogenate of the organisms and excess substrates of the ETS, has 
proved to be useful for estimating the metabolic activity of different organisms (PACKARD 1985, DEL 
GIORGIO 1992, G.-T6TH & AL. 1995). It is simple, rapid and extremely sensitive and has been used 
extensively with marine plankton (KENNER & AHMED 1975, BAMSTEDT 1980, VOSJAN & OLANCZUK-
NEYMAN 1991) and freshwater plankton (BORGMANN 1978, JAMES 1987, DEL GIORGIO 1992), benth-
ic organisms (CAMMEN & al. 1990, MusK6 & al. 1995, 1998), marine sediments (VOSJAN & 
OLANCZUK-NEYMAN 1977, RELEXANS 1996), and freshwater sediments (TREVORS 1984, G.-T6TH & 
AL. 1994). The slow response ofETS activity to short-term variations in environmental factors makes 
the method superior to direct respiratory measurements on incubated animals (BAMSTEDT 1980). It 
is, however, necessary to determine empirically the relation between the highest potential metabolic 
activity and realised oxygen consumption in order to interpret the ETS <lata properly (DEL GIORGIO 
1992). Much has been published on the respiration of chironomids (ERMAN & HELM 1970, 
KONSTANTINOV 1971, BAIRLEIN 1989, HAMBURGER & DALL 1990, HAMBURGER & AL. 1994), but there 
is no study conceming their ETS activity. 
Previous investigations have shown that the chironornid larvae are the dominant group of the 
macrozoobent_hos in mountain lakes in NW Slovenia (SIMČIČ unpubl. data). These lakes are shallow 
T. Simčič: Respiration rate and respiratory electron transport system ... 85 
and, except the eutrophic lakes Krnsko jezero and Jezero na Planini pri Jezeru, transparent to the bot-
tom. Zoobenthos are reported to have a greater effect on energy flux through secondary production 
in shallow lakes than in deeper ones (J6NASSON & al. 1990, LINDEGAARD 1994). Therefore, chirono-
mids should be taken into account when energy flow through the food webs in these lakes is inves-
tigated. 
The purpose of this paper was to determine ETS activity and respiration rate of four different chi-
ronomid species from mountain lakes, and to calculate the ETS/R ratios of these species in order to 
contribute to the estimation of energy flux through secondary production of benthic organisms. 
Materials and methods 
Sampling of animals: ETS activity and respiration rate were measured in the laboratory for 
Paratanytarsus austriacus (Kieffer) and Procladius (Holotanypus) spp. from the oligotrophic lake 
Zgornje Kriško jezero, Zavrelimyia barbatipes (Kieffer) from the oligotrophic lake Srednje Kriško 
jezero, and Chironomus thummi (Kieffer) from the eutrophic lake Krnsko jezero. Zoobenthos sam-
ples were taken using van Veen grab (Eijkelkamp). It was important to handle the larvae as little as 
possible during identification. Single animals were used for each analysis. Different sets of animals 
were taken to be investigated separately in the respirometric measurements and separately in ETS 
activity measurements in the first part of experjments. To determine relationship between ETS activ-
ity and respiration rate, the individual animal was exarnined firstly by microrespirometer and then by 
ETS-assay. Before experiments animals were rinsed with filtered water to remove bacteria adhering 
to the body surface, and weighed on an electrobalance. The dry mass of animals was calculated using 
factors determined by drying five samples of animals for 24 h at 60°C. 
Respiration measurements: Respiration rate was estimated using a twin-flow microrespirome-
ter (CYCLOBIOS, Innsbruck, Austria) (GNAIGER 1983). In the open-flow system the concentration 
of oxygen was measured before and after the chamber containing the animal. Oxygen consumption 
was calculated from the reduction of oxygen concentration and the flow rate of the water with a com-
puter program Dat.Graf 2.1 Analysis. 
ETS activity measurements: ETS activity was measured using the method proposed by 
PACKARD (1971), modified by OwENS & KING (1975) and improved by G.-T6TH (1999). Each fresh-
ly weighed animal was homogenized in 4 ml of cold homogenization buffer (0.1 M sodium phos-
phate buffer pH = 8.4, 75 µM MgSO4, 0.15% (w/v) polyvinyl pyrrolidone, 0.2% (v/v) Triton-X-100) 
for 2 min. The homogenate was then sonicated using an ultrasound homogenizer (Cole-Parmer) for 
20 sec and centrifuged in refrigerated centrifuge (Sigma) for 4 min at O °C at 8500 x g. Three 0.5 ml 
samples from each homogenate were incubated in 1.5 ml substrate solution (0.1 M sodium phosphate 
buffer pH = 8.4, 1.7 mM NADH, 0.25 mM NADPH, 0.2% (v/v) Triton-X-100) with 0.5 ml of 2.5 
mM 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) solution for 40 min at 
a standard temperature of 20 °C. The reaction was stopped by adding 0.5 ml of stopping solution 
(formalin(conc.):H3POiconc.)=1: 1). Formazan production was determined spectrophotometrically 
from the absorbance of the sample at 490 nm against the blank (1.5 ml substrate solution and 0.5 ml 
INT solution were incubated as for the sam ples, and 0.5 ml of homogenate was added after the stop-
ping). ETS activity was measured as the rate of tetrazolium dye reduction and converted to equiva-
lent oxygen utilised per dry mass (DW) in a given tirne interval as described by KENNER & AHMED 
(1975). 
86 Acta Biologica Slovenka, 47 (2), 2004 
Analysis of data: ETS/R ratios were calculated as the ratio of the calculated maximum oxygen 
consumption (ETS), as measured by in vitro enzymatic rates, to the rate of respiration (R) in vivo 
(OWENS & KING 1975). An analysis of variance (ANOVA) was carried out to test for differences 
between species. Linear regression between dry mass (DW) and the parameters investigated (ETS 
activity, respiration rate, ETS/R ratio), and between ETS activity and respiration rate were calculat-
ed after logarithmic transformation of data, according the power function Y = a Xb (log Y = log a + 
b log X) (DEL GIORGIO 1992). The statistical tests were performed using Microsoft Excel. 
Results 
ETS activity 
ETS activity was found to differ significantly between species (Tab. 1). The highest value (27.6 
µL 0 2 mg· 1 h-1) was measured in Paratanytarsus (Fig. la). Significantly lower values were observed 
in Procladius (18.5 µL 02 mg-1 h· 1), followed by Chironomus (14.0 µL 0 2mg-l h- 1) and Zavrelimyia 
(10.3 µL 0 2 mg· 1 h-1). 
The relationship between ETS activity and body mass is shown in Fig. 2a. The mass-specific 
ETS activity of chironomids decreased with increasing size of the animals (log ETS = 1.06 - 0.18 
log DW; r=-0.53 ; n=68; p<0.001). The b value was 0.82. The body mass of the animals used in the 
experiments differed significantly between species (Tab. 1). The mean values (± SD) were 0.07 ± 
0.04 mg DW for Paratanytarsus, 0.08 ± 0.03 mg DW for Zavrelimyia, 0.35 ± 0.27 mg DW for 
Procladius and 1.06 ± 0.94 mg DW for Chironomus. 
Table 1: Results of analysis of variance applied to ETS activity, respiration rate (R), ETS/R ratio and body 
mass (DW) in chironomids with species as factor; d.f. - degree of freedom. 
Tabela 1: Rezultati analize variance za aktivnost ETS, stopnjo dihanja (R), razmerje ETS/R in telesno maso 
(DW) pri ličinkah trzač z vrsto kot faktorjem; d.f.-stopinje prostosti 
d.f. 
between group 
ETS 3 
R 3 
ETS/R 3 
DW 3 
d.f. 
within group 
64 
39 
38 
75 
F-value 
14.29 
7.94 
15.34 
17.94 
P-value 
<0.001 
<0.001 
<0.001 
<0.001 
T. Simčič : Respiration rate and respiratory electron transport system ... 87 
45 
40 a) 
35 
,-.., 
~ 30 
>, -..... ' ·;; ~ 25 ·.::: Cl 
(.) oIJ 
~ s 20 
E-< N 
i:i:i o 
...l 15 
:i. 
'-' 
10 
5 
o 
Chironomus Paratanytarsus Procladius Zavrelimyia 
20 
18 b) 
16 
- 14 2 -~ -
c<l - 12 ~ ~ 
.8 Cl 10 ..... oIJ _g s 
- - - ---
o. N 8 "' o <l) 
~ ...l 
:i. 6 '-' - - -
4 - + . 
2 
o 
Chironomus P aratanytarsus Procladius Zavrelimyia 
Figure 1: (a) ETS activity and (b) respiration rate of different chironomid larvae (mean ± standard devia-
tion) at 20 °c. 
Slika 1: (a) Aktivnost ETS in (b) stopnja dihanja pri ličinkah različnih vrst trzač (srednja vrednost ± stan-
dardna deviacija) pri 20 °C. 
88 Acta Biologica Slovenica, 47 (2), 2004 
Table 2: The ratios ETS/R for different species of chironomids, measured at 20 °C. 
Tabela 2: Razmerja ETS/R za različne vrste trzač, izmerjena pri 20 °C. 
n ETS/R ratio Standard deviation 
Chironomus thummi 
Paratanytarsus austriacus 
Procladius (Holotanypus) spp. 
'Zavrelimyia barbatipes 
18 
9 
8 
7 
2.73 
2.44 
3.88 
0.98 
60 r------------------, 
50 ~ 
l 
o 
o 0.5 
Body mass (mg) 
o o o o 
o 
o 
a) 
1.5 
60 .--------- ----------, 
b) 
50 
JO 
,, 
o 8 
~d>i,~. ; o 8 o~o o ~ o o 
o 
o 0.5 1.5 
Body mass (mg) 
• Procladius l!I Zavrelimyia i> Paratanytarsus o Chironomus 
0.93 
0.86 
0.75 
0.21 
Figure 2: The relationship between (a) ETS activity and body mass (DW), and (b) respiration rate (R) and 
body mass of different chironomid Iarvae at 20 °c. 
Slika 2: Razmerje med (a) aktivnostjo ETS in telesno maso (DW) ter (b) stopnjo dihanja in telesno maso pri 
ličinkah različnih vrst trzač pri 20 °C. 
T. Simčič: Respiration rate and respiratory electron transport system ... 89 
Respiration rate 
Respiration rate differed significantly between species (Tab. 1). The highest values were obtained for 
Paratanytarsus (12.4 µL 0 2 mg·1 h·1) and 'Zavrelimyia (11.9 µL 0 2 mg·1 h-1), while respiration rates of 
Chironomus (5.7 µL 0 2 mg-1 h-1) and Procladius (4.0 µL 0 2 mg- 1 h- 1) were approximately two and three 
times lower (Fig. 1 b ). The regression between respiration rate and body mass of chironornids was statis-
tically significant (log R = 0.60- 0.34 log DW; r=-0.71; n=43; p<0.001; Fig. 2b); b value was 0.66. 
The ETS/R ratio 
The ratio ETS/R differed significantly between species (Tab. 1). A relatively low value, close to 
1, was determined for 'Zavrelimyia, while the highest ratio of 3.88 was obtained for Procladius (Tab. 
2). Positive correlation between ETS/R ratios and body mass was observed (r=0.61; n=27; p<0.001). 
Respiration rate correlated strongly with ETS activity for all-four species (Fig. 3). Correlation 
coefficients and regression constants for relationships between ETS activity and respiration rate for 
each species are shown in Tab. 3. 
Table 3: Relationship between ETS activity and respiration rate of four chironomid species (<lata were log 
transformed). 
Tabela 3: Razmerje med aktivnostjo ETS in stopnjo dihanja pri štirih vrstah trzač (podatki so bili logarit-
mirani). 
Chi ronomus thummi 
Paratanytarsus austriacus 
Procladius (Holotanypus) spp. 
7.avrelimyia barbatipes 
2 
i 1,5 
§ 
~~ 
~ e 
10,5 
o 
o 0,5 
n 
18 
9 
8 
7 
Correlation b loga P-value 
coefficient (slope) (intercept) 
0.73 0.56 0.72 <0.001 
0.82 1.47 -1.02 <0.01 
0.71 0.41 0.09 <0.05 
0.85 0.74 0.30 <0.05 
1,5 2 
log ETS activity 
• Procladius • Zavrelimyia A Paratanytarsus o Chironomus 
Figure 3: The relationship between ETS activity and respiration rate of different chironomid larvae. 
Correlation coefficients and regression constants are given in Tab. 3. 
Slika 3: Razmerje med aktivnostjo ETS in stopnjo dihanja pri ličinkah različnih vrst trzač. Korelacijski in 
regresijski koeficienti so podani v Tab. 3. 
90 Acta Biologica Slovenica, 47 (2), 2004 
Discussion 
Results of the present study revealed that ETS activity and respiration rate differed significantly 
between chironomid species (Fig. 1; Tab. I ). Chironomid species vary significantly in body size 
(Tab. 1) and thus characters related to body size would be expected to vary between species. 
KONSTANTINOV (1971) reported that the mass-specific respiration rate of chironomid larvae was 
inversely related to body size. An effect of size on ETS activity was also found in previous investi-
gations, with larger species having lower ETS activities (SIMČIČ & BRANCELJ 1997, 2001). The larg-
er average body sizes of Chironomus and Procladius were probably one of the reasons for the lower 
metabolic activity than in the smaller larvae of Zavrelimyia and Paratanytarsus. The reasons for 
higher metabolic activity in smaller animals are well discussed in PETERS (1983). 
The significant correlations between body mass and ETS activity and between body mass and 
respiration rate confirmed the effect of body size on metabolic activity in chironomids (Fig. 2). A b 
value of 0.82 was found in the case of ETS activity and 0.66 for respiration rate. HAMBURGER & DALL 
(1990) found a value of 0.73 for respiration rate for four different species of chironomids. Similar 
values of ETS activity were reported for the benthic macrofaunal species Corophium volutator, 0.86 
(CAMMEN & al. 1990), for Nereis virens, 0.84 (CAMMEN & al. 1990), for Chelicorophium 
curvispinum, 0.66 (MusK6 & al. 1995), and for Anostraca Chirocephalus croaticus, 0.787 (SIMČIČ & 
BRANCELJ 2000). In general, b values ranged from 0.58 to 0.96 (see LAMPERT 1987). However, the 
distribution of ETS activity of Zavrelimyia compared to similar sized individuals of Paratanytarsus, 
Chironomus and Procladius (Fig. 2a) indicated that other factors, beside body size, had influenced 
on ETS activity. Larvae of Zavrelimyia are considered as cold-stenotherms (FERRARESE 1983, 
FITTKAU & ROBACK 1983). Therefore, one of the reasons for Iow ETS activities of Zavrelimyia was 
probably respiratory enzyme systems with narrow temperature optima which caused their inactiva-
tion at temperature of 20 °C. Contrary to ETS activities, respiration rates of Zavrelimyia were close 
to those of similar-sized individuals of Paratanytarsus. 
ETS activity correlated significantly with respiration race in all species investigated (Fig. 3). 
These results are in accord with those of KING & PACKARD (1975) and SIMČIČ & BRANCELJ (2001) 
who also found a correlation between ETS activity and respiration rate in invertebrate species. 
However, ETS activity is a direct enzymatic process, depending on the concentration (BAMSTEDT 
1980) and characteristics (PACKARD 1971) of the enzymes, whereas respiration is a complex physi-
ological process. It means that respiration is also influenced by the intact intracellular environment, 
substrate concentrations, and structure and properties of intact lipid membranes (WITHERS 1992). As 
investigated species differed in body size (Tab. 1) and their ecological tolerance (FITTKAU & R0BACK 
1983, PINDER & RE1ss 1983), the differences in the ETS activity-respiration rate relationship between 
them were expected. 
One of the purposes of the present study was to obtain an ETS/R ratio that could be used for esti-
mating actual metabolism in chironomids from ETS activity. As shown in Tab. 2 the values of this 
ratio differed significantly between cbironomid species (Tab. 1). In the present experiments, the basal 
metabolism and expenditure on locomotion were measured. The expenditure on feeding and specif-
ic dynamic action (SDA) was minimal, because the animals were not fed just prior to or during the 
experiments. The low ETS/R ratio in Zavrelimyia means tbat this species exploited 100% of its meta-
bolic potential for basal metabolism and locomotion. Procladius had a much higher ETS/R ratio, 
wbich means less intensive exploitation of metabolic potential for basal metabolism and activity 
(-25% ). These ratios are similar to the values found in other studies conceming invertebrate species. 
T. Simčič ; Respiration rate and respiratory electron transport system ... 91 
Ratios approaching 2 are characteristic of zooplankton (BAMSTEDT 1980, JAMES 1987, SIMČIČ & 
BRANCELJ 1997) but, in the case ofbenthic species, higher values were determined. MusK6 & al. (1995) 
found that the arnphipod Chelicorophium curvispinum exhibited an ETS/R ratio of 4.07 at 20 °C. 
CAMMEN & al. (1990) reported an ETS/R ratio for Corophium volutator of 2.38, and for the poly-
chaete Nereis volutator 11 .11 (measured at 10 °C). The latter extremely high value was explained by 
N. volutator 's low activity. ETS/R ratio was also influenced by the size of organisms. Present results 
are in accord with those of CAMMEN & al. (1990) who found that larger animals have higher ETS/R 
ratios. The reason for higher ETS/R ratios is the greater decrease in respiration relative to ETS activ-
ity with increasing body size. Previous studies have also shown that the ETS/R ratio differs between 
related species having different ecological tolerance and preference, and consequently they exploit 
their metabolic potential differently (MUSK6 & AL. 1995, SIMČIČ & BRANCELJ 1997). FANSLOW & al. 
(2001) reported that, in organisms with high ratios, the capacity for elevated metabolism is main-
tained so that maintaining the enzyme machinery for increased metabolic activity is an advantage. 
Investigated chironomid species have different temperature and food preference and tolerance 
(FITTKAU & R0BACK 1983, PINDER & REISS 1983), and it is reasonable to assume that ETS/R ratio is 
influenced by these differences as well. Low ETS/R ratio of Zavrelimyia suggest that its energy 
metabolism is less adaptable to environmental changes compared with the more widely distributed 
Chironomus, Paratanytarsus and Procladius. However, further studies will provide additional data 
in order to obtain detailed conclusions related to relationship between ETS/R ratio and different eco-
logical demands of chironomid larvae. Nevertheless, as ETS/R ratio differed significantly between 
chironomid species, the energy flux through macrozoobentos has to be estimated by using different 
conversion factors. Preliminary studies on the estimation of energy flow through respiration in 
mountain lakes revealed that contribution of chironomids to total respiration differed between shal-
low oligotrophic and eutrophic lakes. 
Conclusions 
l. ETS activity and respiration rate, measured at a standard temperature of 20 °C, differed 
between chironomid species; the body size of species is one of the factors that affected metabolic 
activity; b value was 0.82 for ETS activity and 0.66 for respiration rate. This indicates the greater 
decrease in respiration rate relative to ETS activity with increasing body size. 
2. The ETS-assay is shown to be a convenient method for estimating respiration rate in chirono-
mid larvae. 
3. ETS/R ratios differ between species, so the use of different conversion factors to calculate res-
piration rate from ETS activity in different chironomid species is recommended. The differences can 
be explained by different species-specific body size, and different ecological tolerance and prefer-
ence of chironomid species. 
Povzetek 
Ličinke trzač (Chironomidae) so široko razširjena skupina, ki ima pomembno vlogo pri pretoku 
energije skozi prehranjevalne splete, še zlasti v plitvih jezerih. Predhodne raziskave so pokazale, da 
ličinke trzač številčno prevladujejo v makrozoobentoški združbi gorskih jezerih SZ Slovenije, zato 
92 Acta Biologica Slovenica, 47 (2), 2004 
je pri oceni pretoka energije v jezeru potrebno upoštevati tudi njihov delež. Ker je neposredna 
določitev stopnje dihanja običajno zamudna in nepraktična, so razvili biokemijsko metodo, s katero 
merimo aktivnost dihalnega elektronskega transportnega sistema (ETS), ki predstavlja metabolni 
potencial organizmov. Številne raziskave so pokazale, da obstaja med aktivnostjo ETS in dihanjem 
pozitivna korelacija, tako da lahko na osnovi izmerjene aktivnosti ETS s pomočjo razmerja ETS/R 
hitro ocenimo intenzivnost dihanja organizmov. Stopnjo dihanja in aktivnost ETS smo določali pri 
ličinkah štirih različnih vrst trzač, ki smo jih nabrali v treh gorskih jezer: ličinke Chironomus thum-
mi v Krnskem jezeru, ličinke Paratanytarsus austriacus in Procladius (Holotanypus) spp. v 
Zgornjem Kriškem jezeru in ličinke Zavrelimyia barbatipes v Srednjem Kriškem jezeru. Aktivnost 
ETS in stopnja dihanja, ki smo jo določali pri standardni temperaturi 20 °C, sta se značilno razliko-
vali med vrstami. Aktivnost ETS in stopnja dihanja trzač sta se zmanjševali z naraščajočo velikostjo 
živali, kar kaže na vpliv telesne velikosti na intenziteto metabolizma. Opazili smo pozitivno 
korelacijo med stopnjo dihanja in aktivnostjo ETS pri vseh štirih vrstah, kar pomeni, da je metoda 
ETS primerna za oceno dihanja tudi pri ličinkah trzač. Raziskava je pokazala značilne razlike v 
razmerju ETS/R med preiskovanimi vrstami trzač. Različna razmerja so posledica razlik v vrstno-
specifični telesni velikosti trzač ter njihove različne ekološke preference in tolerance. 
Acknowledgements 
I would like to thank Dr. Pain for linguistic improvement of the manuscript and two anonymous 
reviewers for helpful comments. This work was financially supported by the Slovenian Ministry of 
Education, Science and Sport (Project SLO-Alpe2). 
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ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2004 Vol. 47, Št. 2: 95-111 
Sprejeto (accepted): 2004-09-17 
Diverziteta dnevnih metuljev (Lepidoptera: Rhopalocera) v 
Regijskem parku Škocjanske jame 
Diversity of Bytterfly Fauna (Lepidoptera: Rhopalocera) in the 
Skocjanske jame Regional Park 
Tatjana ČELIK 
Jovan Hadži Institute of Biology, Scientific Research Centre of the Slovenian 
Academy of Sciences and Arts, Novi trg 2, SI - 1000 Ljubljana, Slovenia; 
fax : +386 1 425 77 97; e-mail: tcelik@zrc-sazu.si 
Izvleček. V letih 2001 in 2002 smo v Regijskem parku Škocjanske jame 
popisovali favno dnevnih metuljev v 9 habitatnih tipih: suhi travniki, polsuhi 
travniki, zaraščajoči suhi travniki, gojeni travniki, njive, gozdni robovi, gozdne 
poti, melišča in naselja. Zabeležili smo 90 vrst dnevnih metuljev, kar je 50 % favne 
dnevnih metuljev Slovenije. Prevladujejo vseevropske (33 %), južnoevropske (20 
% ) in južnovzhodnoevropske (17 % ) vrste. Favno sestavljajo travniške (56 vrst) , 
grmovne (16 vrst), gozdne (8 vrst) in ubikvitame (10 vrst) vrste. Med travniškimi 
vrstami je 34 (39 %) mezofilnih in 22 (24 %) kserotermofilnih vrst. 18 (20 %) reg-
istriranih vrst je evropsko in nacionalno ogroženih. Sestava favne metuljev v habi-
tatnem tipu je odvisna od vrstne sestave in strukturiranosti vegetacije v habitatnem 
tipu ter zgradbe krajinskega mozaika, ki vpliva na disperzijo vrst. Alfa diverziteta 
je največja na košenih (62 vrst) in zaraščajočih (60 vrst) suhih travnikih. Razloga 
za velike vrednosti beta diverzitet in visoko gama diverziteto sta prisotnost 
ekološko specializiranih vrst in heterogenost območja. Z vidika ohranjanja vrstne 
raznolikosti in ogroženih vrst dnevnih metuljev so v parku najpomembnejši nasled-
nji habitatni tipi: suhi travniki, polsuhi travniki, zaraščajoči suhi travniki, gozdne 
poti in gozdni robovi ter melišča. 
Ključne besede: regijski park Škocjanske jame, dnevni metulji (Rhopalocera), 
razširjenost, favnistični elementi, vrstna diverziteta, ogrožene vrste 
Abstract. In 2001 and 2002 butterfly diversity were sampled in Škocjanske 
jame Regional Park, across 9 habitat types: dry grasslands, semi-dry grasslands, 
unmanaged dry grasslands (dry grasslands in the early successional stages), culti-
vated grasslands, abandoned fields, woodland margins, woodland paths, tracks and 
rides, screes and settlements. A total of 90 species of butterflies were recorded, 
which represent 50% of Slovenian butterfly fauna. All-European (33%), south-
96 Acta Biologica Slovenica, 47 (2), 2004 
European (20%) and southeast-European (17%) species are prevailing. The butterfly fauna consists 
of grassland (56 spp.), seminemoral (16 spp.), nemoral (8 spp.) and ubiquitous (10 spp.) species. 
Among grassland species are 34 (39%) mesophilous and 22 (24%) xerothermophilous species. A 
total of 18 (20%) species are threatened at a European and a national leve!. Species composition of 
habitat type depends on floristic composition and structure of vegetation in habitat type, and land-
scape structure, which affects dispersal of butterfly species. The highest alfa diversity was found in 
dry grasslands (62 spp.) and unmanaged dry grasslands (60 spp.). The large proportions of special-
ist species and habitat heterogeneous area result in high values of beta diversity between habitat 
types and high gama diversity. For preserving the high species richness and threatened species in 
studied area, the most important habitat types are dry grasslands, semi-dry grasslands, unmanaged 
dry grasslands, woodland margins, woodland paths and screes. 
Keywords: Škocjanske jame Regional Park, butterflies (Rhopalocera), distribution, faunal ele-
ments, species diversity, threatened species 
Uvod 
Regijski park Škocjanske jame je bil ustanovljen 1.10.1996 z namenom varovati izjemne geomorfo-
loške, geološke in hidrološke znamenitosti, redke in ogrožene rastlinske in živalske vrste, paleontološka 
in arheološka najdišča, etnološke in arhitekturne značilnosti ter kulturno krajino (Ur.!. RS št. 57 /96) na 
območju Škocjanskih jam, ki so od leta 1986 vpisane v seznam svetovne dediščine pri UNESCO. Park 
se nahaja v občini Divača, na skrajnem jugovzhodnem obrobju mezoregije Kras (PERKO & KLADNIK 
1998). To je prehodno območje med apnencem matičnega krasa in flišnim svetom severozahodnega dela 
Brkinov (PARK ŠKOCJANSKE JAME 2003). Meja zavarovanega območja parka poteka na zahodu ob magis-
tralni cesti Divača-Kozina, na severu južno od naselij Dolnje Ležeče, Gradišče pri Divači in Brežec pri 
Divači, na severovzhodu po vrhnjem robu sten desnega brega reke Reke, na vzhodu južno od vasi Famlje 
do mostu čez Reko med Vremskim Britofom in Škofljami, na jugovzhodu po vrhnjem robu sten levega 
brega Reke, vključno s spodnjim delom iztočne soteske potoka Sušice, do vasi Naklo ter na jugu po 
obrobju kraške planote nad udornicama Sokolak in Globočak. Površina parka, ki obsega območje jam, 
površino nad jamami, sistem udornic in sotesko Reke do mostu pri Škofljah, je 4,13 km2 (Ur.!. RS št. 
57/96). Razgibano površje se nahaja na nadmorskih višinah med 300 in 450 metri. Na zavarovanem 
območju parka so tri manjša naselja s tipično kraško arhitekturo: Škocjan, Betanja in Matavun. 
Regijski park Škocjanske jame leži na vzhodnem robu dela Slovenije, ki ima submediteransko 
podnebje (OGRIN 1995). Povprečna letna temperatura zraka je med 10 in 12 °C, povprečna januars-
ka med O in 2 °C ter povprečna julijska temperatura med 16 in 18 °C (MAROLT 2003a). Povprečna 
letna količina padavin je 1400-1500 mm (MAROLT 2003b). 
Upoštevaje zoogeografsko regionalizacijo Slovenije po CARNELUTTiju (1992) sodi regijski park 
Škocjanske jame v primorsko regijo. WRABER (1969) uvršča Škocjanske jame z okolico v submedit-
eransko fitogeografsko območje, ZUPANČIČ & al. (1987) ter ZUPANČIČ & ŽAGAR (1995) pa jih uvrščajo 
v Kraško-vipavski distrikt severnega obalnega sektorja jadranske province mediteranske regije. 
Razgiban relief s sistemom udornic in vrtač (npr. Velika dolina, Mala dolina, Dol Globočak, Dol 
Sokolak, Dol Lisičina, Dol Jablanc, Sapendol, Lesen) in raznolik mikroklimatski režim na območju 
parka sta razloga za zelo raznovrstno rastlinstvo. Prevladuje kraška flora, ki jo sestavljajo termofilne 
ilirske, dinarske, balkanske in submediteranske vrste, manjši je delež srednjeevropskih, alpinskih in 
mediteranskih vrst (MARTINČIČ 2001). Avrikelj (Primula auricula), skorjasti kamnokreč (Saxifraga 
T. Čelik : Diverziteta dnevnih metuljev ... 97 
crustata), dvocvetna vijolica (Viola biflora) in skalna kernerjevka (Kernera saxatilis), značilne 
alpinske vrste, katerih pojavljanje na tem območju je pogojeno s poletno temperaturno inverzijo v 
udornicah, so ostanki ledenodobne flore (glacialni relikti). Topli zrak, ki izhaja pozimi iz podzeml-
ja, ustvarja specifičen zimski temperaturni režim, ki omogoča uspevanje mediteranskih vrst, kot so 
npr. venerini laski (Adiantum capillus-veneris), rdečeplodni brin (Juniperus oxycedrus) in ostrolist-
ni beluš (Asparagus acutifolius) . Te predstavljajo v Škocjanskih jamah termofilne relikte, ki so tu 
ostali iz borealnega obdobja holocena ali iz terciarja. (MARTINČIČ 2001) 
Primarna oblika vegetacije na območju Škocjanskih jam je klimatozonalna gozdna združba črnega 
gabra in puhastega hrasta Ostryo-Quercetum pubescentis (Ht 1950) Trinajstic 1974 (MARTINČIČ 2001). 
Zaradi izsekavanja gozda je v sedanjem času večinoma uničena. Kot degradacijske oblike primarne aso-
ciacije so razširjene sekundarne gozdne združbe, grmišča, travišča, obdelovalne in urbane površine. 
Pregled prispevkov o flori in vegetaciji Škocjanskih jam, objavljenih do konca prejšnjega stolet-
ja, je podal MARTINČIČ (1973, 2001). Kasneje so v okviru projekta Flora, favna in vegetacija regi-
jskega parka Škocjanske jame floro in gozdne združbe raziskovali DAKSKOBLER & al. (2002), veg-
etacijo gozdnih robov in grmišč je proučeval ČARNI (2002), traviščne in meliščne združbe, vegetaci-
jo zidov in skalnih razpok ter vodno in obvodno vegetacijo je opisal SELIŠKAR (2002). 
O favni regijskega parka Škocjanske jame je razmeroma malo podatkov in objav. Na poljuden način 
je favno podzemeljske Reke na območju Škocjanskih jam predstavil SKET (1999). POLENEC (1965, 
1968) je pisal o pajkih v okolici Divače. Inventarizacijo ptic na območju Škocjanskih jam sta napravila 
POLAK & TRONTELJ (neobjavljeno). Pregled raziskav in objav o mehkužcih, hroščih in metuljih na 
območju parka so zbrali SLAPNIK (2002), DROVENIK & PIRNAT (2002) in ČELIK (2002). O metuljih na 
območju Škocjanskih jam in okolice je izredno malo objavljenih virov. V preglednem delu » Verzeichnis 
der bisher in Krain beobachteten Grossschmeterlinge«, ki je najpopolnejša favna o metuljih Kranjske do 
l. svetovne vojne, HAFNER (1909) navaja le podatke iz okolice Senožeč. POLAK (1991) v kratkem 
prispevku o zooloških zanimivostih iz okolice Škocjanskih jam omenja tudi eno vrsto metulja. 
V prispevku predstavljamo rezultate proučevanj favne dnevnih metuljev v Regijskem parku 
Škocjanske jame. Glavni cilji so bili inventarizirati doslej neraziskano favno dnevnih metuljev tega 
zavarovanega območja, raziskati vrstno sestavo in diverziteto v različnih habitatnih tipih in opredeli-
ti tiste, ki so najpomembnejši za ohranitev redkih in ogroženih vrst. 
Metode dela 
Terenski popis 
Dnevne metulje v Regijskem parku Škocjanske jame smo proučevali v letih 2001 in 2002. Vrste 
smo popisovali na območjih, kjer so prisotni habitatni tipi, pomembni za prisotnost dnevnih metul-
jev na območju parka. Na vsakem območju smo opravili terenski popis vsaj enkrat v vsakem mese-
cu od aprila do oktobra. Čas, potreben za enkraten popis posameznega območja, je bil odvisen od 
zahtevnosti terena in raznolikos,ti habitatnih tipov. Vrste smo determinirali z opazovanjem odraslih 
osebkov, le posamezne primerke težje določljivih vrst smo ujeli z metuljnico. Po determinaciji smo 
jih večinoma izpustili, nekatere pa shranili za kasnejšo determinacijo v laboratoriju. 
Klasifikacija, opis in razširjenost proučevanih habitatnih tipov 
Za dnevne metulje primerne življenjske prostore smo razvrstili v devet habitatnih tipov: suhi 
kraški travniki (SuTr), polsuhi travniki (PSuTr), zaraščajoči suhi travniki (ZSuTr), gojeni travniki 
98 Acta Biolog: ica Slovenica, 47 (2), 2004 
(GoTr), opuščene njive (Nj), gozdni robovi (GoR), gozdne poti (GoP), melišča (Me) in naselja (Na). 
Tekoče vode (reka Reka), kali in jame so za dnevne metulje neprimerni življenjski prostori. 
Vegetacija suhih in polsuhih travnikov večinoma pripada redu toploljubnih submediteransko-
ilirskih suhih travišč. Travnike kosijo enkrat v letu. Suhi travniki so razširjeni na plitkih tleh, pred-
vsem na uravnavah med udornicami in vrtačami. Polsuhi travniki poraščajo globlja tla v večjih 
depresijah in manjših vrtačah. Zaraščajoči suhi travniki so opuščena suha travišča, ki jih ne kosijo. 
Zaraščajo se z grmovnimi in drevesnimi vrstami gozdnega roba ter dajejo pokrajini značilno podobo 
kraške gmajne. Gojene travnike gnojijo in kosijo dvakrat na leto. Razširjeni so predvsem v dnu vrtač 
in na rahlo nagnjenih pobočjih. Opuščene njive se pojavljajo večinoma na dnu vrtač. Zaraščajo se s 
plevelno in ruderalno vegetacijo. Gozdne poti na območju parka so neasfaltirane sprehajalne in kole-
sarske steze ter kolovozi, speljani skozi gozd in zaraščajočo kraško gmajno. Zaradi vrzelastega 
sklepa krošenj so poti večinoma svetle, z razgibano zgradbo (drevesna, grmovna, zeliščna vegetaci-
ja, skale, gola tla) in pestro rastlinsko sestavo. Poti na obeh straneh mejijo na svetle gozdove (izje-
ma sestoji črnega bora in mezofilne gozdne združbe na pobočjih velikih udornic), ki jih ponekod 
prekinjajo zadnji sukcesijski stadiji zaraščanja nekoč suhih kraških travišč. V primerjavi s potmi so 
gozdni robovi ob traviščih strukturno in vrstno osiromašeni, ker travnike pokosijo popolnoma do 
gozda. Kjer gozd meji na melišče, je zeliščna vegetacija gozdnega roba še revnejša, saj manjkajo 
traviščne vrste. Melišča so razširjena na strmih pobočjih udornic. Zaradi grobe strukture tal so po 
floristični sestavi zelo revna. Za metulje primerne površine v naseljih so predvsem vrtovi, zidovi, 
groblje in druge ruderalne površine. 
Habitatne tipe smo primerjali glede na prisotnost vrst dnevnih metuljev. Numerično analizo 
podobnosti habitatnih tipov smo izvedli s programskim paketom SYN-TAX 2000 (PODANI 2001), z 
metodo hierarhične klasifikacije (Metoda popolnega povezovanja= Complete Linkage Clustering). 
Kot mero različnosti smo uporabili Sorensenov koeficient podobnosti. 
Opredelitev zoogeografske in ekološke pripadnosti vrst 
V saki vrsti smo določili zoogeografsko pripadnost po kriterijih evropske horizontalne 
razširjenosti vrst (CARNELUTII 1981). 
Na podlagi empirično pridobljenih avtekoloških podatkov na terenu in objavljenih virov o ekoloških 
potrebah vrst (npr. EBERT & RENNWALD 1993a, EBERT & RENNWALD 1993b, ScHWEIZERISCHER BUND FOR 
NATURSCHUTZ 1991, T0LMAN & LEWINGT0N 1997, WEIDEMANN 1995), smo vsako vrsto uvrstili v 
ekološko skupino. To sestavljajo vrste, ki imajo podobne ekološke potrebe in na določenem območju 
poseljujejo iste habitatne tipe (BLAB & KUDRNA 1982; KuDRNA 1986). Prednost ekološke klasifikacije 
vrst je predvsem v naravovarstvenem smislu, saj so v posamezni ekološki skupini združene vrste, ki so 
na določenem območju izpostavljene enakim antropogenirn dejavnikom. Pri razvrščanju v ekološke 
skupine srno upoštevali naslednje ekološke potrebe vrst: dejanske in potencialne hranilne rastline gosenic 
in metuljev ter mesta za počivanje in terrnoregulacijo odraslih osebkov. 
Ogroženost vrst 
Ogroženost vrst v evropskem in nacionalnem merilu smo povzeli po Rdečem seznamu evropskih 
dnevnih metuljev (van SwAY & WARREN 1998), Direktivi sveta Evropske skupnosti 92/43/E.E.C (= 
Habitatna direktiva) (van der MADE & WYNH0FF 1996), Konvenciji o varstvu prosto živečega 
evropskega rastlinstva in živalstva ter njunih naravnih življenjskih prostorov(= Bernska konvencija) 
(Ur.l. RS 17/55) ter Pravilniku o uvrstitvi ogroženih rastlinskih in živalskih vrst v rdeči seznam (Ur.!. 
RS 82/2002). 
T. Čelik : Diverziteta dnevnih metul jev ... 99 
Vrstna diverziteta in naravovarstveni pomen habitatnih tipov 
Naravovarstveno vrednost proučevanih habitatnih tipov smo opredelili na podlagi (1) vrstne 
diverzitete dnevnih metuljev ter (2) števila in razporeditve ogroženih vrst po posameznih habitatnih 
tipih. 
Vrstno diverziteto dnevnih metuljev smo opredelili na podlagi analize alfa (a), beta (f3) in gama 
(o) diverzitete inventariziranih vrst v letih 2001 in 2002. Alfa diverziteto smo ocenili kot število vrst 
v habitatnem tipu (KRYŠTUFEK 1999, MORENO & HALFFTER 2001). 
Beta diverziteta je merilo raznolikosti vrstne sestave med različnimi habitatnimi tipi (MORENO & 
HALFFTER 2001, HARRISON & lNOUYE 2002). Izračunali smo jo kot odstotek vrst, ki so prisotne le v 
enem od obeh habitatnih tipov, ki ju primerjamo (COLWELL & CODDINGTON 1994 cit. po MORENO & 
HALFFTER 2001) : 
B = (S;+Sr2V;i)/(S;+Si-V;)*l00, 
pri čemer je S; in Si število vrst v habitatnih tipih; in i; V;i je število vrst, ki se pojavljajo v obeh 
habitatnih tipih. Beta diverziteta ima vrednosti med O (vrstna sestava v obeh habitatnih tipih je enaka) 
in 100 (habitatna tipa poseljujejo različne vrste) (MORENO & HALFFTER 2001). 
Gama diverzitetaje število vrst na raziskovanem območju (MORENO & HALFFrER 2001). 
Nomenklaturni vir 
Za metulje smo nomenklaturo in sistematiko povzeli po KARSHOLT & RAZOWSKI (1996), za rast-
line po MARTINČIČ & al. (1999) in za vegetacijo po ZUPANČIČ (1997). 
Rezultati in razprava 
V regijskem parku Škocjanske jame smo v letih 2001 in 2002 zabeležili 90 vrst dnevnih metul-
jev (Tab. 1), kar je 50 % vseh vrst metuljev iz skupine Rhopalocera, ki živijo v Sloveniji. 
Tabela l . Seznam vrst dnevnih metuljev, opaženih v letih 2001 in 2002 v Regijskem parku Škocjanske jame, 
njihova ekološka pripadnost in ogroženost. Legenda: RSS_Pravilnik o uvrstitvi ogroženih rastlinskih in žival-
skih vrst v rdeči seznam, Priloga 16: Rdeči seznam metuljev (Lepidoptera) (Ur. l. RS 82/2002): E- prizade-
ta vrsta, V - ranljiva vrsta; RSE_Rdeči seznam evropskih dnevnih metuljev (van SWAY & WARREN 1998): EN 
- prizadeta vrsta, VU - ranljiva vrsta; FFH_Direktiva sveta Evropske skupnosti 92/43/E.E.C (= Habitatna 
Direktiva) (van der MADE & WYNHOFF 1996): II - vrsta s seznama Aneksa II k Habitatni Direktivi, IV - vrsta 
s seznama Aneksa IV k Habitatni Direktivi; BK_Konvencija o varstvu prosto živečega evropskega rastlinst-
va in živalstva ter njunih naravnih življenjskih prostorov(= Bernska konvencija) (Ur. l. RS 17/55): II - vrsta 
s seznama Aneksa II k Bernski konvenciji. (*razlaga okrajšav: glej Ekološka pripadnost vrst) 
Table I. List of butterflies found in Škocjanske jame Regional Park in 2001 and 2002, their ecological require-
ments and threat status. Legend: RSS_Slovenian Red List: Appendix 16: Red list of butterflies and moths 
(Lepidoptera) (Ur. l. RS 82/2002);: E - endangered species, V - vulnerable species; RSE_Red Data Book of 
European Butterflies (Rhopalocera) (van SwAY & WARREN 1998): EN - endangered species, VU - vulnerable 
species; FFH_Council Directive 92/43/E.E.C (= Habitats Directive) (van der MADE & WYNHOFF 1996): II -
species in Annex II, IV - species in Annex IV; BK_Convention on the conservation of European wildlife and 
natura) habitats (= Bern Convention) (Ur. l. RS 17/55): II - species in Annex II. (*for explanation see chapter 
Ekološka pripadnost vrst) 
100 Acta Biolog ica Slovenica, 47 {2}, 2004 
Vrsta 
Ekološka 
RSS RSE FFH BK 
sku ina* 
E1ynnis tages (Linnaeus, 1758) MezTr 
Spialia sertorius (Hoffmannsegg, 1804) KseTr v 
Pyrgus carthami (Hiibner, 1813) KseTr v 
Pyrgus malvae (Linnaeus, 1758) MezTr 
Pyrgus armoricanus (Oberthiir, 1910) KseTr v 
Pyrgus alveus (Hiibner, 1803) MezTr v 
Heteropterus morpheus (Pallas, 1771) KseTr 
Carterocephalus palaemon (Pallas, 1771) MezTr 
Thymelicus lineola (Ochsenheimer, 1808) MezTr 
Thymelicus sylvestris (Poda, 1761) MezTr 
Hesperia comma (Linnaeus, 1758) MezTr 
Ochlodes venata (Bremer & Grey, 1853) MezTr 
Zerynthia polyxena (Denis & Schiffermiiller, 1775) KseTr v IV II 
Parnassius mnemosyne (Linnaeus, 1758) MezTr v IV II 
Iphiclides podalirius (Linnaeus, 1758) KseGr 
Papilio machaon (Linaeus, 1758) MezTr 
Leptidea sinapis (Linnaeus, 1758) MezTr 
Anthocharis cardamines (Linnaeus, 1758) MezTr 
Aporia crataegi (Linnaeus, 1758) KseGr 
Pieris brassicae (Linnaeus, 1758) Ubikv 
Pieris rapae (Linnaeus, 1758) Ubikv 
Pieris napi (Linnaeus, 1758) MezTr 
Pontia edusa (Linnaeus, 1758) Ubikv 
Colias croceus (Fourcroy, 1785) Ubikv 
Co/is alfacariensis (Ribbe, 1905) MezTr 
Gonepteryx rhamni (Linnaeus, 1758) MezGo 
Hamearis /ucina (Linnaeus, 1758) MezGr 
Lycaena phlaeas (Linnaeus, 1758) MezTr 
Lycaena tityrus (Poda, 1761) MezTr 
Callophrys rubi (Linnaeus, 1758) MezGr 
Satyrium spini (Denis & Schiffermiiller, 1775) MezGr 
Satyrium ilicis (Esper, 1779) MezGr 
Satyrium acaciae (Fabricius, 1787) MezGr 
Leptotes pirithous (Linnaeus 1767) Ubikv 
Cupido minimus (Fuessly, 1775) MezTr 
Everes argiades (Pallas, 1771) MezTr 
Everes alcetas (Hoffmannsegg, 1804) MezTr 
Celastrina argiolus (Linnaeus, 1758) MezGr 
Scolitantides orion (Pallas, 1771) KseTr v vu 
Glaucopsyche a/exis (Poda, 1761) MezTr vu 
Plebeius argus (Linnaeus, 1758) MezTr 
Plebeius idas (Linnaeus, 1761) MezTr v 
Plebeius argyrognomon (Bergstriisser, 1779) MezTr v 
Aricia agestis (Denis & Schiffermiiller, 1775) MezTr 
Cyaniris semiargus (Rottemburg, 1775) MezTr 
Polyomatus amandus (Schneider, 1792) KseTr 
Polyomatus thersites (Cantener, 1835) KseTr E 
T. Čelik: Diverziteta dnevnih metuljev ... 101 
Vrsta 
Ekološka 
RSS RSE FFH BK sku ina* 
Polyomatus icarus (Rottemburg, I 775) Ubikv 
Meleageria bellargus (Rottemburg, 1775) KseTr 
Meleageria coridon (Poda, I 761) KseTr 
Libythea celtis (Laicharting, 1782) Ubikv 
Argynnis paphia (Linnaeus, 1758) MezGo 
Argynnis aglqja (Linnaeus, 1758) MezGr 
Argynnis adippe (Denis &Schiffermiiller) MezGr 
Jsoria lathonia (Linnaeus, 1758) MezTr 
Brenthis daphne (Denis & Schiffermiiller, 1775) KseGr 
Brenthis hecate (Denis & Schiffermiiller, I 775) KseTr 
Clossiana euphrosyne (Lynnaeus, 1758) MezGo 
Clossiana dia (Linnaeus, 1767) MezTr 
Vanessa atalanta (Linnaeus, 1758) Ubikv 
Vanessa cardui (Linnaeus, 1758) Ubikv 
Jnachis io (Linnaeus, I 758) Ubikv 
Polygonia c-album (Linnaeus, 1758) MezGo 
Euphydryas aurinia (Rottemburg, 1775) KseTr v vu II II 
Melitaea cinxia (Linnaeus, 1758) KseTr 
Me/itaea trivia (Denis & Schiffermiiller, 1775) KseTr v 
Me/itaea phoebe (Denis & Schiffermuller, 1775) KseTr 
Melitaea didyma (Esper, 1778) KseTr 
Melitaea aure/ia (Nickerl, 1850) KseTr v vu 
Melitaea britomartis (Assmann, 1847) KseTr v vu 
Melitaea athalia (Rottemburg, 1775) MezTr 
Limenitis camil/a (Linnaeus, 1758) MezGo 
Pararge aegeria (Linnaeus, 1758) MezGo 
Lasiommata megera (Linnaeus, 1767) MezTr 
Lasiommata maera (Linnaeus, 1758) MezGr 
Lopinga achine (Scopoli, 1763) MezGo vu IV II 
Coenonympha arcania (Linnaeus, 176 I) MezGr 
Coenonympha glycerion (Borkhausen, 1788) MezTr 
Coenonympha pamphi/us (Linnaeus, 1758) MezTr 
Pyronia tithonus (Linnaeus, 1767) MezGr 
Aphantopus hyperantus (Linnaeus, 1758) MezGr 
Maniola jurtina (Linnaeus, I 758) MezTr 
Erebia aethiops (Esper, 1777) MezGo 
Erebia medusa (Dennis & Schiffermuller, 1775) MezTr vu 
Melanargia galathea (Linnaeus, 1758) MezTr 
Minois dryas (Scopoli, 1763) KseTr 
Hipparchiafagi (Scopoli, 1763) KseGr 
Hyparchia semele (Linnaeus, 1758) KseTr v 
Arethusana arethusa (Denis & Schiffermiiller, 1775) KseTr 
Brintesia circe (Fabricius, 1775) KseTr 
102 Acta Biologica Slovenica, 47 (2), 2004 
Zoogeografska pripadnost vrst 
Na obravnavanem območju prevladujejo vseevropsko razširjene vrste (33 %), kijih je v Sloveniji 
le 19 % (Sl. 1). Geografska lega, mikroklimatske razmere in apnenčasta geološka podlaga razisko-
vanega območja se odražajo v večjem deležu južnoevropskih (20 % ) in južnovzhodnoevropskih (17 
%) vrst ter manjšem deležu vzhodnoevropskih (16 %) in srednjeevropskih vrst glede na zastopanost 
omenjenih skupin v Sloveniji. Odsotnost severnoevropskih vrst na območju parka je pričakovana 
glede na razširjenost osmih predstavnikov te skupine v Sloveniji : šest vrst živi le v Alpah ter sub-
alpinskem in montanskem pasu predalpske in dinarske regije, dve vrsti pa sodita v skupino dom-
nevno izumrlih vrst. Od osmih zahodnoevropskih vrst v Sloveniji, v parku Škocjanske jame živita le 
dve (Colias alfacariensis, Spialia sertorius). Vzrok je redkost preostalih šest vrst: dve živita le v 
osrednjem delu Julijskih Alp, ena v skrajnem jugozahodnem delu primorske regije, tri vrste pa so v 
Sloveniji domnevno izumrle. 
20% 
(a) 
z 
2% 
v 
20% 
J 
19% 
(b) 
Slika l. Porazdelitev dnevnih metuljev po kriterijih evropske horizontalne razširjenosti (CARNELUITI 1981) v 
Regijskem parku Škocjanske jame (a) in v Sloveniji (b) (Vs: vseevropske vrste, S: severnoevropske vrste, Sr: 
srednjeevropske vrste, J: južnoevropske vrste, V: vzhodnoevropske vrste, JV: južnovzhodnoevropske vrste, 
Z: zahodnoevropske vrste). 
Figure 1. Partitioning of butterl1ies according to european horizontal distribution (CARNELUITJ 1981) in Škoc-
janske jame Regional Park (a) and Slovenia (b) (Vs: all-European species, S: north-European species, Sr: 
rniddle-European species, J: south-European species, V: east-European species, JV: southeast-European 
species, Z: west-European species). 
Ekološka pripadnost vrst 
V Regijskem parku Škocjanske jame živijo vrste, ki pripadajo naslednjim ekološkim skupinam 
(Sl. 2): 
• mezofilne travniške vrste (MezTr): poseljujejo ne preveč intenzivno obdelane travnate, bogato 
cvetoče odprte površine (polsuhi travniki, vrtovi, z gosto vegetacijo zarasle opuščene njive, gozdni 
robovi in poti). 34 vrst; 
• mezofilne grmovne vrste (MezGr): poseljujejo bogato cvetoče gozdne robove, mejice in 
zaraščajoče travnike. 12 vrst; 
T. Čelik: Diverziteta dnevnih metul jev ... 103 
• mezofilne gozdne vrste (MezGo): poseljujejo svetle gozdove, gozdne jase, robove in poti. 8 
vrst; 
• kserotermofilne travniške vrste (KseTr): poseljujejo suha in topla travišča, peščene, kamnite in 
skalnate predele. 22 vrst; 
• kserotermofilne grmovne vrste (KseGr): poseljujejo suhe in tople grmiščne predele (robovi 
svetlih toploljubnih gozdov, zaraščajoča suha travišča, skalnati predeli in melišča). 4 vrste; 
• ubikvitarne vrste (Ubikv): poseljujejo bogato cvetoče predele vseh življenjskih prostorov 
primernih za metulje. V to skupino uvrščamo tudi migratome vrste. 10 vrst. 
KseTr 
24% 
Ublkv 
11% 
MezGo 
9% 
MezGr 
13% 
39% 
Slika 2. Porazdelitev dnevnih metuljev Regijskega parka Škocjanske jame po ekoloških skupinah (MezTr: 
mezofilne travniške vrste, MezGr: mezofilne grmovne vrste, MezGo: mezofilne gozdne vrste, KseTr: 
kseroterrnofilne travniške vrste, KseGr: kseroterrnofilne grmovne vrste, Ubikv: ubikvitarne vrste). 
Figure 2. Partitioning of butterflies of Škocjanske jame Regional Park among ecological forrnations (MezTr: 
mesophilous grassland species, MezGr: mesophilous seminemoral species, MezGo: mesophilous nemoral 
species, KseTr: xerotherrnophilous grassland species, KseGr: xerotherrnophilous serninemoral species, 
Ubikv: ubiquists). 
V favni dnevnih metuljev Regijskega parka Škocjanske jame prevladujejo travniške vrste (56 
vrst, 62 % ), mezofilnih (39 % ) je več kot kserotermofilnih (24 % ). Grmovnih vrst je 16 (17 % ), gozd-
nih pa le 8 (9 %). 
Vrstna sestava in ekološke značilnosti favne metuljev v habitatnih tipih 
Na podlagi števila in porazdelitve vrst določene ekološke skupine v posameznih habitatnih tipih 
(Sl. 3) lahko opredelimo habitatni tip, ki je najpomembnejši za obstoj izbrane skupine vrst (ekološki 
specialisti). Kserotermofilne travniške vrste so specialisti, za njihovo preživetje na obravnavanem 
območju so najpomembnejši suhi travniki (19 vrst, 86 %), zaraščajoči suhi travniki (15 vrst, 68 %) 
in svetle gozdne poti (12 vrst, 55 %) (Sl. 3). Mezofilne travniške vrste imajo širšo ekološko valenco 
kot kserotermofilne travniške vrste, saj jih več kot polovica prisotnih v petih habitatnih tipih: suhi 
travniki (25 vrst, 74 %), zaraščajoči suhi travniki (22 vrst, 65 %), polsuhi travniki (18 vrst, 53 %), 
gozdni robovi ( 17 vrst, 50 % ) in gozdne poti ( 17 vrst, 50 % ). Gozdne poti so ključnega pomena za 
obstoj mezofilnih gozdnih vrst, saj se v omenjenem habitatnem tipu pojavljajo vse vrste te ekološke 
104 Acta Biologica Slovenica, 47 (2), 2004 
skupine (8 vrst, 100 % ). Za mezofilne grmovne vrste imajo na obravnavanem območju največji 
pomen zaraščajoči suhi travniki (9 vrst, 75 %), za kserotermofilne grmovne vrste pa gozdni robovi 
(4 vrste, 100 %). 
25 
20 
15 
število vrst 
10 
5 
o 
Slika 3. Število vrst posamezne ekološke skupine v habitatnih tipih Regijskega parka Škocjanske jame (razla-
ga okrajšav: glej Metode dela in Ekološka pripadnost vrst). 
Figure 3. Number of species of each ecological formation in habitat types of Škocjanske jame Regional Park 
(for explanation see chapter Metode dela and Ekološka pripadnost vrst). 
Mobilnost odraslih osebkov je razlog, da se ekološko specializirana vrsta lahko pojavlja v različnih 
habitatnih tipih, vendar je stopnja ekološke navezanosti vrste na posamezen habitatni tip zelo različna 
zaradi različnih ekoloških potreb posameznih ontogenetskih stadijev. Larvalni stadiji monofagne 
(gosenice se hranijo z eno rastlinsko vrsto) ali oligofagne (gosenice se hranijo le z nekaj vrstami rastlin) 
vrste lahko živijo Je v enem habitatnem tipu, medtem ko se metulji iste vrste, če niso prehranski spe-
cialisti, lahko hranijo povsod, kjer so prisotne hranilne rastline odraslih osebkov. Vzrok za pojav stenek-
ih vrst v različnih habitatnih tipih je tudi spreletavanje odraslih osebkov, ki iščejo partnerje ter primer-
na mesta za počivanje in uravnavanje telesne temperature. Na podlagi rezultatov analize podobnosti 
habitatnih tipov glede na prisotnost vrst dnevnih metuljev (sl. 4) sklepamo, da je favna metuljev 
posameznega habitatnega tipa odraz vrstne sestave in strukturiranosti vegetacije. 
Po sestavi vrst sta si najbolj podobni združbi metuljev na suhih travnikih (SuTr) in zaraščajočih 
suhih travnikih (ZSuTr) v zgodnjih sukcesijskih stadijih (sl. 4). To je posledica podobnih ekoloških 
razmer v obeh habitatnih tipih, kar je razvidno iz razmerja med številom mezofilnih in kserotermofil-
nih travniških vrst (SuTr: 1,3; ZsuTr: 1,5). V primerjavi s košenimi suhimi travniki se na zaraščajočih 
pojavlja manj travniških vrst in več mezofilnih grmovnih in gozdnih vrst (sl. 3), razmerje med njimi 
je 4,9 (SuTr) oziroma 2,9 (ZSuTr). Favni metuljev obeh tipov suhih travnikov je najbolj podobna 
favna na gozdnih poteh. Mozaik svetlih gozdov, grmišč in zadnjih sukcesijskih stadijev nekdanjih 
T. Čelik: Diverziteta dnevnih metul jev ... 
0,95 
0,9 
0,85 
0,3 
0,75 
0,7 
0,65 
>- 0,6 
'š 0,55 
] 0,5 
:@ 0,45 
Q 0,4 
0,35 
0,3 
0,25 
0,2 
0,15 
0,1 
0,05 
o 
1 
1 
_L 
SuTr ZSuTr GoP 
105 
1 
--
PSuTr GoR GoTr Ni Me No 
Slika 4. Dendrogram podobnosti proučevanih habitatnih tipov Regijskega parka Škocjanske jame glede na vrst-
no sestavo dnevnih metuljev (SuTr: suhi travniki, PSuTr: polsuhi travniki, ZSuTr: zaraščajoči suhi travniki, 
GoTr: gojeni travniki, Nj: opuščene njive, GoR: gozdni robovi, GoP: gozdne poti, Me: melišča, Na: naselja). 
Figure 4. Dendrogram of researched habitat types of Škocjanske jame Regional Park according to the butter-
fly species composition (SuTr: dry grasslands, PSuTr: semi-dry grasslands, ZSuTr: unmanaged dry grass-
lands (dry grasslands in the early successional stages), GoTr: cultivated grasslands, Nj: abandoned fields, 
GoR: woodland margins, GoP: woodland paths, tracks and rides, Me: screes, Na: settlements). 
suhih travišč ob poteh, bogata zeliščna vegetacija in velik delež osvetljenih golih tal so razlogi, da je 
razmerje med številom mezofilnih in kserotermofilnih travniških vrst podobno (1,4) kot na obeh tipih 
suhih travnikov. Na gozdnih poteh je število mezofilnih grmovnih in gozdnih vrst večje kot na 
košenih in zaraščajočih suhih travnikih, zato je razmerje med travniškimi ter mezofilnimi grmovni-
mi in gozdnimi vrstami nižje (2,1). Na polsuhih travnikih je razmerje med mezofilnimi in kseroter-
mofilnimi travniškimi vrstami (4,5) trikrat večje kot na košenih in zaraščajočih suhih travnikih. Višja 
in gostejša zeliščna vegetacija, v kateri je več mezofilnih rastlinskih vrst kot na obeh tipih suhih 
travnikov, preprečuje pojavljanje nekaterih kserotermofilnih travniških vrst metuljev (S. orion, P. 
thersites, M. bellargus, M. coridon, M. didyma, M. aurelia, M. dryas, A. arethusa, B. circe). 
Vegetacija gozdnih robov je strukturno in vrstno revna, saj večinoma meji na travnike, ki jih pokosi-
jo do gozda (izjema zaraščajoči suhi travniki). Zato je v združbi metuljev razmerje med travniškimi 
ter mezofilnimi grmovnimi in gozdnimi vrstami še nižje (1,8) kot v vseh že omenjenih habitatnih 
tipih. V favni metuljev gojenih travnikov in opuščenih njiv prevladujejo ubikvitarne in splošno 
razširjene mezofilne travniške vrste (Sl. 4). Opuščene njive so bolj ugoden habitat za kserotermofilne 
travniške vrste metuljev kot gnojeni in košeni travniki. Na njivah, ki niso v rabi, cvetijo ruderalne 
rastlinske vrste, ki so pomemben vir nektarja, neporasla osončena tla pa so primerna mesta za ter-
moregulacijo odraslih osebkov. Zaradi majhnega števila opaženih vrst, se melišča in naselja po vrst-
ni sestavi favne metuljev najbolj razlikujejo od ostalih habitatnih tipov. 
106 Acta Biologica Slovenica, 47 (2), 2004 
Naravovarstveni pomen proučevanih habitatnih tipov 
V Regijskem parku Škocjanske jame je prisotnih 18 evropsko in nacionalno ogroženih vrst 
dnevnih metuljev (Tab. 1), ki predstavljajo 10 % favne dnevnih metuljev Slovenije. Med njimi je 11 
kserotermofilnih travniških vrst, 6 mezofilnih travniških vrst in ena mezofilna gozdna vrsta. Največ 
ogroženih vrst (13) živi na suhih travnikih, ki imajo tudi največjo alfa diverziteto (Tab. 2). 
Tabela 2. Alfa (a) diverziteta in število ogroženih vrst dnevnih metuljev v proučevanih habitatnih tipih regi-
jskega parka Škocjanske jame. 
Table 2. Alpha ( a) diversity and number of threatened butterfly species in researched habitat types of Škoc-
janske jame Regional Park. 
Habitatni Alfa (a) Stevilo 
tie diverziteta ogroženih vrst 
Suhi travniki 62 13 
Polsuhi travniki 32 4 
Zaraščajoči suhi travniki 60 9 
Gojeni travniki 18 1 
Opuščene njive 18 1 
Gozdni robovi 38 2 
Gozdne poti 51 6 
Melišča 5 1 
Naselja 3 o 
Na gojenih travnikih, opuščenih njivah, meliščih in v naseljih je število vrst najmanjše (Tab. 2). 
Velika razlika v številu vrst med omenjenimi in preostalimi habitatnimi tipi je razlog za visoke vred-
nosti beta diverzitete (nad 60 % vrst) med temi pari habitatnih tipov (Tab. 3). 
Tabela 3. Beta diverziteta (zgoraj, desno) dnevnih metuljev in število skupnih vrst (spodaj, levo) med 
proučevanimi habitatnimi tipi regijskega parka Škocjanske jame. 
Table 3. Beta diversity (upper, right) of butterfly species and the number of species in common (lower, left) 
between pairs of habitat types of Škocjanske jame Regional Park. 
SuTr PSuTr ZSuTr GoTr Nj GoR GoP Me Na 
SuTr 55,4 39,5 75,0 75,0 64,9 45,2 93,7 95,2 
PSuTr 29 54,0 61,1 75,0 67,9 56,9 84,4 100 
ZSuTr 46 29 74,2 76,2 60,0 45,8 93,4 95,0 
GoTr 16 14 16 50,0 75,6 76,8 95,5 95,0 
Nj 16 10 15 12 80,9 74,6 90,5 95,0 
GoR 26 17 28 11 9 56,5 89,7 97,5 
GoP 40 25 39 13 14 27 92,3 98,1 
Me 4 5 4 1 2 4 4 100 
Na 3 o 3 1 1 1 1 o 
T. Čelik: Diverziteta dnevnih metuljev ... 107 
MORENO & HALFFTER (2001) navajata, da so strukturiranost krajine, mobilnost in ekološka spe-
cializacija vrst pomembni dejavniki, ki vplivajo na beta diverziteto. Avtorja sta mnenja, da so nizke 
beta diverzitete na določenem območju posledica zelo mozaične pokrajine z veliko raznolikimi habi-
tatnimi krpami, ki delujejo kot koridorji za razširjanje vrst, ter velikega števila zelo mobilnih evriek-
ih vrst. V parku Škocjanske jame se melišča, naselja, gojeni travniki in opuščene njive po vrstni ses-
tavi metuljev zelo razlikujejo (visoka~ diverziteta) od ostalih habitatnih tipov (Tab. 3). To je posled-
ica velikega števila ekološko specializiranih vrst, ki živijo na suhih in polsuhih travnikih ter gozdnih 
robovih in poteh, ter načina gospodarjenja. Na gojenih travnikih, njivah in v naseljih se pojavlja 26 
vrst, od teh je 19 vrst iz skupin mezofilnih travniških in ubikvitarnih vrst, 7 vrst iz ostalih ekoloških 
skupin so predstavniki bolj mobilnih vrst. Večina vrst, ki se pojavljajo na gojenih travnikih, 
opuščenih njivah, meliščih in v naseljih je torej bolj mobilnih ali so generalisti in živijo tudi v ostal-
ih habitatnih tipih (Tab. 2, Tab. 3). Gojeni travniki, njive in površine v naseljih so prehranjevalni 
habitat odraslih osebkov teh vrst, saj imajo zaradi pogostih človekovih posegov v omenjenih habi-
tatnih tipih vrste manj možnosti, da izpeljejo celotne življenjske cikle. Predpostavko, da ti habitatni 
tipi nimajo ključnega pomena za vrstno diverziteto dnevnih metuljev v parku potrjuje tudi ugo-
tovitev, da 98 % (88) vrst živi v štirih drugih habitatnih tipih: košeni in zaraščajoči suhi travniki, 
gozdni robovi in poti. Vrstno bogastvo pa ni nujno najboljši pokazatelj naravovarstvene vrednosti 
območja: območje, v katerem živi malo vrst, ki so redke ali ogrožene ima večji naravovarstveni 
pomen kot območje, ki ga poseljujeje veliko število splošno razširjenih in pogostih vrst (STORK & al. 
2003). Ogroženo vrsto Parnassius mnemosyne smo na območju parka opazovali le na meliščih in 
polsuhem travniku na vznožju melišč udornice Sokolak. Druga vrsta, ki je v štirih, za vrstno bogast-
vo parka najpomembnejših habitatnih tipih (SuTr, ZSuTr, GoP, GoR) nismo našli, je modrinček 
Plebeius argyrognomon. Vrsta je v Sloveniji redka, na raziskovanem območju smo jo zabeležili le v 
Dolu Jablanc, na cvetočem gojenem travniku. Ta habitatni tip za obstoj vrste ni odločilen, saj vrsta 
živi predvsem na suhih zagrmičenih travnikih, gozdnih robovih in jasah, redkeje v opuščenih vino-
gradih, kamnolomih in na pustih, ruderalnih površinah. Najpogostejši hranilni rastlini gosenic sta 
pisana šmarna detelja (Coronilla varia) in sladki grahor (Astragalus glacyphyllos), včasih pa samice 
odlagajo jajčeca tudi na lucerno (Medicago sativa), ranjak (Anthyllis spp.), detelje (Trifolium spp.), 
turške detelje (Onobrychis spp.) in kozjo deteljo (Lembotropis nigricans) (SCHWEIZERISCHER BUND 
FOR NATURSCHUTZ 1991). Velika množina hranilnih rastlin gosenic na omenjenem travniku je razlog 
za prisotnost vrste v tem habitatnem tipu. Vse ostale ogrožene vrste (16), ki živijo v parku, najdemo 
v treh habitatnih tipih: na košenih in zaraščajočih suhih travnikih ter gozdnih poteh. 
Gama vrstna diverziteta dnevnih metuljev na raziskovanem območju je večja (90 vrst) kot 
največja alfa diverziteta (SuTr: 62 vrst) (Tab. 2). To pomeni, da vrstno bogastvo metuljev na območju 
parka ni odvisno le od števila vrst, ki živijo v habitatnem tipu z največjo alfa diverziteto, temveč od 
topografske in ekološke heterogenosti krajine. 
Zaključki 
Vrstna sestava in diverziteta dnevnih metuljev v regijskem parku Škocjanske jame je odraz 
geografske lege, relifne razgibanosti in ekološke heterogenosti območja. Prisotnost različnih habitat-
nih tipov je razlog za pojavljanje vrst iz šest ekoloških skupin: mezofilne travniške, mezofilne 
grmovne, mezofilne gozdne, kserotermofilne travniške, kserotermofilne grmovne in ubikvitarne 
vrste. Vrstno najbogatejši habitatni tipi so suhi travniki, zaraščajoči suhi travniki in gozdne poti. V 
108 Acta Biologica Slovenica, 47 (2), 2004 
njih se pojavlja 16 od 18 evropsko in nacionalno ogroženih vrst, ki živijo v parku. Sestava favne 
metuljev v habitatnem tipu je odvisna od vrstne sestave in strukturiranosti vegetacije v habitatnem 
tipu ter zgradbe krajinskega mozaika, ki vpliva na disperzijo vrst. Zaradi velike raznolikosti habitat-
nih tipov na majhnem območju (4 km2), mobilnosti odraslih osebkov ter razlik v ekoloških potrebah 
gosenic in metuljev, se nekatere ekološko specializirane vrste pojavljajo tudi v habitatnih tipih, ki 
nimajo ključnega pomena za njihov obstoj (GoTr, Nj, Na, Me) in v njih večinoma (izjema melišča) 
ni drugih, za te habitatne tipe specifičnih vrst. Ti habitatni tipi imajo v primerjavi z ostalimi nizke 
alfa diverzitete, kar je vzrok za visoke bete diverzitete med omenjenimi pari habitatnih tipov. Visoka 
gama diverziteta je odraz heterogenosti raziskovanega območja, ki ga poseljujejo ekološke special-
izirane vrste, vezane na določene habitatne tipe. Ugotovitvi, da se ogrožena vrsta P. mnemosyne 
pojavlja izključno v habitatnih tipih z nizko alfa diverziteto in da je gama diverziteta večja od mak-
simalne alfa diverzitete, pomenita, da je pri naravovarstvenem vrednotenju raziskovanega območja 
in oblikovanju naravovarstvenih smernic potrebno upoštevati dva dejavnika: alfa diverziteto in raz-
poreditev ogroženih vrst po habitatnih tipih. Z vidika ohranjanja vrstne raznolikosti in ogroženih vrst 
dnevnih metuljev so v parku najpomembnejši naslednji habitatni tipi: suhi travniki, polsuhi travniki, 
zaraščajoči suhi travniki, gozdne poti in gozdni robovi ter melišča. 
Summary 
Because of their extraordinary significance for the world's natural heritage, the Škocjanske jame 
were included in UNESCO's World Heritage List in 1986. The Škocjanske jame Regional Park was 
established on October l" 1996 with the intention of preserve exceptional geomorphological, geo-
logical and hydrological remarkablenesses, rare and threatened plant and animal species, paleonto-
logical and archeological sites, ethnological and arhitectural features, and traditional landscape (Ur.!. 
RS št. 57 /96). 
The Škocjanske jame Regional Park is situated in the south-western part of Slovenia, in the 
Divača municipality. It is placed on south-eastern part of the region Kras, on the passage from fly-
sch to limestone. It extends over an area of 413 hectares and encompasses the area of the caves, the 
surface above the caves, the system of collapsed dolines and the Reka river gorge to the bridge in 
Škoflje (Ur.I. RS št. 57/96). The sub-Mediterranean climate (OGRIN 1995) with very diverse micro-
climatic conditions in the dolines and their surroundings account for the presence of Mediterranean 
thermophilous flora and Alpine species (glacial relicts) (MARTINČIČ 2001). 
In this region of Slovenia, primary vegetation is climax forest association Ostryo-Quercetum 
pubescentis (Ht. 1950) Trinajstic 1974 (MARTINČIČ 2001). Because of forest clearing in past cen-
turies, above ali for pastures and meadows, the primary vegetation was destroyed. Abandonment of 
pasturing and stockbreeding, which was the phenomenon most common in the second half of the 201h 
century, reflects in mosaic of secondarily developed forest associations, scrub and dry grasslands in 
different succesional stages. 
Still, despite its conservation status, this area is relatively poorly known in entomofaunistical 
terms, and particularly with respect to its fauna of Lepidoptera. Thus, in the present study, we inves-
tigated species composition of the area, species richness and diversity in different habitat types, and 
significance of habitat types for preserving threatened species. 
In 2001 and 2002 butterfly diversity were sampled in Škocjanske jame Regional Park, across 9 
habitat types: dry grasslands, semi-dry grasslands, unmanaged dry grasslands, cultivated meadows, 
T. Čelik: Diverziteta dnevnih metuljev ... 109 
abandoned fields, woodland margins, woodland paths, tracks and rides, screes and settlements. 
Zoogeographical partitioning of registered butterfly species based upon European horizontal distri-
bution according to CARNELUTII (1981) was made. All species were sorted into ecological formations 
considering ecological classification ofEuropean butterflies (BLAB & KUDRNA 1982; KUDRNA 1986). 
Threat status of each species was determined allowing for Slovenian Red List of butterflies and 
moths (Lepidoptera) (Ur.!. RS 82/2002), Red Data Book ofEuropean Butterflies (Rhopalocera) (van 
SWAY & WARREN 1998), Council Directive 92/43/E.E.C (van der MADE & WYNHOFF 1996) and 
Convention on the conservation of European wildlife and natura! habitats (Ur. l. RS 17 /55). The sim-
ilarity in the species composition of the butterfly fauna of different habitat types was estimated using 
the programme package SYN-TAX (PODANI 2001). Nature conservation value of each habitat type 
and researched area asa whole was assessed on the basis of (1) alpha, beta and gama butterfly diver-
sity, (2) number of species of each ecological formation in each habitat type and (3) number and dis-
tribution of threatened species among habitat types. 
A total of 90 species of butterflies were recorded, which represent 50% of Slovenian butterfly 
fauna. All-European (33% ), south-European (20%) and southeast-European (17%) species are prevail-
ing. A total of 18 (20%) species are threatened at a European and a national level. The butterfly fauna 
consists of mesophilous grassland species (34 spp.), mesophilous seminemoral species (12 spp.), 
mesophilous nemoral species (8 spp.), xerothermophilous grassland species (22 spp.), xerother-
mophilous seminemoral species (4 spp.) and ubiquists (10 spp.). Species composition of habitat type 
depends on floristic composition and structure of vegetation in habitat type, and landscape structure, 
which affects dispersal of butterfly species. The highest alfa diversity was found in dry grasslands (62 
spp.) and unmanaged dry grasslands (60 spp.), between which the smallest difference in butterflies 
assemblages occurred. Both habitat types are also the most important for the existence of xerother-
mophilous grassland, mesophilous grassland and mesophilous seminemoral species in the researched 
area. Woodland paths and margins are of the greatest significance for mesophilous nemoral and 
xerothermophilous seminemoral species. A total of 88 (98%) registered species were found in four 
habitat types: dry grasslands, unmanaged dry grasslands, woodland paths and woodland margins. Two 
remaini,ng species, Pamassius mnemosyne and Plebeius argyrognomon, were registered on screes, 
semi-dry and cultivated grasslands, each of them in only one locality. Cultivated meadows, abandoned 
fields and settlements not contribute to species richness of the area (gama diversity). In these habitat 
types the additional nectar sources and suitable sites for termoregulation are the main reasons for 
appearance of imagoes of generalist and more vagile specialist species. The large proportions of spe-
cialist species and heterogeneous landscape result in high values of beta diversity between habitat 
types, and high gama diversity. Due to the facts, that threatened species Pamassius mnemosyne lives 
only in habitat types with very low alpha diversity and that gama diversity is higher than maximum 
alpha diversity, conservation management must base upon two measures: alpha diversity and distribu-
tion of threatened species among habitat types. For preserving the high species richness and threatened 
species in Škocjanske jame Regional Park, the most important habitat types are dry grasslands, semi-
dry grasslands, unmanaged dry grasslands, woodland margins, woodland paths and screes. 
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ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2004 Vol. 47, Št. 2: 113-115 
JUBILEJI 
Profesor dr. Miroslav Zei - devetdesetletnik 
Kazimir TARMAN in Jože ŠTIRN 
Profesor Miroslav Zei se je rodil 25. julija 1914 v Nabrežini pri Trstu. Po italijanski zasedbi Trsta 
se je družina Zeijevih umaknila v Slovenijo. Nov dom so našli v Mariboru. Tu je mladi Miro leta 
1932 maturiral. Po maturi se je vpisal na Filozofsko fakulteto v Ljubljani. Odločil se je za študij 
biologije, še posebno ga je pritegnila zoologija. Že leta 1936 je diplomiral in takoj začel pripravljati 
doktorsko tezo. Minila so le štiri leta po diplomi in že je obranil disertacijo:«Prispevek k morfologi-
ji in sistemu jadranskih vrst družine Maenidae (giric)« in bil potem promoviran za doktorja filozof-
skih ved. 
Kot, da bi dečku rojenemu ob morju, vile sojenice že pri rojstvu naklonile moč odkrivanja 
skrivnosti morskih širjav in globin, se je lotil raziskovanja ribjega sveta. Sprejel je izziv morja in se 
zaposlil kot asistent na Oceanografskem inštitutu v Splitu. Od samega začetka mu je stal ob strani 
mentor, ljubljanski prof. dr. Jovan Hadži, ki je bil tudi sam raziskovalno povezan z raziskovanjem 
podmorskega življenja. Spoprijel se je z ihtiološko problematiko. Lotil se je proučevanja giric (rod 
Maena), njihove neznane biologije in ekologije. S primerjalno anatomskimi proučevanji in eksperi-
mentalno metodo umetne oploditve jajčec ter vzgojo mladic, je dokazal obstoj treh vrst giric v 
Jadranskem morju. 
Splitsko obdobje je bilo zelo pomembno za njegovo osebno znanstveno pot in delovanje. Hkrati 
tudi zelo pomembno za širšo zoološko in biološko oceanografsko stroko. Z Zeijem začenja na 
Jadranu nov pristop v raziskavah ribjega bogastva, zlasti uvajanje ekološko in populacijsko urejene-
ga ribolova. Njegove tedanje raziskave in znanstvene objave o pridnenih ribjih naseljih so bile pio-
nirsko delo v sodobno biologijo rib in praktično uporabo tega znanja v ribolovu, tako na Jadranu kot 
v drugih območjih Sredozemskega morja. Prav te raziskave so ga napeljale na že omenjeno odkri-
vanje vrst pri giricah in za tiste čase biološke neznanke - njihovega menjavanja spola. Odkritje, ki je 
pozneje prišlo v ihtiološke in zoofiziološke učbenike. V splitskem obdobju je vodil tečaje in 
praktične vaje iz biologije morja za študente tedanjih jugoslovanskih univerz. Po letu 1945 pa je 
postal vršilec dolžnosti direktorja Oceanografskega inštituta v Splitu. 
Leta 1947 gaje povabil na ljubljansko univerzo njegov učitelj in predstojnik Zoološkega inštitu-
ta prof. dr. Jovan Hadži. Svet takratne Filozofske fakultete ga je potem imenoval za izrednega pro-
fesorja. Leta 1954 je postal že redni profesor. Po upokojitvi prof. Hadžija je leta 1956 postal pred-
stojnik Zoološkega inštituta na tedanji Prirodoslovno-matematični fakulteti Univerze v Ljubljani. 
Po prihodu na univerzo je prevzel predmet primerjalno anatomijo vretenčarjev. Kasneje je 
uvedel še predmeta biologija strunarjev in praktični tečaj iz biologije morja na rovinjskem morsko-
biološkem inštitutu JAZU, ki gaje kot direktor uspešno vodil vrsto let (1951 - 1960). S svojim ugle-
114 Acta Biologica Slovenica, 47 (2), 2004 
dom in organizacijsko spretnostjo je privabil na rovinjske tečaje iz morske biologije tudi učitelje in 
študente avstrijskih in nemških univerz. Tu je nastajala, v letih 1960-63, z njegovim sodelovanjem 
odlična knjiga: Fauna und Flora der Adria v redakciji znanega dunajskega profesorja dr. R. Riedla.V 
povojnih časih, ko je primanjkovalo raziskovalne opreme in znanstvene literature, je v zameno za 
ponujene usluge na inštitutu izpopolnil delovno opremo laboratorijev in obogatil, po umiku 
Italijanov, zelo osiromašeno knjižnico. 
Nekdanji učenci se spominjamo prof. Zeija kot odličnega učitelja. Dokaj težko snov iz primer-
jalne biologije vretenčarjev, še posebno manj privlačno osteološko materijo, je predstavljal s 
sproščeno besedo in sliko, zelo nazorno. Zato smo njegova predavanja poslušali z zanimanjem. 
· Filogenetsko podajana poglavja iz primerjalne anatomije so se odvijala kot napet film. Najtesnejši 
stik s profesorjem pa smo imeli študenti na praktičnih vajah iz biologije morja. Vodil je laborato-
rijske vaje in z nami na ladjici Bios križaril po »rovinjskem morju«, ko smo zajemali planktonske 
vzorce in z mrežo vlačnico strgali morsko dno. Ob njegovi razlagi smo spoznavali biotsko razno-
likost podmorskega življenja. V sprehodih po rovinjskem parku pa nam je približal še obalni rastlin-
ski in živalski svet. V vsem njegovem pedagoškem delu je izžarevala sla po predajanju znanja 
mladim. Bil je učitelj v najžlahtnejšem pomenu besede. V spominu nam je ostala tudi pokončna drža 
obeh učiteljev zoologije: prof. J. Hadžija in Zeija, ko sta ostala zvesta genetiki v nevarnih časih poli-
tično podprtega prodiranja šarlatanskega lisenkizma iz Sovjetske zveze tudi na ljubljansko Univerzo. 
Svoj pogled v obrambo genetikov sta posredovala na petkovih seminarskih urah. 
Profesorja Zeija pa poznamo njegovi sodelavci tudi kot prodornega in ustvarjalnega znanstveni-
ka. Zeijevo raziskovalno delo zajema snov iz sistematike, biologije in ekologije rib ter 
mnogoščetincev, morske ekologije in problemov speciacije populacij plazilcev (Lacertidae) na izoli-
ranih istrskih otočkih. Ukvarjal se je tudi z uvajanjem novih raziskovalnih metod in tehnik, n.pr. tedaj 
še s povsem pionirskim delom na podmorski televiziji. O svojih raziskovalnih problemih, največkrat 
povezanih z ribolovno tematiko se je z nami rad pogovarjal. Imel je izvirne zamisli o ekološko-pop-
ulacijskem pristopu k izkoriščanju ribjih naselij v našem morju. Žal je pri tedanjih ustanovah, ki so 
se ukvarjale z morskim ribištvom naletel pogosto na gluha ušesa. Posledice tega, kot je pojav »prelo-
va«, se kažejo pri upravljanju z ribjim bogastvom našega Jadrana še dandanes. Dela je objavljal v 
domačih in tujih revijah. 
Po prevzemu vodstva v Zoološkem inštitutu smo se dnevno srečevali pri kavici v njegovem kabi-
netu. Bili so to nepozabni delovni sestanki. S širokim pogledom na razvoj inštituta in biologije v 
celoti je razvijal zamisli o novih področjih. Tako je spodbujal širitev inštitutskih dejavnosti od 
splošne zoologije, zoološke sistematike in evolucije še na limnologijo in ekologijo živali. S svojimi 
poznanstvi je poskrbel za dopolnilno izobraževanje sodelavcev na univerzah in raziskovalnih inštitu-
cijah po svetu. 
Klic Pozejdona iz morja širjav ga je leta 1962 pozval na afriško morje. Postal je direktor 
oceanografsko-ribiških projektov ZN v Gani. S taksonomskimi, ekološko-populacijskimi in ribiško-
ekonomskimi raziskovanji je postavil osnovo za učinkovitejši ribolov v Gvinejskem zalivu. S podob-
nimi projekti se je kasneje ukvarjal še na FAO v Rimu, v Tuniziji in zahodni Afriki, od Maroka do 
Angole. Vodil je poslanstva Tehnične pomoči ZN v vzhodni in zahodni Afriki. Kot cenjen 
znanstvenik je bil član številnih mednarodnih delegacij in direktor morsko biološke šole pri FAO-ju 
ter UNESCO-ju v Carigradu. S svojimi raziskovalnimi izsledki in izkušnjami je prispeval k učinkovi­
tosti morskega ribištva. Kljub znanemu načelu, da FAO od sodelavcev ne pričakuje znanstvenih 
objav, ampak predvsem rezultate, ki so hitro in neposredno uporabni za razvoj gospodarnega ribištva, 
je prof. Zei objavljal svoje strokovne in znanstvene prispevke tudi v obdobju delovanja pri tej orga-
Jubile ·i 115 
nizaciji. Objavil je dela, ki so pomembna za območje osrednjega Atlantika in za boljše poznavanje 
biologije sardel ter njim sorodnih vrst. Kar 13 polnih let je deloval pri mednarodni organizaciji. S 
pridobljenim ugledom je potem privabil k sodelovanju pri ZN še štiri nekdanje učence, ki so potem 
postali nosilci pomembnih nalog pri FAO in UNESCO. Po upokojitvi pri ZN leta 1975 se je vrnil iz 
Rima v domovino in se naselil v Portorožu. 
Leta 1969 je bila ustanovljena Morska biološka postaja ali kratko MBP pri Inštitutu za biologi-
jo univerze v Ljubljani. S tem dejanjem se je izpolnila dolgoletna želja obeh profesorjev: Hadžija in 
Zeija, da bi tudi Slovenci dobili raziskovalno ustanovo na Jadranu. Po vrnitvi v domovino, je v letih 
1977-1983, prof. Zei prevzel mesto honorarnega predstojnika v tej ustanovi. S svojo avtoriteto je 
pospešil financiranje adaptacije in gradnjo novih prostorov v obmorski stavbi na Fomačah pri Piranu. 
V novo zgradbo se je inštitucija preselila leta 1980. Glede na pridobljene izkušnje je poskušal uvel-
javiti uporabna ribiško-biološka raziskovanja za razvoj marikultur komercialnih ribjih vrst in školjk. 
Tudi po vrnitvi iz Rima je ostal prof. Zei v svoji stroki zelo dejaven. Obnovil je naziv rednega 
univerzitetnega profesorja. Med leti 1976 - 1985 je predaval predmeta ihtiologijo z ribiško biologi-
jo in morsko biologijo. Od leta 1980- 1989 je bil glavni predavatelj cikličnega mednarodnega tečaja 
ribiške biologije in oceanografije za slušatelje afriških, azijskih in Jatino-ameriških držav, ki je 
potekal v organizaciji Zavoda SFRJ za mednarodno sodelovanje na Inštitutu za oceanografijo in 
ribištvo v Splitu. Po nalogu tega zavoda je opravil še misiji tehnične pomoči za razvoj morskega 
ribištva v Sao Tome - Principe in na Zanzibarju ( 1980 - 1982). 
Po letu 1983 se je posvetil pisanju znanstvenih in strokovnih člankov. Zbral in uredil je sloven-
ska imena morskih rib, s čimer je prispeval k Slovarju slovenskega jezika (SAZU, 1970- 1991). 
Prof. Zei je avtor mnogih poljudnoznanstvenih člankov in knjig. Veliko je pisal v naš Proteus. 
Kar 11 knjig je namenil morju, ribam in vretenčarjem. Knjiga Človek in ocean (1950) je še vedno 
edino slovensko delo iz oceanografije. Knjiga Življenje našega Jadrana (1947), ki jo je napisal sku-
paj s prijateljem- zdravnikom dr. J. Zhanelom, je bila dolga leta nepogrešljiva dijaška in študentska 
spremljevalka pri spoznavanju morskega življenja. Kot mojster umetniško zapisane besede se je 
predstavil s knjigo Obrazi morja (1987), kjer razkrije svoje globoko doživljanje in razumevanje 
morja, življenja in človeka. V zaporedju poglavij filozofsko povezuje znanost in resnico z umetnos-
tjo ter mistično domišljijo. Legende o morju se prepletajo s stvarnimi doživetji. Zeije mojster besede 
in razmišljanja. Ker pozna in ljubi morje piše iz srca. 
Na koncu mu avtorja tega zapisa želiva še veliko srečnih let, da bi nam še podarjal znanje in »zei-
jevsko« izpovedoval misli o morju in življenju v njem. Prepričana sva, da prav to želijo našemu jubi-
lantu vsi, ki ga poznajo in še posebno njegovi bivši ter hvaležni učenci . 
V Ljubljani in Portorožu, julija 2004 
NAVODILA AVTORJEM 
l. Vrste prispevkov 
a) ZNANSTVENI ČLANEK je celovit opis originalne raziskave in vključuje teoretični pregled 
tematike, podrobno predstavljene rezultate z diskusijo in sklepe ter literatumi pregled: shema IMRAD 
(Introduction, Methods, Results And Discussion). Dolžina članka, vključno s tabelami, grafi in slika-
mi, na sme presegati 15 strani; razmak med vrsticami je dvojen. Recenzirata ga dva recenzenta. 
b) PREGLEDNI ČLANEK objavi revija po posvetu uredniškega odbora z avtorjem. Število 
strani je lahko večje od 15. 
c) KRATKA NOTICA je originalni prispevek z različnih bioloških področij (sistematike, 
biokemije, genetike, mikrobiologije, ekologije itd.), ki ne vsebuje podrobnega teoretičnega pregleda. 
Njen namen je seznaniti bralca s preliminarnimi ali delnimi rezultati raziskave. Dolžina na sme pre-
segati 5 strani. Recenzira ga en recenzent. 
d) KONGRESNA VEST seznanja bralce z vsebinami in sklepi pomembnih kongresov in posve-
tovanj doma in v tujini. 
e) DRUŠTVENA VEST poroča o delovanju slovenskih bioloških društev. 
2. Originalnost prispevka 
Članek, objavljen v reviji Acta Biologica Slovenica, ne sme biti predhodno objavljen v drugih 
revijah ali kongresnih knjigah. 
3. Jezik 
Teksti naj bodo pisani v angleškem jeziku, izjemoma v slovenskem, če je tematika zelo lokalna. 
Kongresne in društvene vesti so praviloma v slovenskem jeziku. 
4. Naslov prispevka 
Naslov (v slovenskem in angleškem jeziku) mora biti kratek, informativen in razumljiv. Za 
naslovom sledijo imena avtorjev in njihovi polni naslovi (če je mogoče, tudi štev. faxa in e-mail). 
5. Izvleček - Abstract 
Podati mora jedrnato informacijo o namenu, uporabljenih metodah, dobljenih rezultatih in zaključkih. 
Primerna dolžina za znanstveni članek naj bo približno 250 besed, za kratko notico pa 100 besed. 
6. Ključne besede - Keywords 
Število naj ne presega 10 besed, predstavljati morajo področje raziskave, predstavljene v članku. 
Člankom v slovenskem jeziku morajo avtorji dodati ključne besede v angleškem jeziku. 
7. Uvod 
Nanašati se mora le na tematiko, ki je predstavljena v članku ali kratki notici. 
8. Slike in tabele 
Tabele in slike (grafi, dendrogrami, risbe, fotografije idr.) naj v članku ne presegajo števila 10, v 
članku naj bo njihovo mesto nedvoumno označeno. Ves slikovni material naj bo oddan kot fizični 
original (fotografija ali slika). Tabele in legende naj bodo tipkane na posebnih listih (v tabelah naj 
bodo le vodoravne črte). Naslove tabel pišemo nad njimi, naslove slik in fotografij pod njimi. Naslovi 
tabel in slik ter legenda so v slovenskem in angleškem jeziku. Pri citiranju tabel in slik v besedilu 
uporabljamo okrajšave (npr. Tab. 1 ali Tabs. 1-2, Fig. 1 ali Figs. 1-2; Tab. 1 in Sl. 1). 
9. Zakjučki 
Članek končamo s povzetkom glavnih ugotovitev, ki jih lahko zapišemo tudi po točkah. 
10. Povzetek - Summary 
Članek, ki je pisan v slovenskem jeziku, mora vsebovati še obširnejši angleški povzetek. Velja 
tudi obratno. 
11. Literatura 
Uporabljene literaturne vire citiramo med tekstom. Če citiramo enega avtorja, pišemo ALLAN 
(1995) ali (ALLAN 1995), če sta dva avtorja (TRlNAJSTIC & F'RANJIC 1994), če je več avtorjev (PULLIN 
& al. 1995). Kadar navajamo citat iz večih del hkrati, pišemo (HONSIG-ERLENBURG & al. 1992, WARD 
1994A, ALLAN 1995, PULLIN & al. 1995). V primeru, če citiramo več del istega avtorja, objavljenih 
v enem letu, posamezno delo označimo s črkami a, b, c itd. (WARD 1994a,b). Če navajamo dobesed-
ni citat, označimo dodatno še strani : TOMAN (1992: 5) ali (TOMAN 1992: 5-6). Literaturo uredimo po 
abecednem redu, začnemo s priimkom prvega avtorja, sledi leto izdaje in naslov članka, mednarod-
na kratica za revijo (časopis), volumen poudarjeno, številka v oklepaju in strani. Npr.: 
HONSIG-ERLENBURG W., K. KRAINER, P. MILDNER & C. W!ESER 1992: Zur Flora und Fauna des 
Webersees. Carinthia II 182/102 (1): 159-173. 
TRINAJSTic & J. FRANJJC 1994: Ass. Salicetum elaeagno-daphnoides (BR.-BL. et VOLK, 1940) 
M. MOOR 1958 (Salicion elaeagni) in the Vegetation in Croatia. Nat. Croat. 3 (2): 253-256. 
WARD J. V. 1994a: Ecology of Alpine Streams. Freshwater Biology 32 (1): 10-15. 
WARD J. V. 1994b: Ecology of Prealpine Streams. Freshwater Biology 32 (2): 10-15. 
Knjige, poglavja iz knjig, poročila, kongresne povzetke citiramo sledeče: 
ALLAN J. D. 1995: Stream Ecology. Structure and Function of Running Waters, 1st ed. 
Chapman & Hall, London, 388 pp. 
PULLIN A. S., l. F. G. MCLEAN & M. R. WEBB 1995: Ecology and Conservation of Lycaena dis-
par: British and European Perspectives. In: PULLIN A. S. (ed.): Ecology and Conservation of 
Butterflies, 1st ed. Chapman & Hall , London, pp. 150-164. 
TOMAN M. J. 1992: Mikrobiološke značilnosti bioloških čistilnih naprav. Zbornik referatov s 
posvetovanja DZVS, Gozd Martuljek, pp. 1-7. 
12. Format in oblika članka 
Članek naj bo poslan v obliki Word dokumenta (doc) ali kot obogateno besedilo (rtf) v pisavi 
"Times New Roman CE 12" z dvojnim medvrstnim razmakom in levo poravnavo ter s 3 cm robovi 
na A4 formatu. Odstavki naj bodo med seboj ločeni s prazno vrstico. Naslov članka in poglavij naj 
bodo pisani krepko in v velikosti pisave 14. Vsa latinska imena morajo biti napisana ležeče. 
Uporabljene nomenklaturne vire navedemo v poglavju Metode. Tabele in slike so posebej priložene 
tekstu. Vse strani (vključno s tabelami in slikami) morajo biti oštevilčene. Glavnemu uredniku je 
potrebno oddati original, dve kopiji in elektronski zapis na disketi 3,5", na CD-romu ali kot priponko 
elektronske pošte (slednjega odda avtor po opravljenih strokovnih in jezikovnih popravkih). 
13. Recenzije 
Vsak znanstveni članek bosta recenzirala dva recenzenta (en domači in en tuji), kratko notico pa 
domači recenzent. Avtor lahko v spremnem dopisu predlaga tuje recenzente. Recenziran članek, ki 
bo sprejet v objavo, popravi avtor. Po objavi prejme 30 brezplačnih izvodov. V primeru zavrnitve se 
originalne materiale vrne avtorju skupaj z negativno odločitvijo glavnega urednika. 
INSTRUCTIONS FOR AUTHORS 
l. Types of Articles 
a) SCIENTIFIC ARTICLES are comprehensive descriptions of original research and include a the-
oretical survey of the topic, a detailed presentation of results with discussion and conclusion, and a bib-
liography according to the IMRAD outline (lntroduction, Methods, Results, and Discussion). The 
length of an article including tables, graphs, and illustrations may not exceed fifteen (15) pages; lines 
must be double-spaced. Scientific articles shall be subject to peer review by two experts in the field. 
b) REVIEW ARTICLES will be published in the joumal after consultation between the editori-
al board and the author. Review articles may be longer than fifteen (15) pages. 
c) BRIEF NOTES are original articles from various biological fields (systematics, biochemistry, 
genetics, microbiology, ecology, etc.) that do not include a detailed theoretical discussion. Their aim 
is to acquaint readers with preliminary or partial results of research. They should not be longer than 
five (5) pages. Brief note articles shall be subject to peer review by one expert in the field. 
d) CONGRESS NEWS acquaints readers with the content and conclusions of important con-
gresses and seminars at home and abroad. 
e) ASSOCIATION NEWS reports on the work of Slovenc biology associations. 
2. Originality of Articles 
Manuscripts submitted for publication in Acta Biologica Slovenica should not contain previous-
ly published material and should not be under consideration for publication elsewhere. 
3. Language 
Articles and notes should be submitted in English, or as an exception in Slovenc if the topic is 
very local. As a rule, congress and association news will appear in Slovenc. 
4. Titles of Articles 
Titles (in Slovene and English) must be short, informative, and understandable. The title should 
be followed by the name and full address of the author (and if possible, fax number and e-mail 
address). 
5. Abstract 
The abstract must give concise information about the objective, the methods used, the results 
obtained, and the conclusions. The suitable length for scientific articles is approximately 250 words, 
and for brief note articles, 100 words. 
6. Keywords 
There should be no more than ten (10) keywords; they must reflect the field of research covered 
in the article. Authors must add keywords in English to articles written in Slovene. 
7. Introduction 
The introduction must refer only to topics presented in the article or brief note. 
8. lllustrations and Tables 
Articles should not contain more than ten (10) illustrations (graphs, dendrograms, pictures, pho-
tos etc.) and tables, and their positions in the article should be clearly indicated. Ali illustrative mate-
rial should be provided as physical originals (photographs or illustrations). Tables with their legends 
should be submitted on separate pages (only horizontal lines should be used in tables). Titles of 
tables should appear above the tables, and titles of photographs and illustrations below. Titles of 
tables and illustrations and their legends should be in both Slovene and English. Tables and illustra-
tions should be cited shortly in the text (Tab. 1 or Tabs. 1-2, Fig. 1 or Figs. 1-2; Tab. 1 and Sl. 1). 
9. Conclusions 
Articles shall end with a summary of the main findings which may be written in point form. 
10. Summary 
Articles written in Slovene must contain a more extensive English summary. The reverse also 
applies. 
11. Literature 
References shall be cited in the text. If a reference work by one author is cited, we write Allan 
(1995) or (Allan 1995); if a work by two authors is cited, (TrinajstiC & FranjiC 1994); if a work by 
three or more authors is cited, (Pullin & al. 1995); and if the reference appears in severa! works, 
(Honsig-Erlenburg & al. 1992, Ward 1994a, Allan 1995, Pullin & al. 1995). If severa! works by the 
same author published in the same year are cited, the individual works are indicated with the added 
letters a, b, c, etc.: (Ward 1994a,b). If direct quotations are used, the page numbers should be includ-
ed: Toman (1992: 5) or (Toman 1992: 5-6). 
The bibliography shall be arranged in alphabetical order beginning with the surname of the first 
author followed by the year of publication, the title of the article, the international abbreviation for the 
joumal (periodical), the volume (in bold print), the number in parenthesis, and the pages. Examples: 
HONSIG-ERLENBURG w., K. KRAINER, P. MILDNER & C. WIESER 1992: Zur Flora und Fauna des 
Webersees. Carinthia II 182/102 (1): 159-173. 
TRINAJSTic & J. FRANJJc 1994: Ass. Salicetum elaeagno-daphnoides (BR.-BL. et VOLK, 1940) 
M. MOOR 1958 (Salicion elaeagni) in the Vegetation in Croatia. Nat. Croat. 3 (2): 253-256. 
WARD J. V. 1994a: Ecology of Alpine Streams. Freshwater Biology 32 (1): 10-15. 
WARD J. V. 1994b: Ecology of Prealpine Streams. Freshwater Biology 32 (2): 10-15. 
Books, chapters from books, reports, and congress anthologies use the following forms: 
ALLAN J. D. 1995: Stream Ecology. Structure and Function of Running Waters, 1st ed. Chapman 
& Hall, London, 388 pp. 
PULLIN A. S., l. F. G. MCLEAN & M. R. WEBB 1995: Ecology and Conservation of Lycaena dis-
par: British and European Perspectives. In: PuLLIN A. S. (ed.): Ecology and Conservation of 
Butterflies, 1st ed. Chapman & Hall, London, pp. 150-164. 
TOMAN M. J. 1992: Mikrobiološke značilnosti bioloških čistilnih naprav. Zbornik referatov s 
posvetovanja DZVS, Gozd Martuljek, pp. 1-7. 
12. Format and Form of Articles 
Articles should be send as Word document (doc) or Rich text format (rtf) using "Times New 
Roman CE 12" font with double spacing, align left and margins of 3 cm on A4 pages. Paragraphs 
should be separated with an empty line. The title and chapters should be written bold in font size 14. 
Ali scientific names must be properly italicized. Used nomenclature source should be cited in the 
Methods section. Tables and illustrations shall accompany the texts separately. Ali pages including 
tables and figures should be numbered. The original manuscript, two copies, and an electronic copy 
(after all corrections) on a 3.5" computer diskette, on CD-ROM or by e-mail must be given to the 
editor-in-chief. All articles must be proofread for professional and language errors before submis-
sion. 
13. Peer Review 
All Scientific Articles shall be subject to peer review by two experts in the field (one Slovene and 
one foreign) and Brief Note articles by one Slovene expert in the field. Authors may nominate a for-
eign reviewer in an accompanying letter. Reviewed articles accepted for publication shall be correct-
ed by the author. Authors shall receive thirty (30) free copies of the journal upon publication. In the 
event an article is rejected, the original material shall be retumed to the author together with the neg-
ative deterrnination of the editor-in-chief. 
\'ni. -'7: šl. 1 
Članki - .\rlkks: 
Tihor SZERDAIIELYI. Szihia F(rn. Szilard CZ()BEL . .lanos B.\LO(;H, Zoltan :\AG\' & Zoltan TUR\: 
Cocnological and ,ynphysiological investigatio11s on locss grassland \'cgctation (Su!t·io-Fc.,111cct11111 rttJ)icolue) 
dosc !D Giidiillii llills illungaryl ........................................................................................................................ .... 3 
Szihia Ffrrt .. )anus BALOGH. Szilard CZOBEL. Zoltan NAGV, Sandor BARTHA & Zoltan TUBA: 
Scaso1wl :111d daily pattcrn. 1c111poral and ,palial ,ariahility of cL"osy,1c111 C02-eschange in a 1e1npera1e Pannnnian 
lttC-ss gra .... sland .................. ............................................................. .................. ........ . .......... 13 
Ni\cs hTGOI\IČ. Boštjan POKOR:\V, 1\ldita STROPI\IK: Polluti<>n ni" garuen soils and \"egetahb in 
1lll' Sakk ,,ilky ..................................................... ................................................ .. ................................ 11 
Danko DII\IINIC', Boris IIRAŠOVEC. l\lilan CLAVAŠ. Nenad POTOČIC: Hannl"ul impact nf exceptional 
cnld air ,,uthreak in april 1997 on silver l"ir in Crnatia ........................................................................................... 27 
Aleksandra KRl\"OGRAD KLEI\IEN('IČ. Danijel \'RHO\'ŠEK. Nataša SI\IOLAR mul (;orazd KOSI: 
Algae ol" specil"ic- envirnn111ents in Slmrnia ................................................ .. .............................................. ~ 1 
, ·01. -'7: št. 2 
t'lanki - .\rlides: 
i\lalt'ja (;SPA\I. i\largareta \'R'IAČNIK . .lana A\IBROŽIČ DOLINŠEK, i\laja KO\'AČ. 
l\larjana C\\ILOH . .lana ŽEL: Tissue cul!ure or pyrcthrum 1Tanace1u111 cinerariifolium tTrevir.l 
Schu l 11 Bi p. 1 .................................................................................................................................... . .. ...... .-+5 
\lall'ja CERI\I: L1V-B radiation screen :md rcspiratory potcntial in phy1oplank1on in mnuntain lakes ................ 57 
Nina KRŽI('. All·nka GABERŠČIK & l\lall'ja GERI\I: The phenotypic plasticity ol" Glyceria rluitans 
growing o,cr thc \\'atL·r/land gradient ........ . .. ... 65 
Jasna PA R,\DIŽ mul i\lilan LO\'K,\: Pni len grain hinassay for cm in111111en1al con1a111ination hiomoni1<>ring „ 7:', 
Tanja Sl'.\I(:t(:: Rc,piralion rate and respiralory elcctron tran,port s~ ,tem ( ETS) ac1i, i1y of chironomid la1Yac 
rrom 111oun1ain lake, 1 NW Slownia) ..... ................................... .. ........................................ in 
Tatjana ČELIK: Divcrzitl'la dnevnih 111e1uljev t L~pidoptcra: Rhopaloccra) v Regijsl-.em parl-.u Šl-.ocjanske 
jame ............. .. .. .... 9) 
Jubileji: 
Kazimir T.-\ R\IA\I in Jo l' ŠTIRN: Profesor dr. l\lirn,la,· Zei - dcvctdcsetlctnil-. .............................................. 1 1 ~