VOL. 58 [T. 1 LJUBLJANA 2015 ACTA BIOLOGICA SLOVENICA prej/formerly BIOLO[kI VESTNIk ISSN 1408-3671 izdajatelj/publisher UDk 57(497.4) Dru{tvo biologov Slovenije ACTA BIOLOGICA SLOVENICA LJUBLJANA 2015 Vol. 58, [t. 1: 1–60 Acta Biologica Slovenica Glasilo Društva biologov Slovenije – Journal of Biological Society of Slovenia Izdaja – Published by Društvo biologov Slovenije – Biological Society of Slovenia Glavna in odgovorna urednica – Editor in Chief Alenka Gaberščik, e-mail: alenka.gaberscik@bf.uni-lj.si Tehnični urednik – Managing Editor Gregor Zupančič, e-mail: gregor.zupancic@bf.uni-lj.si Uredniški odbor – Editorial Board Robert Zorec (SLO), Matija Gogala (SLO), Alenka Malej (SLO), Livio Poldini (I), Mark Tester (AUS), Nejc Jogan (SLO), Mihael J. Toman (SLO), Franc Janžekovič (SLO), Branko Vreš (SLO), Boris Sket (SLO), Franc Batič (SLO), Hubert Potočnik (SLO), Georg A. Janauer (A), Doekele G. Stavenga (NL) Naslov uredništva – Address of Editorial Office Acta Biologica Slovenica, Večna pot 111, SI-1001 Ljubljana, Slovenija http://bijh.zrc-sazu.si/abs/ Zasnova oblikovanja – Design Žare Vrezec ISSN 1408-3671 UDK 57(497.4) Natisnjeno – Printed on: 2015 Tisk – Print: Tiskarna Pleško d.o.o., Ljubljana Naklada: 400 izvodov Cena letnika (dve številki): 15 € za posameznike, 42 € za ustanove Številka poslovnega računa pri Ljubljanski banki: 02083-142508/30 Publikacijo je sofinancirala Javna agencija za raziskovalno dejavnost Republike Slovenije Acta Biologica Slovenica je indeksirana v – is indexed in: CAB Abstracts, Web of Knowledge – Thomson Reuters ACTA BIOLOGICA SLOVENICA LJUBLJANA 2015 Vol. 58, [t. 1: 3–9 β-lactam antibiotics, recA mutation and SOS response β-laktamski antibiotiki, mutacija recA in odziv SOS Zdravko Podlesek Department of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Večna pot 111, 1000 Ljubljana, Slovenia Correspondence: zdravko.podlesek@bf.uni-lj.si Abstract: The claim that β-lactam antibiotics induce the SOS response, allowing E. coli survival in the presence of low antibiotic concentrations, was evaluated. No association between the recA gene and antibiotic survival rate was found. Disagree- ments with published observations are attributed to discrepancy in minimum inhibitory concentrations and growth characteristics of various strains carrying the recA mutation. Moreover, β-lactam antibiotics do not induce expression of the SOS regulated gene cka, encoding colicin K in wild-type strains. Keywords: SOS response, E. coli, β-lactam antibiotics, antibiotic resistance Povzetek: Preverili smo trditev, da β-laktamski antibiotiki sprožijo odziv SOS, kar naj bi omogočalo bakteriji E.coli preživeti v prisotnosti nizkih koncentracij antibiotikov. Opravljeni poizkusi niso pokazali nobene povezave med delovanjem produkta gena recA in stopnjo preživelosti ob prisotnosti antibiotika. Neskladje z objavljenimi opa- zovanji lahko pripišemo razlikam v minimalni inhibitorni koncentraciji in dramatično različnim lastnostim rasti pri doslej uporabljenih sevih z mutacijo v genu recA. Z uporabo genetsko neokrnjenega seva smo ugotovili, da β-laktamski antibiotiki ne sprožijo izražanje gena za kolicin K (cka), gena, ki je nadzorovan s sistemom SOS. Ključne besede: odziv SOS, E. coli, β-laktamski antibiotiki, odpornost proti antibiotikom Introduction In most bacteria, DNA damage is addressed by a set of genes constituting the SOS response. The response can be triggered by diverse exogenous treatments that elicit DNA damage and physical stress such as high pressure. In addition, stalled replication forks, unrepaired defects following recombination or chromosome segregation, as well as DNA damage caused by metabolic inter- mediates can also induce the SOS response. The LexA and RecA (recombinase A) proteins are its key regulators (Erill et al. 2007). RecA protein responds to a single-stranded DNA formed at sites of the DNA damage and triggers degradation of LexA transcriptional repressor. As a consequence, a number of LexA repressed genes are induced, covering among others synthesis and secretion of 4 Acta Biologica Slovenica, 58 (1), 2015 colicins in E. coli. In 2004, Miller et al. reported that sublethal concentrations of β-lactam antibiotics trigger the SOS response through the DpiBA two- component signal transduction system. Although this effect is generally accepted, several facts question the nature of this induction.. First, the DpiA and DpiB proteins have been shown to be involved in anaerobic citrate metabolism (Kaspar and Bott 2002). DpiB only binds specifically to the promoter regions of cit and mdh operons involved in citrate metabolism. In addition, it was found that DpiBA may also control the hexuronate dis- similation pathway via the exuTR operon, which may possibly be linked to citrate fermentation (Yamamoto et al. 2008). Second, a transcriptome profiling demonstrated that dpiA was not induced as one of the SOS genes after exposure to UV, in fact, it was downregulated (Courcelle et al. 2001). Also, contrary to the observation by Miller et al., dpiA and dpiB were found to be downregulated during ampicillin treatment (Sangurdekar et al. 2006). The aim of this study was to elucidate the mechanism by which arrest of cell-wall synthesis by β-lactam antibiotics triggers the expression of the seemingly unrelated dpi operon. However, neither differences in survival rates of recA mutants nore β -lactam induction of the SOS response were observed when reproducing certain experiments published by Miller et al. (2004). Material and Methods Bacterial strains and plasmids Parental bacterial strains are listed in Table 1. Strains AB1157 recA::Kn, MG1655 recA::Kn and RW118 recA::Kn were constructed by transducing the respective wild-type strains with a P1 lysate, prepared from strain JW2669 carrying a recA deletion mutation, and by selecting for kanamycin resistance. Transductants were verified by PCR with primers flanking the recA::Kn ORF and for mitomycin-C sensitivity. Plasmid pKCT1-Tc, carrying the colicin K gene was constructed as follows. Firstly, a 1.8 kb blunt-ended fragment containing the Tcr gene was obtained after digestion of vector pBR322 with restriction enzymes BsrB I and BseJ I. Secondly, plasmid pKCT1 (Mulec et al.) was digested with Psp1406 I to remove the β-lactamase gene, and the final fragment was treated to generate blunt ends (CloneJet kit; Fermentas). Both fragments were subsequently ligated and used to transform E. coli DH5α. Transformants were selected for tetracycline resistance. Table 1: Bacterial strains and plasmid used in this study. Tabela 1: Uporabljeni bakterijski sevi in plazmidi. Strain Genotype Source/Reference BW25113 lacIq rrnBT14 DlacZWJ16 hsdR514 DaraBADAH33 DrhaBADLD78 (Datsenko and Wanner 2000) JW1889 BW25113 araF::Kn Keio Collection JW2669 BW25113 recA::Kn Keio Collection AB1157 thr-1 leuB6 thi-1 supE44 lacY1 kdgK51 galK2 ara-14 xyl-5 mtl-1 proA2 his-4 argE3 str-31 tsx-33 (Bachmann 1987) MG1655 ilvG rfb-50 rph-1 E. coli Genetic Stock Center (CGSC 6300) RW118 hr-1 araD139 D (gpt-proA)62 lacY1 tsx-33 supE44 galK2 hisG4 rpsL31 xyl-5 mtl-1 argE3 thi-1 sulA211 (Ho et al. 1993) RW464 RW118 recA1 (Ho et al. 1993) Plasmid pKCT1-Tc pKCT1 with Apr gene replaced by Tcr gene (Mulec et al. 2003); this study 5Podlesek: β-lactam antibiotics and SOS response Determination of minimum inhibitory concentration (MIC) LB medium was inoculated with a 1 percent inoculum of an overnight culture of E. coli and incubated at 37 °C until the culture reached a density of 0.5 McFarland’s turbidity standard (optical density at 600 nm (OD600) of approxi- mately 0.1). A portion of the culture was diluted ten-fold with fresh LB medium, incubated for an additional 30 minutes at 37 °C and subsequently used at a 1:1000 dilution to inoculate test tubes with gradually increasing concentrations of antibiotics. Thus, the final cell number was approximately 5 x 104 per milliliter. After 18-24 hours of growth at 37 °C with aeration, the lowest concentration of antibiotic that has inhibited the visible growth of particular strain was determined. Survival rate determination An overnight culture was used to inoculate 40 ml of LB medium and incubated at 37 °C to an OD600 of 0.5. The culture was then divided into four 10 ml aliquots in shake flasks, one was used as a control, while appropriate concentrations of antibiotics were added to the others. Growth was continued with standard aeration at 37 °C. Samples were withdrawn at desired intervals and used for viable cell number determination. It was found that more accurate results of viable cell number were obtained when the antibiotic was removed from the sample prior to analysis by brief pelleting in a bench-top centrifuge. The sample was then resus- pended in the original volume of fresh LB medium, followed by incubation of test tubes at 37 °C for 15 minutes in a water bath and then used for serial dilutions. This additional step greatly eliminated the presence of long filamentous, undivided cells as observed by microscopy (data not shown). All experiments were repeated at least three times. In addition, stock solutions of antibiotics were kept at -80 °C and used immediately after thawing and then discarded. Colicin K induction All RecA+ strains were transformed with the colicin K producing plasmid pKCT1-Tc. Typi- cally, an overnight culture grown at 37 °C was used to inoculate 40 ml of LB medium at a 1% inoculation level, which was incubated until an OD600 of 0.5 was reached. The culture was then divided into four 10 ml aliquots, one served as control, the second one was exposed to 0.5 µg/ml of mitomycin C (Sigma), the third to ampicillin at ¼ x MIC and the fourth to piperacillin at ¼ x MIC. Minimal inhibitory concentrations for particular strain are shown in Table 2. After growth for an- other 4 hrs, a 1 ml sample was removed. The cells were lysed by sonication (3 × 20 s bursts) using a Vibra Cell (Sonics) sonicator and a lysate was prepared by subsequent centrifugation at 20,000 x g for 10 min. Colicin K activity of the lysate was determined by agar diffusion on LB plates, seeded with E. coli DH5α as an indicator strain. A two-fold serial dilutions were used. It was found that the uninduced strain MG 1655 exhibited the lowest colicin K activity and thus, the highest dilution of its lysate still showing inhibition was defined as 1 arbitrary activity unit. Results and Discussion β-lactam survival rate of RecA-/RecA+ strains Prior to comparing the survival of different E. coli mutants against β-lactam antibiotics, the minimum inhibitory concentrations (MIC) of the two antibiotics, ampicillin and piperacillin, was determined. Contrary to the results published by Miller et al. (2004), MICs for both antibiot- ics were substantially different, either lower or higher, in some commonly used isogenic RecA-/ RecA+ strains (Tab. 2). To exclude the influence of possible secondary mutations, unintentionally introduced during preparation of recA defective strains, two fresh recA mutant strains, MG1644 recA::Kn and AB1157 recA::Kn were constructed. Introduction of a recA deletion mutation into strain MG1655, generally considered the least geneti- cally manipulated E. coli strain, revealed that the observed MIC changes were indeed related to the recA mutation. As shown in Table 2, the MIC of ampicillin dropped from 7 µg/ml to 5 µg/ml and that of piperacillin from 2.5 µg/ml to 1.5 µg/ml. The observed strong sensitivity of all recA strains to the DNA damaging agent mitomycin C was as expected. However, MICs for unrelated targets (e.g. 6 Acta Biologica Slovenica, 58 (1), 2015 chloramphenicol) were also significantly reduced. In addition, some growth characteristics were also affected. All recA defective strains exhibited slower growth rates in comparison with the corresponding wild-type strains e.g., as much as twofold in the JW2669 (RecA-) when compared with the isogenic strain JW1889 (RecA+). Furthermore, the maximum cell concentration in a culture was also affected by mutation of recA, again most pronounced in strain JW2669 – the number of cells was reduced by as much as one order of magnitude. The striking difference between this particular strain and other recA mutants is not understood; none of the known mutations in JW1889, its parental strain, seem to be associated with any recA dependent process. Reduced viability of recA mutants was already observed by Miranda and Kuzminov (2003), although a direct comparison is not possible, as they cultivated their cultures under suboptimal conditions (at 28 °C). Next, the survival rate of isogenic RecA-/ RecA+ strains following addition of β-lactam antibiotics in the exponential growth phase was determined. The concentrations used were chosen in accordance with the obtained MIC data and at similar multiples (1/2, 1 and 2) of MICs to those used by Miller et al. (2004). The obtained results are presented in Table 3 for ampicillin and in Ta- ble 4 for piperacillin, respectively. As shown for both antibiotics, no significant difference in the survival rate of the isogenic RecA-/RecA+ strains was found. Clearly, our data did not confirm the previously reported assertion that inactivation of recA increases bacterial susceptibility to ampicillin and piperacillin. SOS induction Colicins, bacteriocins produced by E. coli strains, are typically regulated by the SOS re- sponse. To test the ability of either ampicillin or piperacillin to induce the SOS response, we studied the effect of both antibiotics on colicin K expression. Although the cka gene is transcribed- late during the SOS response, induction resulted in an increase of cka expression spanning three orders of magnitude (Tab. 5). Thus, even a slight change in cka gene activity could be easily iden- tified. Nevertheless, our results (Tab. 5) showed no cka induction by ampicillin or piperacillin at ¼ x MIC. As expected, colicin K expression was fully induced by mitomycin C. In conclusion, inactivation of recA results in important alterations in cell metabolism: (a) reduction of growth rate; (b) reduction of final cell density to only 10 % to 60 % of the wild- Table 2: Growth characteristics of isogenic RecA-/RecA+ strains in presence of antibiotics. Final number of cells is expressed as mean ± standard deviation. Tabela 2: Značilnosti rasti izogenih sevov RecA-/RecA v prisotnosti antibiotikov. Končno število celic je izraženo kot povprečna vrednost ± standardna deviacija. Parent strain Mutant strains MIC for ampicillin (µg/ml) MIC for piperacillin (µg/ml) MIC for chloramphenicol (µg/ml) MIC for mitomycin (µg/ml) Doubling time (min) Final number of cells (x 109) BW25113 w.t. 8.0 2.5 6.0 5.0 23 2.9 ± 0.6 recA::Kn 5.0 1.5 3.5 0.2 40 0.3 ± 0.06 MG1655 w.t. 7.0 2.5 5.5 6.0 22 3.5 ± 0.9 recA::Kn 5.0 1.5 4.0 0.4 29 2.1 ± 0.3 AB1157 w.t. 10.0 4.0 5.5 3.0 28 2.5 ± 0.2 recA1 7.0 2.0 5.5 0.45 33 1.1 ± 0.2 recA::Kn 7.0 2.5 5.5 0.4 37 1.2 ± 0.4 RW118 w.t. 9.0 2.5 3.5 6.0 25 2.6 ± 0.3 recA1 7.0 2.0 3.0 0.25 40 0.75 ± 0.15 7Podlesek: β-lactam antibiotics and SOS response Table 3: Survival of various isogenic RecA-/RecA+ strains following treatment with ampicillin. Percentage of survivors resembles the ratio of cell count before and and after the addition of antibiotic and is expressed as mean ± standard deviation. Tabela 3: Preživetje različnih izogenih sevov RecA-/RecA+ po izpostavljenju ampicilinu. Odstotek preživetih je razmerje med številom celic pred in po dodatki antibiotika ter je izražen kot povprečna vrednost ± standardna deviacija. Parent strain Mutant strain % of survivors 1/2 x MIC 1 x MIC 2 x MIC 1 h 4 h 1 h 4 h 1 h 4 h BW25113 w.t. 32.8 ± 18.0 3.5 ± 1.0 10.2 ± 3.7 1.0 ± 0.7 4.1 ± 1.4 0.6 ± 0.3 recA::Kn 80.1 ± 32.7 35.5 ± 1.5 8.1 ± 1.4 5.2 ± 2.3 1.7 ± 1.3 0.4 ± 0.3 MG1655 w.t. 61.6 ± 25.7 44.9 ± 27.8 22.8 ± 13.5 0.8 ± 0.6 1.8 ± 0.8 0.42 ± 0.39 recA::Kn 75.9 ± 14.6 40.4 ± 22.4 32.7 ± 7.6 0.55 ± 0.28 2.1 ± 0.9 0.14 ± 0.03 AB1157 w.t. 105.7 ± 19.7 32.7 ± 15.0 51.1 ± 14.4 4.4 ± 1.9 20.6 ± 7.7 0.1 ± 0.06 recA1 105.1 ± 27.5 17.0 ± 2.2 41.2 ± 8.1 1.9 ± 0.8 35.3 ± 4.2 0.6 ± 0.4 recA::Kn 90.8 ± 11.6 65.1 ± 22.0 38.5 ± 19.0 7.9 ± 4.8 33.9 ± 18.6 3.3 ± 1.1 RW118 w.t. 48.8 ± 25.3 5.2 ± 2.9 17.1 ± 8.5 0.7 ± 0.56 1.5 ± 0.8 0.32 ± 0.2 recA1 40.2 ± 10.3 12.6 ± 11.0 13.0 ± 3.7 0.9 ± 1.3 0.9 ± 0.6 0.08 ± 0.05 Table 4: Survival of various isogenic RecA-/RecA+ strains following treatment with piperacillin. Percentage of survivors resembles the ratio of cell count before and and after the addition of antibiotic and is expressed as mean ± standard deviation. Tabela 4: Preživetje različnih izogenih sevov RecA-/RecA+ po izpostavljenju piperacilinu. Odstotek preživetih je razmerje med številom celic pred in po dodatki antibiotika ter je izražen kot povprečna vrednost ± standardna deviacija. Parent strain Mutant strain % of survivors 1 x MIC 2 x MIC 4 x MIC 1 h 4 h 1 h 4 h 1 h 4 h BW25113 w.t. 33.1 ± 12.1 12.4 ± 7.4 21.8 ± 12.9 6.5 ± 4.8 16.5 ± 8.2 1.4 ± 1.0 recA::Kn 39.8 ± 12.3 33.9 ± 7.0 27.5 ± 15.7 19.9 ± 10.8 16.3 ± 3.3 10.7 ± 5.2 MG1655 w.t. 29.9 ± 11.9 8.4 ± 5.5 13.6 ± 4.9 6.9 ± 3.8 15.8 ± 6.1 4.9 ± 3.9 recA::Kn 56.5 ± 10.9 3.4 ± 2.2 32.5 ± 7.9 3.3 ± 2.5 35.6 ± 8.0 2.8 ± 2.3 AB1157 w.t. 82.4 ± 16.5 7.5 ± 3.1 64.0 ± 19.5 9.0 ± 5.5 43.0 ± 21.6 4.2 ± 0.5 recA1 68.0 ± 34.0 14.5 ± 4.5 52.2 ± 21.7 2.8 ± 1.3 23.5 ± 3.1 2.2 ± 1.6 recA::Kn 59.1 ± 15.5 7.1 ± 2.2 37.2 ± 12.0 4.4 ± 1.3 27.2 ± 13.8 3.8 ± 1.4 RW118 w.t. 42.2 ± 7.1 7.5 ± 3.8 29.9 ± 11.9 2.1 ± 1.0 30.9 ± 8.4 2.5 ± 1.7 recA1 34 ± 10.5 12.3 ± 5.3 24.9 ± 11.3 6.3 ± 3.8 19.3 ± 12.0 3.9 ± 1.6 8 Acta Biologica Slovenica, 58 (1), 2015 type strain, depending on the genetic background (Tab. 2); and (c) increased sensitivity to different antibiotics with unrelated mode of action. In this respect, the interpretation of an isolated phenom- enon, such as the response to β -lactams, could be misleading if based solely on the comparison between RecA-/RecA+ strains. Summary Due to the claim that β-lactam antibiotics in- duce the SOS response in E. coli, the characteristics of various recA mutants were analyzed. It was shown that inactivation of the recA gene severely altered cell metabolism, reflected in its growth rate, final population density and its sensitivity to unrelated antibiotics. By using genetically “clean” wild-type strains, it was possible to show that there is no link between the presence of β-lactam antibiotics and the SOS response. The observations presented here urge the reinterpretation of the published results based on the genetically over-manipulated strains. Povzetek Zaradi trditve, da β-laktamski antibiotiki sprožijo odziv SOS pri E. coli, smo analizirali značilnosti nekaterih mutant recA. Pokazalo se je, da inaktivacija tega gena izzove dramatične spremembe v celičnem metabolizmu, ki se odražajo v stopnji rasti, končni populaciji celic in občutljivosti za različne, nesorodne antibiotike. Uporaba genetsko “čistega” seva je pokazala, da ni nobene povezave med β-laktamskimi antibiotiki in odzivom SOS. Predstavljena opazovanja kličejo po nujni re- interpretaciji marsikaterega objavljenega rezultata, izvedenega na genetsko (pre)obremenjenih sevih. Acknowledgement This work was supported by the Slovenian Research Agency (ARRS). References Bachmann, B. J., 1987. Derivation and genotypes of some mutant derivatives of Escherichia coli K-12. In: Neidhardt, F.C., Ingraham, J.L., Low, K.B., Magasanik, B., Schaechter, M., Um-barger, H.E., (ed.): Escherichia coli and Salmonella typhimurium. Cellular and Molecular Biology, American Society for Microbiology, Washington, D.C., pp. 190-1219. Table 5: The activity of colicin K synthesized in E.coli RecA+ strains of different genetic backgrounds following addition of ampicillin (1/4 of MIC), piperacillin (1/4 of MIC) or mitomycin C (0.5 µg/ml), respectively. Tabela 5: Aktivnost kolicina K sintetiziranega v sevih E. coli RecA+ različnega genetskega ozadja po dodatku bodisi ampicilina (1/4 MIC), piperacilina (1/4 MIC) ali mitomicina C (0.5 µg/ml). Strain Colicin K activity (arbitrary units) no inducer/ control ampicillin piperacillin mitomycin C JW1889 2 2 1 1280-2560 MG1655 1 1 1-2 1280 AB1157 8 8 8 1280 RW118 8 8 4 2560-5120 9Podlesek: β-lactam antibiotics and SOS response Courcelle, J., Khodursky, A., Peter, B., Brown, P.O., Hanawalt, P.C., 2001. Comparative gene expres- sion profiles following UV exposure in wild-type and SOS-deficient Escherichia coli. Genetics, 158, 41-64. Datsenko, K.A., Wanner, B.L., 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA, 97, 6640-6645. Erill, I., Campoy, S., Barbe, J., 2007. Aeons of distress: an evolutionary perspective on the bacterial SOS response. FEMS Microbiol. Rev., 31, 637-656. Ho, C., Kulaeva, O.I., Levine, A.S., Woodgate, R., 1993. A rapid method for cloning mutagenic DNA repair genes: isolation of umu-complementing genes from multidrug resistance plasmids R391, R446b, and R471a. J. Bacteriol., 175, 5411-5419. Kaspar, S., Bott, M., 2002. The sensor kinase CitA (DpiB) of Escherichia coli functions as a high- affinity citrate receptor. Arch. Microbiol., 177, 313-321. Miller C., Thomsen L.E., Gaggero C., Mosseri R., Ingmer H., Cohen, S.N., 2004. SOS response in- duction by ß-Lactams and bacterial defense against antibiotic lethality. Science, 305, 1629-1631. Miranda A., Kuzminov, A., 2003. Chromosomal lesion suppression and removal in Escherichia coli via linear DNA degradation. Genetics, 163, 1255-1271. Mulec J., Podlesek Z., Mrak P., Kopitar A., Ihan A. and Žgur-Bertok, D., 2003. A cka-gfp transcrip- tional fusion reveals that the colicin K activity gene is induced in only 3 percent of the population. J. Bacteriol., 185, 654-659. National Committee for Clinical Laboratory Standards, 2000. Methods for dilution antimicrobial su- sceptibility tests for bacteria that grow aerobically. Approved standard, 5th ed. NCCLS document M7-A5. National Committee for Clinical Laboratory Standards, Wayne, Pa. Sangurdekar, D.P., Srienc, F., Khodursky, A.B., 2006. A classification based framework for quantita- tive description of large-scale microarray data. Genome Biol., 7, R32. Yamamoto K., Matsumoto F., Oshima T., Fujita N., Ogasawara N., Ishihama, A., 2008. Anaerobic regulation of citrate fermentation by CitAB in Escherichia coli. Biosci. Biotechnol. Biochem., 72, 3011-3014. Taxonomy, phytogeography and phytosociology of Laserpitium krapfii Crantz. in Slovenia Taksonomska, fitogeografska in fitocenološka oznaka vrste Laserpitium krapfii Crantz. v Sloveniji Tinka Bačiča, Marko Accettob, Branko Vrešc , Igor Dakskoblerd,* aDepartment of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, SI-1000 Ljubljana, Slovenia bTrnovski pristan 18, SI-1000 Ljubljana, Slovenia cJovan Hadži Institute of Biology ZRC SAZU, Novi trg 2, SI-1000 Ljubljana dJovan Hadži Institute of Biology ZRC SAZU, Regijska raziskovalna enota Tolmin, Brunov drevored 13, SI-5220 Tolmin and Biotechnical Faculty, Biotehniška fakulteta, Department for Forestry and Renewable Resources, Večna pot 83, SI-1000 Ljubljana *correspondence: igor.dakskobler@zrc-sazu.si Abstract: The article discusses the occurrence, distribution and phytosociological affinity of Laserpitium krapfii in Slovenia. According to some literature sources (Tutin 1968, Fischer et al. 2008) and the distribution patterns, two subspecies of L. krapfii are be expected in Slovenia: L. krapfii subsp. krapfii and L. krapfii subsp. gaudinii. The revision of the Slovene herbarium material in LJU and LJS herbaria confirmed only the occurrence of its type subspecies. It has a Dinaric pattern of distribution (NW-SE) in the Alpine, Prealpine, Dinaric and Predinaric phytogeographical regions, with most of its known localities in the hills south of Ljubljana, in the Snežnik mountains, in the Kočevje region with the Kolpa Valley and in the Gorjanci mountains. Since the spe- cies mostly thrives in the mountain beech forests, it can be considered as a diagnostic (differential) species of the Illyrian alliance Aremonio-Fagion. Key words: Laserpitium krapfii subsp. krapfii, taxonomy, phytogeography, Aremonio-Fagion, Slovenia Izvleček: V članku obravnavamo pojavljanje, razširjenost in fitocenološko na- vezanost vrste Laserpitium krapfii v Sloveniji. Glede na literaturne navedbe (Tutin 1968, Fischer et al. 2008) in vzorca razširjenosti, bi pri nas lahko pričakovali uspe- vanje dveh podvrst: L. krapfii subsp. krapfii in L. krapfii subsp. gaudinii. Z revizijo herbarijskega gradiva v LJU in LJS smo za Slovenijo potrdili le pojavljanje podvrste L. krapfii subsp. krapfii. Razširjena je v dinarski smeri (severozahod-jugovzhod), v alpskem, predalpskem, dinarskem in preddinarskem fitogeografskem območju, z največjo gostoto nahajališč v hribovju južno od Ljubljane, v Snežniškem pogorju, na Kočevskem s Kolpsko dolino in na Gorjancih. Največ nahajališč je v montanskih bukovih gozdovih, zato jo lahko štejemo za diagnostično (razlikovalno) vrsto ilirske zveze Aremonio-Fagion. Ključne besede: Laserpitium krapfii subsp. krapfii, taksonomija, fitogeografija, Aremonio-Fagion, Slovenija ACTA BIOLOGICA SLOVENICA LJUBLJANA 2015 Vol. 58, [t. 1: 11–23 12 Acta Biologica Slovenica, 58 (1), 2015 Introduction Laserpitium krapfii is a member of the Umbel- liferae family (Apiaceae). The identification of the members of this family is relatively difficult, especially since fully ripe fruits and flowers are very important for their identification. Among other members of the Laserpitium genus, L. krapfii is well characterized by its ovoid to oblong, dentate leaf-segments, 0-5 bracts, which are glabrous, and 5-15 rays of the umbel, which are distinctly of different lengths; flowers are usually greenish (Martinčič 2007). The distribution of L. krapfii in Slovenia was discussed in Fleischmannn (1844), Plemel (1862), Paulin (1904), Neugebauer (1932), Mayer (1952), T. Wraber (1966), Thellung (1975) etc.; contemporary knowledge is summarized by Martinčič (2007). According to this literature source, L. krapfii thrives in the Slovenian Alpine (Tolminsko), Dinaric and Predinaric (Gorjanci mountain range) phytogeographical region. In the work Gradivo za Atlas flore Slovenije (Jogan & al. 2001), the distribution map of L. krapfii has accidentally been omitted. According to Flora Europaea (Tutin 1968) there are two subspecies of L. krapfii: L. krapfii subsp. krapfii (syn. L. marginatum Waldst. & Kit.; incl. L. alpinum Waldst. & Kit.) and L. krapfii L. subsp. gaudinii (Moretti) Thell. (L. gaudinii Moretti). The type subspecies has a Dinaric-Carpathian pattern of distribution: it thrives in the northern part of Balkan Peninsula, extending to NE Italy and in Carpathians (Tutin 1968). The other sub- species, L. krapfii subsp. gaudinii has an Alpine distribution. According to Flora Europaea (Tutin 1968), it grows in W Austria, E Switzerland, N Italy, and in the northwestern part of the former Yugoslavia, which implicitly include Slovenia. Similarly, Fischer et al. (2008) also indicate that L. krapfii L. subsp. gaudinii thrives in Slovenia (with its main distribution area in E Switzerland, N Italy and N Tyrol). However, according to Martinčič (2007) there is only one subspecies present in Slovenia, that is the type species L. krapfii subsp. krapfii. There are no older Slovenian data refering to L. krapfii L. subsp. gaudinii. Only L. krapfii subsp. krapfii (sin. L. marginatum) is considered by the authors (f. e. Paulin 1904, Neugebauer 1932, Mayer 1952). It should be added, that in Austria, L. krapfii subsp. gaudinii is very rare. It is included in the red list of threatened species (Niklfeld and Schratt-Ehrendorfer; 1999), but not mentioned in Verbreitungsatlas der Farn- und Blütenpflanzen Kärntens (Hartl et al. 1992). In adjacent parts of Italy (Friuli Venezia Giulia) the species doesn’t occur (Poldini 2002). On the other hand, according to the distribution map in Flora Alpina (Aeschimann and al. 2004), the type subspecies has an eastern-Alpine-Illyrian pattern of distribution; it grows in Alpine areas of N Italy, Switzerland, W Austria, and (surprisingly) in Dinaric mountains, but not in Slovenia. Morphologically, the two subspecies are well characterized (see Table 1) and they also differ in ecological preferences. L. krapfii subsp. gaudinii thrives in low-nutrient meadows, glacial gravel and bushes, in mountaine and subalpine belt (Fischer et al. 2008), while the type subspecies prefers forests (Thellung 1975, Martinčič 2007). Since Slovenia lies between the Alps and the Dinarides, we believe that the occurrence of both subspecies is expected. That was the reason why we revised all the available herbarium material to verify the taxonomic status of Slovene populations. The distribution map of L. krapfii has not yet been published, so we systematically gathered floristic data to produce an up-to-date distribution map. We also investigated the occurrence of the species in different forest communities and investigated its phytosociological preferences. Material and methods We reviewed all the available herbarium material of this species in Herbarium of the Lju- bljana University (LJU) (22 herbarium sheets) and Herbarium of the Institute of Biology ZRC SAZU (LJS) (13 herbarium sheets). Data on the revised herbarium specimens (Specimina visa) are in Appendix. We checked the discriminative characters, provided by Pignatti 1982, Tutin 1968 and Thellung 1975 (Table 1). 13Bačič et al.: Laserpitium krapfii in Slovenia Table 1: Discriminative characters for Laserpitium krapfii subsp. krapfii and L. krapfii subsp. gaudinii (Pignatti 1982, Tutin 1968, Thellung 1975). Preglednica 1: Razlikovalni znaki med podvrstama Laserpitium krapfii subsp. krapfii in L. krapfii subsp. gaudinii (Pignatti 1982, Tutin 1968, Thellung 1975). Character L. krapfii subsp. krapfii L. krapfii subsp. gaudinii pruinosity of the stem slightly pruinose usually strongly pruinose similarity of the upper cauline leaves with lower leaves similar markedly different the shape of leaf-segments of upper cauline leaves ovate, usually dentate oblong, usually entire roughness of the umbel rays rays rough or shortly hispid on inner side rays glabrous and smooth on inner side presence of setae on primary ridges of fruit ridges with short setae ridges glabrous To get further insight in the discrimination of the two subspecies, we also compared Slovene material with the available material of L. krapfii subsp. gaudinii from abroad. Distributional data were obtained from the database of Centre for Cartography of Fauna and Flora and the FloVegSi database of the Institute of Biology of the SRC SASA. We used standard botanical and phytosociological methods (Ehren- dorfer and Hamann 1965, Jalas and Suominen 1967, Braun-Blanquet 1964). Nomenclature source for the names of the taxa is Mala flora Slovenije (Martinčič et al. 2007) and for the names of the syntaxa (Šilc and Čarni 2012). The distribution map was made with the application FloVegSi (Seliškar et al. 2003). Results and discussion The herbarium revision in the determination of the subspecies We found that all of the examined specimens, collected in the territory of Slovenia, belong to the type subspecies. Despite the fact that for now we do not have confirmation of the occurence of L. krapfii subsp. gaudinii in Slovenia, we need to be aware of this possibility during the fieldwork in the northern part of the country, especially on screes, low-nutrient dry meadows and in tall herb and scrub communities of the mountain and subalpine belt. Subspecies can reliably be identified, if the plants are flowering or fruiting, that is from mid- July onwards. The fruits are fully developed at the beginning of August, but for the determination of the subspecies they are not required. Especially useful characters are the roughness of the umbel rays (prickle trichomes on the inner side of the rays – Figure 1) and the presence of short setae on primary ridges of the fruit (Figure 2). Both are present only in the type subspecies. When observing these characters, we need to use a stronger magni- fying glass (20-30 x magnification). In herbarium material, the characters pruinosity of the stem and the similarity of the upper cauline leaves with the lower ones proved hard to assess. The leaves seemed rather variable and there were not enough material of L. krapfii subsp. gaudinii to evaluate the differences in shapes of the leaf-segments of upper cauline leaves and their dentation in both of the subspecies. 14 Acta Biologica Slovenica, 58 (1), 2015 Figure 1: Prickle trichomes on the inner side of the rays present in L. krapfii subsp. krapfii (top) and absent in L. krapfii subsp. gaudinii (below). Slika 1: Žarki kobula z bodičkami na notranji strani pri L. krapfii subsp. krapfii (zgoraj) in gladki kobulovi žarki pri L. krapfii subsp. gaudinii (spodaj). Figure 2: Short setae on primary ridges of the fruit in L. krapfii subsp. krapfii. Slika 2: Ščetine na rebrih plodu pri L. krapfii subsp. krapfii. 15Bačič et al.: Laserpitium krapfii in Slovenia The distribution of species in Slovenia The distribution of L. krapfii subsp. krapfii in the Alpine and Prealpine phytogeographical region According to the results of our study, in the Alpine phytogeographical region, the occurrence of L. krapfii is limited to the southern extensions of the Tolmin-Bohinj ridge of the Julian Alps. The localities, that are closest to this ridge, are on the slopes of the peak Žabijski Kuk above the Razor pasture. Most localities lie on the slopes of the side-lying, south-oriented ridge Žabijski Kuk– Vrh nad Sopotom– Krikov vrh– Tolminski Triglav– Kobilja glava – Jalovnik, above the valleys of streams Zadlaščica and Kneža (Knežica) with its right tributary Lipovšček (Dakskobler 1991, 1994, 2001, 2002, 2003, 2006, 2015). The reports for L. krapfii in extensions of the Savinja Alps (plateau Krašica above Šmartno at Dreta) were published by Diaci (1997). There are no other reports of L. krapfii in this mountain range and Diaci’s records are not documented with herbarium material. We assess that these data need verification and that is the reason why we marked them as ‘questionable’ in our distribution map (Figure 3). In the phytosociological tables published by Piskernik (1977), there are two additional records of L. krapfii for Alpine-Pealpine part of Slovenia. The first relates to Breginjski Stol. Although the flora of this area is relatively well-studied, L. krapfii was not observed there by other botanists so far (see Čušin 2006). The second record is from Paški Kozjak above Spodnji Dolič that is in the Prealpine phytogeographic region. These two localities are also marked as ‘questionable’, since the vaucher herbarium material is not available. We here publish some new data from herbarium LJS (revision by Dakskobler and Vreš, February 2015) for Alpine and Prealpine region (Bača and Idrijca Valleys: 9749/4, 9849/1, 9848/4 – for details see Appendix). There are also reliable records of L. krapfii in Prealpine phytogeographical region, on the Zaplana plateau between Vrhnika and Logatec (T. Wraber 1996, Rozman 2000). The distribution of L. krapfii in Dinaric and Predinaric phytogeographical region The localities of L. krapfii in the northwestern- most part of the Dinaric mountains – in Slovenian Dinaric phytogeographical region, were published by Głowacki and Arnold (1870). This publica- tion was summarized by Neugebauer (1932). According to these authors, the species thrived in Vojščica on the Vojskarska planota plateau in the Idrija mountains. We confirmed these records in the nearby hill Hudournik, on the edge of the plateau above the valleys of Kanomlja and Hotenja (Dakskobler 2001). As far as we know, Paulin’s (1904) report about locality under Čaven at Predmeja (Dol) has no recent confirmations. Paulin also reported that the species presumably grows near Idrija (“angeblich bei Idria”). Since surroundings of Idrija extend over several MTB quadrants, this record is not precise enough to put it in our distribution map. The localities in SW part of the Trnovski gozd between Otlica and Col (under Kovk) (Dakskobler 1997, phytocoenological table 4 and herbarium LJU and LJS) and on the western edge of the plateau Nanos above the valley of Bela and at Podkraj (Dakskobler (1997), M. Wraber and Žigon, 16. 10. 1970 – personal notes of Wraber M. and J. Žigon) were recently confirmed. The localities of L. krapfii on the hills south of Ljubljana (Krim, Mokrc, Rakitna plateau, Iška basin) have already been known to botanists for over 170 years (Fleischmann 1843, Paulin 1904). These populations are well documented with herbarium material LJU (authors Budnar, Mayer, Dolšak, Paulin, Zrimec – see Appendix). There are also other recent reports for this area (Robič 1960a, b and Accetto 2010, 2013) – the systematic map- ping of flora was carried out particularly in Iška. The records for the Snežnik mountains have been contributed by Paulin (1904), Justin (1923, LJU), Tregubov (1957), T. Wraber (1966, also herbarium material in LJU), Zupančič (1972, LJU), Piskernik (1977, 1991), Marinček (1996) and Surina and Rakaj (2007) – our observations during floristic fieldwork in this area showed the same (unpublished data BV). Older data referring to the presence of L. krapfii in Kočevsko and Kolpa valley (Fleischmann 1844, Plemel 1862, LJU, Paulin 1904) are recently con- firmed with numerous records: Martinčič (1958, LJU), Peterlin (1960, LJU), M. Wraber (1962, 1963, in litt.), Štimec (1982), Hočevar et al. (1985, 1995), Accetto (1995, 1999a, b, 2002a, 2003, 2007a,b, 2008), Accetto et al. (1996), Frajman (2001, LJU), 16 Acta Biologica Slovenica, 58 (1), 2015 Trčak et al. (2002) and our own unpublished data (BV). According to these reports, the taxon is widespread in the Kočevsko: it thrives in Bela stena near Ribnica, Fridrihštajn, Podstene at Ko- privnik in the Kočevski Rog, Goteniški Snežnik, Goteniška gora, the mountains of Borovška gora with Firstov rep, Krokar and Krempa and in their ravines above the Kolpa valley, Stružnica, Mirna gora near Semič, the slopes above the Čabranka valley (Strma reber, Belica) etc. The presence of L. krapfii in the Gorjanci mountain range was noticed already by Paulin (1904). In the following decades, the plants were collected by Rataj (1954, LJU), Martinčič (1954, LJU), Strgar (1960, LJU), T. Wraber (1992, LJU) and recorded by Košir (1979), Hočevar et al. (1985) and Accetto (2002b). In Gorjanci, we recorded the species in forest reserve Kobile (Dakskobler et Grah, 2013, 2014, in litt.). Distribution map According to the distribution map (Figure 3), L. krapfii s. str. has a Dinaric pattern of the distribution, extending from NW to SE of Slo- venia. It thrives is the Alpine, Prealpine, Dinaric and Predinaric phytogeographical region, with a concentration of its localities in the hills south from Ljubljana, the Snežnik Mountains, the Kočevje region with the Kolpa valley and in the Gorjanci mountain range. Figure 3: Distribution of Laserpitium krapfii subsp. krapfii in Slovenia. Slika 3: Razširjenost vrste Laserpitium krapfii subsp. krapfii v Sloveniji. Phytosociological characteristics of L. krapfii According to Aeschimann et al. (2004), L. krapfii subsp. gaudinii is predominantly a scree taxon, a character species of the alliance Petasi- tion paradoxi. Zupančič (1999) characterizes L. krapfii as character species of the alliance Vaccinio-Piceion or the order Vaccinio-Piceetalia, while Accetto (2010, 2013) treats L. krapfii to be character species of the class Erico-Pinetea. 17Bačič et al.: Laserpitium krapfii in Slovenia Košir (1979) selected L. krapfii (= L. marginatum) as character species for the association Arunco- Fagetum. After this species, Vukelić et al. (2010) named the altimontane-subalpine spruce forest in northern part of the Velebit range – Laserpitio krapfii-Piceetum. In the Snežnik Mts., Tregubov (1957) considers L. krapfii as a differential species for the subassociation Calamagrostio-Abietetum piceetosum. Surina and Rakaj (2007) found the species in the same area in the stands of the subassociation Polysticho lonchitis-Fagetum rhododendretosum hirsuti. In the stands of the association Polystycho lonchitis-Fagetum under Snežnik, L. krapfii was recorded by T. Wraber (1966) and a few decades later also by Marinček (1996). In the Snežnik mountains, Marinček and Čarni (2010) recorded this species also in the stands of the syntaxon Ranunculo platanifolii-Fagetum var. geogr. Calamintha grandiflora typicum var. Helleborus niger. In the southern Julian Alps, L. krapfii thrives in the stands of the following syntaxa: Seslerio autumnalis-Fagetum, Ostryo-Fagetum (Dakskobler 1991), Arunco-Fagetum (Dakskobler 1994, 2004), Ranunculo platanifolii-Fagetum, Luzulo-Fagetum (Dakskobler 2001), Homogyno sylvestris-Fagetum (Dakskobler 2002), Rhododendro hirsuti-Fagetum (Dakskobler 2003), Rhodothamno-Laricetum ostryetosum (Dakskobler 2006) and Rhododendro hirsuti-Ostryetum (Dakskobler 2015). In the sum- mer 2004, we found the species on the north-west slopes of the peak Žabijski Kuk above the Razor pasture (ca. 1350 m – 1400 m a. s. l.), in the Alpine dwarf pine stands (Rhodothamno-Pinetum mugo). On the northern edge of the Vojsko plateau and on the edges of the Trnovski gozd and Nanos plateaus, we recorded the species in the stands of the associations Omphalodo-Fagetum, Ra- nunculo platanifolii-Fagetum, Arunco-Fagetum, Rhododendro hirsuti-Fagetum and Seslerio autumnalis-Fagetum. Near Zaplana, T. Wraber (1996) recorded the species in the stand of the association Ostryo- Fagetum. On Mokrc, Robič (1960) observed it in the stands of the associations Arunco-Fagetum and Omphalodo-Fagetum. In Iška basin, Accetto (2013) found the spe- cies in the stands of the associations Omphalodo- Fagetum and Ostryo carpinifolii-Piceetum, but it is also present in the stands of the association Arunco-Fagetum. In the Gorjanci mountain range, Accetto (2002b) recorded the species in the stands of the association Tanaceto clusii-Fagetum. In the Kočevsko region, Zupančič and Accetto (1994) reported L. krapfii for the stands of the associa- tion Ribeso alpini-Piceetum, while Accetto (1995, 1999 a, b, 2002a, 2003, 2007b, 2008) observed the species in the stands of the syntaxa Carici sempervirentis-Pinetum nigrae, Aconito lycoctoni- Fagetum, Omphalodo-Fagetum, Lamio orvalae- Fagetum, Allio victorialis-Fagetum, Rhododendro hirsuti-Fagetum, Arunco-Fagetum var. geogr. Acer obtusatum and in the stands of two non-forest asso- ciations, Seslerio kalnikensis-Arabidetum muralis and Neckero crispae-Campanuletum justinianae. Most of the localities are in the montane belt, between 400 m and 1200 m n. m. The highest localities of the species in the Julian Alps are in the alpine dwarf pine stands below the peak Žabijski Kuk, about 1400 m a. s. l., while in the Dinaric Mountains, the vertical distribution of L. krapfii reaches (current) upper timberline below Snežnik, at an altitude of about 1600 m. The localities are mostly on calcareous bedrock (dolomite, dolomite limestone and limestone), occasionally with admixture of chert marlstone or claystone. The soils are shallow (mainly rendzina), sometimes due to the admixture of chert somewhat acidic. Although this species occasionally thrives in some spruce, black pine and alpine dwarf pine stands, we ascertained that most localities are in the Illyrian beech forests and therefore we may reasonably consider it as diagnostic (differential) species of the Illyrian alliance Aremonio-Fagion. Taking into account its habitat-type preferences in Slovenia, the species doesn’t seem to have strong affinity to communities of the alliance Vaccinio- Piceion, except for the fact that it predominantly grows on shallow and often rocky ground with moder rendzina. If (when) we rank it among the character spe- cies of spruce forests, in our opinion it should be associated with the suballiance Abieti-Piceenion. 18 Acta Biologica Slovenica, 58 (1), 2015 Conclusions According to some literature sources (Tutin 1968, Fischer et al. 2008) and the distribution patterns, two subspecies of L. krapfii would be expected in Slovenia: L. krapfii subsp. krapfii and L. krapfii subsp. gaudinii. The revision of Slovene herbarium material in LJU and LJS confirmed only the presence of the type subspecies. However, we need to be aware of the possibility of finding the other subspecies, during the fieldwork in the northern part of the country, especially on screes, low-nutrient dry meadows, in tall herb and scrub communities of the mountain and subalpine belt. In Slovenia, L. krapfii subsp. krapfii has a Di- naric pattern of distribution, extending from NW to SE of Slovenia. It has scattered distribution in the Alpine, Prealpine, Dinaric and Predinaric phy- togeographical region, with a concentration of its localities in the mountains south of Ljubljana, the Snežnik Mountains, the Kočevje region with the Kolpa valley and in the Gorjanci mountain range. The north-westernmost localities in the whole dis- tribution area of this taxon are in the southern Julian Alps (the southern extension of the Tolmin-Bohinj ridge), while the localities in the pre-Alpine region include those above the valleys of Bača and Idrijca and on the Zaplana plateau between the towns of Vrhnika and Logatec. Most of its localities are in beech forests ex- tending from the submontane to the subalpine belt (300 m to 1600 m a.s.l.), on calcareous bedrock (dolomite, limestone, in places mixed with chert, marlstone or claystone) and on shallow soil (moder rendzina). The species was observed also in several spruce communities, in Dinaric black pine com- munity, in Alpine dwarf pine and hop hornbeam communities, as well as in communities of moist rock crevices and screes. Phytosociologists treat L. krapfii as a character species of either spruce or pine forests. With most of its localities in the montane beech forests it can le- gitimately be considered as a diagnostic (differential) species of the Illyrian alliance Aremonio-Fagion. Povzetek Vrsta L. krapfii je kobulnica, ki uspeva pri nas predvsem v dinarskih gozdovih. Od ostalih vrst tega rodu jo ločimo po jajčastih do podolgastih listnih segmentih, golem, malolistnem ogrinjalu in le 5-15 kobulovih žarkih, ki so izrazito različno dolgi. Zbirna evropska floristična dela (npr. Tutin 1968, Pignatti 1982, Thellung 1975, Fischer et al. 2008) navajajo dve podvrsti krapfovega jelenovca: L. krapfii subsp. gaudinii (L. gaudinii) in L. krapfii subsp. krapfii (L. krapfii s. str., sin. L. marginatum). Prvi takson ima predvsem alpsko razširjenost, drugi dinarsko-karpatsko. Glede na lego Slovenije in nekatere navedbe (npr. Fischer 2008, Tutin 1968) naj bi pri nas uspevali obe. Da bi to preverili, smo pregledali ves dostopni herbarijski material te vrste v herbariju LJU in LJS. Opazovali smo razlikovalne znake, ki jih navajajo Pignatti (1982), Tutin (1968) in Thellung (1975). Ugotovili smo, da pregledani herbarijski material pripada tipski podvrsti, kar je v skladu z navedbami iz domače literature (npr. Mayer 1952, Martinčič 2007), po katerih naj bi pri nas uspevala le L. krapfii subsp. krapfii. Iz podatkov, zbranih v podatkovnih bazah Centra za kartografijo favne in flore (CKFF) in FloVegSi Biološkega inštituta Jovana Hadžija ZRC SAZU, literaturnih podatkov in podatkov iz herbarijev LJU in LJS smo izdelali zemljevid znane razširjenosti Krapfovega jelenovca v Sloveniji. Ugotavljamo, da je v Sloveniji razširjen v alp- skem, predalpskem, dinarskem in preddinarskem fitogeografskem območju. Najbolj severozahodna nahajališča v njegovem celotnem arealu so v južnih Julijskih Alpah. Glavnina nahajališč leži v dinarski smeri severozahod-jugovzhod, z največjo gostoto nahajališč v hribovju južno od Ljubljane, v Snežniškem pogorju, na Kočevskem s Kolpsko dolino in na Gorjancih. Največ nahajališč je v mon- tanskih bukovih gozdovih, zato Krapfov jelenovec lahko štejemo za diagnostično vrsto ilirske zveze Aremonio-Fagion. Vrsta največkrat raste v bukovih gozdovih od submontanskega do subalpinskega pasu (300 m do 1600 m n. m.), na karbonatni podlagi (dolomit, apnenec, ponekod s silikatno primesjo) in plitvih tleh (prhninasta rendzina). Popisali smo ga tudi v nekaterih smrekovih združbah, v dinarskem črnoborovju, v alpskem ruševju in črnogabrovju ter v združbah vlažnih skalnih razpok in melišč. 19Bačič et al.: Laserpitium krapfii in Slovenia Acknowledgements For friendly consultations and literature we sincerely thank doc. dr. Boštjan Surina. For send- ing us his doctoral dissertation with the reports of L. krapfii in extensions of the Savinja Alps we thank prof. dr. Jurij Diaci. We would also like to thank all the botanists who have contributed their herbarium sheets in the herbaria LJS and LJU. References Accetto, M., 1995. Neckero crispae-Campanuletum justinianae ass. nova v Sloveniji. Razprave 4. razreda SAZU, 36 (2), 31–48. Accetto, M., 1999a. Novo in neznano o rastlinstvu in rastju z območja nad Srebotnikom ob Kolpi. Gozdarski vestnik, 57 (9), 368–380. Accetto, M., 1999 b. Asociacija Carici sempervirentis-Pinetum nigrae (Accetto 1996) Accetto 1999 nom. nov. v Sloveniji (ob stoletnici rojstva prvega slovenskega fitocenologa univ. prof. Gabrijela Tomažiča). Zbornik gozdarstva in lesarstva, 60, 197–151. Accetto, M., 2002a. Pragozdno rastlinje rezervata Krokar na Kočevskem. Gozdarski vestnik, 60 (10), 419–444. Accetto, M., 2002b. Nova spoznanja o rastlinstvu in rastju Gorjancev. Gozdarski vestnik, 60 (4), 192–205. Accetto, M., 2003. Posebnosti rastlinstva in rastja v soteskah Potoka in Modrega potoka v dolini Kolpe. Gozdarski vestnik, 61(3), 115–131. Accetto, M., 2007a. Laserpitium krapfii. In: N. Jogan (ed.): Nova nahajališča 20. Hladnikia, 20: 42. Accetto, M., 2007 b. Arunco-Fagetum Ž. Košir 1962 var. geogr. Acer obtusatum var. geogr. nov. v dolini zgornje Kolpe. Gozdarski vestnik, 65 (9), 422–440. Accetto, M., 2008. Floristične in vegetacijske zanimivosti ob vznožju previsne stene s spodmolom nad Ribjekom ob Kolpi. Hladnikia, 21: 3–17. Accetto, M., 2010. Rastlinstvo Iškega Vintgarja. Praprotnice in semenke. Folia biologica et geologica, 51 (4), 5–149. Accetto, M., 2013. Rastlinstvo in deloma rastje soteske Zale v zgornjem porečju Iške. Zbornik gozdarstva in lesarstva, 99: 3–149. Accetto, M., Babij, V., Carnelutti, J., Čelik, T., Drovenik, B., Seliškar, A., Trpin, D., Vreš, B., 1996. Inventarizacija flore, vegetacije in favne na predvideni trasi ceste Dragarji–Čačiči in naravovarstve- no mnenje (elaborat). Biološki inštitut ZRC SAZU, Ljubljana. 30 pp. Aeschimann, D., Lauber, K., Moser, D. M., Theurillat, J.-P., 2004. Flora alpina. Bd. 1: Lycopodiaceae- Apiaceae. Haupt Verlag, Bern, Stuttgart, Wien. 1159 pp. Braun-Blanquet, J., 1964. Pflanzensoziologie. Grundzüge der Vegetationskunde. 3. Auflage. Springer, Wien – New York. 865 pp. Čušin, B., 2006. Rastlinstvo Breginjskega kota. Založba ZRC, ZRC SAZU, Ljubljana. 198 pp. Dakskobler, I., 1991. Gozd bukve in jesenske vilovine – Seslerio autumnalis-Fagetum (Ht. 1950) M. Wraber (1957) 1960 v submediteransko-predalpskem območju Slovenije. Scopolia, 24: 1–53. Dakskobler, I., 1994. Zapiski o rastlinstvu doline Zadlaščice v južnih Julijskih Alpah. Proteus, 56 (7), 251–257. Dakskobler, I., 1997. Geografske variante asociacije Seslerio autumnalis-Fagetum (Ht.) M. Wraber ex Borhidi 1963. Razprave 4. razreda SAZU, 38 (8), 165–255. 20 Acta Biologica Slovenica, 58 (1), 2015 Dakskobler, I., 2001. Laserpitium krapfii,. In: N. Jogan (ed.): Nova nahajališča – New localities, Hladnikia, 11: 46–47. Dakskobler, I., 2002. Jelovo-bukovi gozdovi v dolinah Kneže, Zadlaščice in Tolminke (južne Julijske Alpe, zahodna Slovenija). Razprave 4. razreda SAZU, 43 (3), 111–165. Dakskobler, I., 2003. Asociacija Rhododendro hirsuti-Fagetum Accetto ex Dakskobler 1998 v zahodni Sloveniji. Razprave 4. razreda SAZU, 44 (2), 5–85. Dakskobler, I., 2006. Asociacija Rhodothamno-Laricetum (Zukrigl 1973) Willner & Zukrigl 1999 v Julijskih Alpah. Razprave 4. razreda SAZU, 47 (1), 117–192. Dakskobler, I., 2015. Phytosociological description of black hornbeam (Ostrya carpinifolia) and flowering ash (Fraxinus ornus) communities in the Julian Alps and in the northern part of the Dinaric Alps (NW and W Slovenia, NE Italy). Hacquetia, 14 (2), 175–247. Diaci, J., 1997. Experimentelle Felduntersuchungen zur Naturverjüngung künstlicher Fichtenwälder auf Tannen-Buchenwaldstandorten (Homogyno sylvestris-Fagetum) in den Savinja-Alpen (Slowe- nien) mit besonderer Berücksichtigung der Ansamungsphase und unter dem Einfluß der Faktorn Licht, Vegetation, Humus und Kleinsäuger, (Diss. ETH, nr. 11357), (Beiheft zur Schweizerischen Zeitschrift für Forstwesen, 80). Schweizerischer Forstverein, Zürich. 197 pp. Ehrendorfer, F., Hamann, U., 1965. Vorschläge zu einer floristischen Kartierung von Mitteleuropa. Ber. Deutsch. Bot. Ges., 78, 35–50. Fischer M. A., Adler, W., Oswald, K., 2008. Exkursionsflora von Österreich, Liechtenstein und Südtirol. Land Oberösterreich, Biologiezentrum der OÖ Landesmuseen, Linz. 1391 pp. Fleischmann, A., 1844. Übersicht der Flora Krain’s. Ann. Landwirth.-Ges. Krain 6: 103–246 (separ. 1–144), Ljubljana. Głowacki, J., Arnold, F.,1870, Flechten aus Krain und Küstenland. Verh. Zool.-Bot. Ges. Wien, Bd., 20, 431–466. Hartl, H., Kniely, G., Leute, G. H., Niklfeld, H., Perko, M. 1992. Verbreitungsatlas der Farn- und Blütenpflanzen Kärntens. – Naturwisenschaftlicher Verein für Kärnten, Klagenfurt. Hočevar, S., Batič, F., Martinčič, A., Piskernik, M.,1985. Preddinarski gorski pragozdovi: Trdinov vrh in Ravna gora na Gorjancih, Kopa v Kočevskem Rogu in Krokar na hrbtu pogorja Borovška gora - Planina nad Kolpo (mikoflora, vegetacija in ekologija). Strokovna in znanstvena dela 76. Univerza Edvarda Kardelja v Ljubljani VDO Biotehniška fakulteta VTOZD za gozdarstvo, Inštitut za gozdo in lesno gospodarstvo, Ljubljana. 267 pp. Hočevar, S., Batič, F., Piskernik, M., Martinčič, A. 1995. Glive v pragozdovih Slovenije. III. Dinarski gorski pragozdovi na Kočevskem in v Trnovskem gozdu. (Strokovna in znanstvena dela 117). Gozdarski inštitut Slovenije, Ljubljana. 320 pp. Jalas, J., Suominen, J., 1967. Mapping the distribution of Europaean vascular plants. Memoranda Soc. pro Fauna Flora Fennica, 43, 60–72. Jogan, N., Bačič, T., Frajman, B., Leskovar, I., Naglič, D., Podobnik, A., Rozman, B., Strgulc–Krajšek, S., Trčak, B., 2001. Gradivo za Atlas flore Slovenije. Center za kartografijo favne in flore, Miklavž na Dravskem polju. 443 pp. Košir, Ž., 1979. Ekološke, fitocenološke in gozdnogospodarske lastnosti Gorjancev v Sloveniji. Zbornik gozdarstva in lesarstva, 17 (1), 1–242. Marinček, L., 1996. Subalpine Buchenwälder in den Westlichen Dinariden. Atti del 24o Simposio della Societa Estalpino-Dinarica di Fitosociologia. Ann. Mus. Civ. Rovereto. Sez.: Arch., St., Sc. nat. Suppl. II, vol. 11 (1995). pp. 197–208. Marinček, L., Čarni, A., 2010. Altimontanski bukovi gozdovi podzveze Saxifrago-Fagenion (Aremonio- Fagion). Scopolia, 69. 1–107. Martinčič, A., 2007. Apiaceae – kobulnice. In: A. Martinčič (ed.): Mala flora Slovenije. Ključ za določanje praprotnic in semenk. Tehniška založba Slovenije, četrta, dopolnjena in spremenjena izdaja, Ljubljana. pp. 379–412. 21Bačič et al.: Laserpitium krapfii in Slovenia Martinčič, A., Wraber, T., Jogan, N., Podobnik, A., Turk, B., Vreš, B., Ravnik, V., Frajman, B., Strgulc Krajšek, S., Trčak, B., Bačič, T., Fischer, M. A., Eler, K., Surina, B., 2007. Mala flora Slovenije. Ključ za določanje praprotnic in semenk. Četrta, dopolnjena in spremenjena izdaja. Tehniška založba Slovenije, Ljubljana. 967 pp. Mayer, E., 1952. Seznam praprotnic in cvetnic slovenskega ozemlja. Dela IV. razreda SAZU 5 (Inštitut za biologijo 3), Ljubljana. 427 pp. Neugebauer, H., 1932. Morphologiscch-geographische Studie über Laserpitium Krapfii Crantz amplify. Thellung.Österreichische botanische Zeitschrift (Wien) 81 (4), 241–275. Niklfeld H., Schratt-Ehrendorfer, L., 1999. Rote Listen gefährdeter Pflanzen Österreichs 2., neu be- arbeitete Auflage – Farn- und Blütenpflanzen. Grüne Reihe des Bundesministeriums für Umwelt, Jugend und Familie, Band 10. Verlag, austria medienservice, Graz. 291 pp. Paulin, A., 1904. Schedae ad Floram exsiccatam Carniolicam 2. Centuria V et VI. Beiträge zur Kenntnis der Vegetationsverhältnisse Krains. III. Samozaložba, Ljubljana. pp. 215–308. Pignatti, S., 1982. Flora d’Italia, Vol. 2. Edagricole. Bologna. p. 244. Piskernik, M., 1977. Gozdna vegetacija Slovenije v okviru evropskih gozdov. Zbornik gozdarstva in lesarstva, 15 (1), 1–236. Piskernik, M., 1991. Gozdna, travniška in pleveliščna vegetacija Primorske. Strokovna in znanstvena dela, 106, Inštitut za gozdno in lesno gospodarstvo, Ljubljana. 241 pp. Plemel, V., 1862. Beiträge zur Flora Krains. Drittes Jahresheft d. Ver. Krain. Landesmus., Ljubljana. pp. 120–164. Poldini, L. (in cooperation with Oriolo, G. and Vidali, M.), 2002. Nuovo Atlante corologico delle piante vascolari nel Friuli Venezia Giulia. Regione Autonoma Friuli Venezia Giulia, Azienda Parchi e Foreste Regionali & Università degli Studi di Trieste, Dipartimento di Biologia, Udine. 529 pp. Robič, D., 1960a. Gozdna vegetacija Mokreca. Diplomsko delo. Univerza v Ljubljani, Biotehniška fakulteta, Oddelek za gozdarstvo. Robič, D., 1960b. Priloge h gozdnogospodarskemu načrtu za desetletje 1962–1971. Karte in opisi gozdnovegetacijskih tipov za gozdne predele Turjak, Medvedica in Mokrec. Elaborat. Gozdno gospodarstvo Ljubljana. Rozman, B., 2000. Flora okolice Zaplane 0051/1. Diplomska naloga. Biotehniška fakulteta. Oddelek za biologijo. Seliškar, T., Vreš, B., Seliškar, A., 2003. FloVegSi 2.0. Računalniški program za urejanje in analizo bioloških podatkov. Biološki inštitut ZRC SAZU, Ljubljana. Surina, B., Rakaj, M., 2007. Subalpine beech forests with Hairy Alpenrose (Polysticho lonchitis- Fagetum rhododendretosum hirsuti subass. nova) on Mt. Snežnik (Liburnian Karst, Dinaric Mts). Hacquetia, 6 (2), 195–208. Šilc, U., Čarni, A., 2012. Conspectus of vegetation syntaxa in Slovenia. Hacquetia, 11 (1), 113–164. Štimec, I., 1982. Flora osnovnega polja 0454 Cerk. Diplomska naloga. Biotehniška fakulteta, Oddelke za biologijo. Thellung, A., 1975. Laserpitium krapfii Crantz. In: G. Hegi: Flora von Mittel-Europa. 5 (2), Verlag Paul Parey, Berlin und Hamburg. pp. 1481–1486. Tregubov, V., 1957. Gozdne rastlinske združbe. In: V. Tregubov & M. Čokl (eds.): Prebiralni gozdovi na Snežniku., Inštitut za gozdno in lesno gospodarstvo, Strokovna in znanstvena dela 4, Ljubljana. pp. 23–65. Trčak, B., Frajman, B., Rozman, B., Jogan, N., 2002. Poročilo o delu floristične skupine. V: Gergeli, A. (ur.): Raziskovalni tabor študentov biologije Semič 2001. Zveza za tehnično kulturo Slovenije, Ljubljana. pp. 11–18. Tutin, T. G. 1968. Laserpitium L. In: T. G. Tutin & al.: Flora Europaea, Vol. 2 (Rosaceae to Umbel- liferae). Cambridge University Press. pp. 368–370. Vukelić, J., Alegro, A., Šegota, V., 2010. Altimontansko – subalpska smrekova šuma s obrubljenim gla- decem (Laserpitio krapfii-Piceetum abietis ass. nova) na sjevernom Velebitu (Hrvatska). Šumarski list (Zagreb) 134 (5–6), 211–228. 22 Acta Biologica Slovenica, 58 (1), 2015 Wraber, T., 1966. Floristične novosti z Notranjskega Snežnika. Varstvo narave, 4 (1965), 43–49. Wraber, T., 1996. Blagayev volčin (Daphne blagayana Freyer) v okolici Vrhnike. Vrhniški razgledi (Vrhnika), 1, 31–42. Wulfen, F. X., 1858. Flora norica phanerogama. (Herausg. E. Fenzl & R. Graf). Wien. XIV + 816 pp. Zupančič, M., 1999. Smrekovi gozdovi Slovenije (Spruce forests in Slovenia). Dela 4. razreda SAZU 36, Ljubljana. 212 pp. + tab. Zupančič, M., Accetto, M.,1994. Ribeso alpini-Piceetum ass. nova v Dinarskem gorstvu Slovenije. Razprave 4. razreda SAZU, 35, 152–175. Appendix Data on the revised herbarium specimens (Specimina visa) of Laserpitium krapfii subsp. krapfii 0051/2 Slovenija: In silvis lucidis declintalis septentrionali - orientalis montis Ulovka supre opp. Vrhnika, s. dolom., 610 m s. m.; leg. T. Wraber, 18. 7. 1994 (LJU10032497). 0052/4 Slovenija: Carniolia. In silvaticis et dumetosis montis Krim (ditio Labacensis), s. calc.; 800 m, leg. Dolšak F., 6. 7. 1925 (LJU10032509). 0052/4 Slovenija: Carniolia. In silvaticis et dumetosis montis Krim (ditio Labacensis), s. calc.; 800 m, leg. Dolšak F., 7. (LJU10032510). 0052/4 Slovenija: Krim. leg. Budnar, 8. 8. 1948 (LJU10032500). 0052/4 Slovenija: Carniola. In pratis et silvaticis lapidosis montis Krim prope Labacum; solo calcareo; 1000 m s. m. (Flora exsiccata Carniolica); leg. Paulin A., 7. (LJU10032496). 0149/2 Slovenija: Primorska, Otlica, Kovk: bukov gozd (Seslerio autumnalis-Fagetum), ekspozicija: NE, nagib: 30°; 740 m n. m.; leg. I. Dakskobler, 13. 7. 1989 (LJS03258). 0149/2 Slovenija: Primorska, Otlica, Kovk: bukov gozd (Seslerio autumnalis-Fagetum), ekspozicuija: N, nagib: 30°. 830 m n.m.; leg. I. Dakskobler, 13.7.1989 (LJS03259) 0152/2 Slovenija: Ljubljanska okolica: Iški Vintgar - in fruticosis, solo calcareo, cca 330 m s. m., leg. E. Mayer, 19.8.1954 (LJU10032507). 0152/2 Slovenija: Rakiška planota JZ od Krima nad Ljubljano, ob umetnem jezeru J od vasi Rakitna; listnat gozd, 800 m n. m.; leg. A. Zrimec, 22.7.1991 (LJU10032498). 0153/1 Slovenija: Dolenjska, Mokrec pri Igu, tik pod vrhom: bukov gozd (Omphalodo-Fagetum (Treg. 1957) Marinček et al. 1993), ekspozicija: W, nagib: 25°, rendzina, apnenec. 972 m n.m.; leg. V. Babij, det. D. Robič, 17. 6. 1997 (LJS05987). 0158/3 Slovenija, Gorjanci: ob poti s Polma na Mirčev grič, leg. V. Strgar, 29. 6. 1960 (LJU10032506). 0257/2 Slovenija: Gorjanci: Gospodična - pr. Miklavž, gozd, 920 m, leg J. Rataj, 23. 6. 1954 (LJU10032490). 0257/2 Slovenija: In silvis umbrosis humidis prope refugium alpinum Paderšičeva koča in monte Gorjanci, solo dolomitico, 850 m s. m., A. Martinčič, 24. 6. 1954 (LJU10032508). 0257/2 Slovenija, Gorjanci: In silvis prope locum Gospodična dictum supra vicum Gabrje. 830 m s. m.; leg. T. Wraber, 26. 6. 1992 (LJU10032501). 0257/2 Slovenija: Dolenjska, Gorjanci, Gabrje (Novo mesto), Gospodična: v bukovem gozdu (Arunco-Fagetum), karbonat, ca. 900 m n.m.; leg. I. Dakskobler, 23. 8. 1989 (LJS03257). 0352/4 Slovenija: Snežnik, Peklo: solo dolomitico; 1230 m s. m., leg. M. Zupančič, 31. 8. 1972 (LJU10032502). 0353/1 Slovenija: Kranjsko-notranjska flora: Biva med grmovjem ob cesti pod Mašunom pod Snežnikom, leg. R. Justin, 15. 7. 1923 (LJU10032511). 0356/3 Slovenija: Podstene v Kočevskem Rogu pri Koprivniku pri Kočevju, leg. V. Plemel, 2. 8. 1849 (LJU10032495). 23Bačič et al.: Laserpitium krapfii in Slovenia 0356/3 Slovenija: Podstene v Kočevskem Rogu pri Koprivniku pri Kočevju, leg. V. Plemel, 2. 8. 1849 (LJU10032499). 0356/4 Slovenija: Kočevski Rog, Semič, Planina - Mirna gora, 800-1000 m n. m. v.; suh, topel travnat pas pod el. daljnovodom (RTŠB Semič 01), leg. B. Frajman, 26. 7. 2001 (LJU10130873). 0452/2 Slovenija, Notranjski Snežnik: Veliki Snežnik, in fagetis /Fagetum subalpinum/ declivitatis septentrionalis; 1550 n s. m.; leg. T. Wraber, 12. 8. 1965 (LJU10032503). 0454/1 Slovenija, Kočevsko: Goteniški Snežnik, gozdna jasa, 1100 m n. m.; leg. I. Štimec, 16. 7. 1981 (LJU10032491). 0454/1 Slovenija: Dolenjska, Bezgovica (Osilnica), Bezgarska planina: travnik, opuščen pašnik, 894 m n. m.; leg. B. Vreš B. & T. Čelik, 13. 7. 2013 (LJS11851). 0454/2 Slovenija: Kočevsko: pobočje Krempe, med grmovjem, 900 m n.m. (Flora osnovnega polja 0454 Cerk), leg. I. Štimec, 4. 7. 1982 (LJU10032494). 0454/2 Slovenija: Kočevsko: Ravne, na gozdni poseki ob poti v Krokarski pragozd, 840 m n. m. (Flora osnovnega polja 0454 Cerk), leg. I. Štimec, 17. 7. 1981 (LJU10032493). 0454/4 Slovenija: In silvis montis Krempa supra vallem fluvii Kolpa, 800 m s. m. leg. A, Martinčič, 9. 7. 1958 (LJU10032504 ). 0454/4 Slovenija: Potok nad Mitroviči v dolini Kolpe, peščeno apnenčasto pobočje; 270 m n. m.; leg. S. Peterlin, 5.-10. 8. 1960 (LJU10032505). 9748/4 Slovenija: Primorska, Julijske Alpe, Krikov vrh: Arunco-Fagetum, 1150 m n.m.; leg. I. Dakskobler, 29. 7. 1992 (LJS03164). 9748/4 Slovenija: Primorska, Julijske Alpe, Krikov vrh: Arunco-Fagetum, 1150 m n. m.; leg. I. Dakskobler, 30. 7. 1992 (LJS03171). 9748/4 Slovenija: Julijske Alpe, Krikov vrh, pobočja nad Mirno grapo: bukov gozd (Arunco- Fagetum), strmo gruščnato pobočje, dolomit z rožencem, rendzina, ekspozicija: NE, nagib: 40°, 940 m n.m.; leg. I. Dakskobler, 21. 7. 1989 (LJS03260). 9748/4 Slovenija: Primorska, dolina Zadlaščice, Pod Sopotom: listopadni gozd (Rhododendro hirsuti-Ostryetum), strmo kamnito pobočje, ki se prelomi v steno, ob lovski poti, ekspozicija: NNW, nagib: 40°, rendzina, apnenec z rožencem. 780 m n. m.; leg. I. Dakskobler, 21. 7. 1993 (LJS03261). 9749/4 Slovenija: Baška dolina, Podbrdo, Batava, pobočje Robarjevega griča, 760 m n. m., bukov gozd, Arunco-Fagetum s. lat., leg. I. Dakskobler, 24. 6. 1990 (LJS, study collection). New locality in Prealpine phytogeographical region. 9848/4 Slovenija: dolina Idrijce, Slap ob Idrijci, osojna pobočja Špehovega brda, Vresnica nad domačijo Bukovca, 350 m n. m., bukov gozd, Arunco-Fagetum; leg. I. Dakskobler, 14. 5. 2002, (LJS, study collection) New localiy in Prealpine phytogeographical region, but actually in northwe- sternmost edge of Trnovski gozd plateau (Dinaric mountains). 9849/1 Slovenija: Baška dolina, vznožje Koriške gore nad cesto Humar-Hudajužna, 390 m in 470 m n. m., bukov gozd, Ostryo-Fagetum; leg. I. Dakskobler, 29.6. 1989 (LJS, study collection) (new locality in Alpine phytogeographical region). 9849/1 Slovenija: Baška dolina, vznožje Kobilice nad čuvajnico ob železniški progi Grahovo ob Bači-Hudajužna, okoli 400 m n. m., bukov gozd, Ostryo-Fagetum; leg. I. Dakskobler, 29.6. 1989 (LJS, study collection) New locality in Prealpine phytogeographical region. Cytological analysis of Fallopia japonica and Fallopia ×bohemica shoots during growth season Citološka analiza poganjkov japonskega in češkega dresnika med rastno sezono Jasna Dolenc Koce*, Katarina Šoln, Brina Stančič, Jon Bančič, Timotej Čepin, Aleš Kladnik University of Ljubljana, Biotechnical Faculty, Department of Biology, Večna pot 111, SI-1000 Ljubljana *correspondence: jasna.dolenc.koce@bf.uni-lj.si Abstract: Fallopia japonica and Fallopia ×bohemica are two very invasive plant species in Europe and North America. Their main mode of spread is vegetative repro- duction. In spring new shoots emerge from the overwintering rhizome, grow rapidly and develop broad leaves which shade undergrowth plants. We studied cell size and starch accumulation in three stem regions at five sampling times during one growth season to determine possible differences in growth dynamics of both Fallopia species. On average F. ×bohemica had somewhat larger cells than F. japonica but the differences were not signi- ficant, except in the internodes of the middle stem region with differentiating cells. Also, cell growth dynamics of both species was similar and the only difference was detected at the 2nd sampling when cells of F. ×bohemica were more elongated. F. ×bohemica also accumulated starch earlier in the growth season and in younger tissues than F. japonica. Keywords: Fallopia japonica, Fallopia ×bohemica, cell size, starch, growth Izvleček: Japonski (Fallopia japonica) in češki dresnik (F. ×bohemica) sta v Evropi in Severni Ameriki zelo invazivni tujerodni rastlinski vrsti. Nespolno razmnoževanje je glavni način njunega razširjanja in vsako pomlad iz korenike požene brst, ki hitro zraste in razvije široke liste, ki zasenčijo podrast. V raziskavi smo preučevali velikost celic in tvorbo škrobnih zrn v treh območjih stebla pri petih vzorčenjih, ki smo jih opravili v eni rastni sezoni in tako ugotavljali morebitne razlike v dinamiki rasti pri obeh vrstah dre- snika. V povprečju so bile celice češkega dresnika nekoliko večje kot celice japonskega dresnika, vendar razlike niso bile statistično značilne razen v členkih srednjega dela stebla z diferencirajočimi se celicami. Tudi dinamika celične rasti je bila podobna pri obeh vrstah dresnika razen pri 2. vzorčenju, ko so bile celice češkega dresnika bolj podaljšane. Češki dresnik je v primerjavi z japonskim kopičil škrob prej in v mlajših tkivih. Ključne besede: japonski dresnik, češki dresnik, velikost celic, škrob, rast ACTA BIOLOGICA SLOVENICA LJUBLJANA 2015 Vol. 58, [t. 1: 25–33 Introduction Fallopia japonica (Houtt.) Ronse Decr. (Po- lygonaceae) and its close relatives in the section Reynoutria are well known and problematic invasive plant species worldwide. In Slovenia are present octoploid Japanese knotweed (F. japonica var. japonica (Houtt.) Ronse Decr.), tetraploid giant 26 Acta Biologica Slovenica, 58 (1), 2015 knotweed (F. sachalinensis (F. Schmidt) Ronse Decr.) and their hybrid hexaploid Bohemian knot- weed (F. ×bohemica (Chrtek and Chrtková) J. P. Bailey) (Strgulc Krajšek and Jogan 2011). Japanese knotweed is cytologically and genetically uniform in Europe and has successfully spread since its early introduction in Europe in the 19th century. Now it is considered as one of the most invasive plant species. On the other hand, giant knotweed has limited genetic variation and is less invasive. Their hybrid Bohemian knotweed exhibits the highest genetic variation (Bailey et al. 2009) and recent study shows that it also has higher invasive potential than Japanese knotweed (Parepa et al. 2014). All three knotweed species are approx. 2-4 m tall herbaceous perennials with high growth rate and broad leaves which shade undergrowth plants (Herpigny et al. 2012). The main competitive traits of knotweeds against the native flora were shown to be their vegetative reproduction with rhizomes and high regeneration success, but allelopathy and genome plasticity also account. All these character- istics enable knotweeds successful colonisation of new habitats, especially ruderal habitats and river banks (Parepa et al. 2014). Japanese knotweed is up to 2 m high and has up to 15 cm long leaves, giant knotweed is higher, from 2 to 4 m, and has up to 30 cm long leaves while Bohemian knotweed is intermediate in size (Vreš 2007, Bailey et al. 2009). The size and shape of organs are related to cell division, cell elonga- tion and cell differentiation which take place in the meristematic and submeristematic tissues. Environmental (water and nutrient supply) as well as genetic factors control organ growth (Mizukami 2001, John and Qi 2008, Krizek 2009). Cell size can be correlated also to the ploidy level (Kondorosi et al. 2000, Sugimoto-Shirasu and Roberts 2003) and has been used in the taxonomic studies to estimate nuclear DNA content and ploidy level by the size of the stomatal guard cells (Šturm and Bačič 2013) even in the fossil plants (Masterson 1994). It was also shown that polyploidization can represent a mean to accelerate the growth of the plant species in niches that require and support fast development (Barow and Meister 2003) and can facilitate plant invasions (te Beest et al. 2012). The aim of our study was to compare the growth of Japanese and Bohemian knotweed during one season. Different cytological traits (cell size, presence of starch grains) were determined and the following questions were asked: (i) Are the cells in the octoploid Japanese knotweed larger cells than in the hexaploid Bohemian knotweed? (ii) Is the cell size different in the meristematic, young and mature regions of the stem, which would lead to different growth dynamics in both Fallopia species? (iii) Is the starch accumulation species specific and correlated to cell size dynamics? Material and methods Knotweed sampling Shoot samples of five plants of Japanese knot- weed (F. japonica var. japonica (Houtt.) Ronse Decr.) and Bohemian knotweed (F. ×bohemica (Chrtek and Chrtková) J. P. Bailey) were collected in Ljubljana, Slovenia (46° 2′ 33.98″ N, 14° 27′ 0.91″ E and 46° 3′ 0.3″ N, 14° 28′ 44″ E, respec- tively) from April to October 2013. According to the developmental stage of the shoot, apical meri- stem (upper region, 1st internode), young (medium region, 5th internode) and mature internodes (lower region, 10th and 15th internode) were collected for further cytological analyses (Table 1). Table 1: Sampling data. Tabela 1: Potek vzorčenja. Sampling Date Collected material Developmental stage 1 15.4.2013 Shoot apex Emerging shoot bud from the rhizome 2 24.4.2013 Shoot apex, internode 5 Young plants approx. 50 cm high 3 5./10.7.2013 Internodes 1, 5, 10 Fully grown plants approx. 2 m high 4 29.8.2013 Internodes 5, 10, 15 Flowering plants 5 25.10.2013 Internodes 5, 10, 15 Senescent plants at the end of the season 27Dolenc Koce et al: Cytological analysis of Fallopia shoots Fixation and preparation of microscopic slides Approx. 1 cm long tissue samples were cut from the stem and fixed in FAA (3.7% formalde- hyde, 50% ethanol, 5% glacial acetic acid) for at least 24 hours at 4°C. For longer storage samples were transferred to 70% ethanol at -20°C. Thin transversal and longitudinal sections of fixed material were hand-cut using a razorblade. Sections were put in a drop of distilled water on the objective glass, covered and analysed with a light microscope (Axioskop 2 MOT, Carl Zeiss, Germany) combined with a colour digital camera (AxioCam MRc, Carl Zeiss, Germany) and AxioVi- sion 4.8 software (Carl Zeiss, Germany). For better resolution of cell walls, autofluorescence (at UV excitation 365/12 nm band pass and emission 397 nm long pass) was also recorded when necessary. Starch identification The presence of starch grains was determined by staining the sections with the indicator iodine solution (3% KI/I2 (w/v)). Image analysis and statistics In the longitudinal sections, width and length of cells (Fig. 1) were measured for at least 20 cells using ImageJ software (Rasband, 1997-2014). The obtained data were used to calculate cell volume according to the formula V=πr2l (r – half of the cell width, l - cell length). Mean values and stan- dard errors were calculated and the samples were compared by t-test and ANOVA (GraphPad Prism). The level of significance was set at p-value < 0.05. Results Cell size of F. japonica and F. ×bohemica gradually increased during the growth season from the smallest meristematic cells at the shoot apex (average width 40.73 μm, average volume 30.95×103 μm3) to the largest mature cells of the 10th and 15th internode (average width 64.38 μm, average volume 593.05×103 μm3). Cell width in the middle shoot region increased to 123% and to 158% in the lower mature region when compared to the meristematic cells in the upper shoot region (Fig. 2A). Cell volume increased more intensely indicating higher level of cell elongation than expansion of cell width; the volume increased to 758% in the middle shoot region and to 1917% in the lower mature region when compared to the meristematic cells in the upper region (Fig. 2B). The width and volume of the cells in both Fallopia species were not significantly different except for the volume of the cells in the middle shoot region (p=0.020). Dynamics of the cell growth during the season was estimated by a trend line and was similar in case of cell width and cell volume expansion for both Fallopia spe- cies (cell width trend line equation for F. japonica y=14.10x, R2=-0.86; for F. ×bohemica y=15.18x, R2=0.15; cell volume trend line equation for F. japonica y=99.76x, R2=0.31; for F. ×bohemica y=110.13x, R2=0.16). Upper shoot region In the upper shoot region cells of the shoot apical meristem and the youngest internodes (1st) were measured. These cells were collected only during first three samplings. Later in the growth season at the 4th and 5th sampling, the apical region developed in reproductive tissues without the shoot apical meristem (Tab. 1). The cell width was very similar for both Fal- lopia species (p=0.974) but the difference was more pronounced in cell volume (p=0.351) where cells of F. ×bohemica were on average larger (39.66×103 μm3) than of F. japonica (25.31×103 μm3) (Fig. 3, Suppl. Tab. 2). In this region, starch granules were present in F. x bohemica at all sampling times while in F. japonica starch occured only at the 3rd sampling when plants were fully developed (Fig. 4). 28 Acta Biologica Slovenica, 58 (1), 2015 Figure 1: Transversal (left) and longitudinal (right) sections of F. japonica and F. ×bohemica shoots. A – Sections of F. japonica meristematic region at the 1st sampling; B - Sections of F. japonica mature shoot region at the 3rd sampling. C - Sections of F. ×bohemica meristematic region at the 1st sampling. D - Sections of F. ×bohemica mature shoot region at the 3rd sampling. Measurement of width (w) and length (l) of cells is indicated by lines in panels C and D. Bar represents 100 μm. Slika 1: Prečni (levo) in vzdolžni (desno) prerez poganjka pri F. japonica in F. ×bohemica. A – Prereza zgornjega meristemskega območja pri F. japonica pri 1. vzorčenju. B - Prereza spodnjega zrelega območja pri F. japonica pri 3. vzorčenju. C - Prereza zgornjega meristemskega območja pri F. x bohemica pri 1. vzorčenju. D - Prereza spodnjega zrelega območja pri F. x bohemica pri 3. vzorčenju. Meritev širine (w) in dolžine (l) celic je prikazana z oznakami na slikah C in D. Merilo predstavlja 100 μm. 29Dolenc Koce et al: Cytological analysis of Fallopia shoots Figure 2: Dynamics of cell growth of F. japonica (FJ) and F. ×bohemica (FB) during growth season. Data present A - cell width and B - cell volume (N=5-25). Trendline is a linear regression line (intercept set at 0.0). Slika 2: Dinamika rasti celic pri F. japonica (FJ) in F. ×bohemica (FB) med rastno sezono. Rezultati prikazujejo A - širino in B - prostornino izmerjenih celic (N=5-25). Trendna črta je linearna regresijska premica (izhodišče nastavljeno na 0,0). Table 2: Cell size in the upper shoot region/meristem of F. japonica and F. ×bohemica. Mean value ± standard error are presented (N=5-10). Tabela 2: Velikost celic v zgornjem/ meristemskem delu stebla pri F. japonica in F. ×bohemica. Rezultati prika zujejo povprečno vrednost ± standardno napako (N=5-10). Sampling F. japonica F. ×bohemica Volume (×103 μm3) Width (μm) Volume (×103 μm3) Width (μm) 1 19.33±6.16 2.46±5.56 9.64±0.49 42.66±3.83 2 19.76±6.06 33.35±2.18 7.57±3.77 35.82±1.44 3 43.88±17.26 50.62±6.30 93.23±33.39 42.91±3.12 30 Acta Biologica Slovenica, 58 (1), 2015 Middle shoot region In the middle shoot region, differentiating cells of the intermediate (5th) internode were measured. These cells were collected during four samplings and were missing only at the 1st sampling because plants were too small (Tab. 1). The width of F. x bohemica cells was on average larger (52.95 μm) than of F. japonica cells (47.51 μm) and the difference was almost significant (p=0.067). On the other hand the cell volume was significantly (p=0.020) larger in F. ×bohemica (321.48×103 μm3) than in F. japonica (178.75×103 μm3) mostly because of the differ- ences at the beginning of the growth season at the 2nd and the 3rd sampling. Later (4th and 5th sampling) the volume size was similar in both Fallopia spe- cies (Fig. 3, Suppl. Tab. 3). In the middle region, there was no difference in the accumulation of the starch granules which were present in cells of both Fallopia species from the 3rd sampling on. Figure 3: Cell size of F. japonica (FJ) and F. ×bohemica (FB) in the upper (U), medium (M) and lower (L) shoot region; A - cell width, B - cell volume. * statistically significant (p<0.05) difference between F. japonica and F. ×bohemica. Slika 3: Velikost celic pri F. japonica (FJ) in F. ×bohemica (FB) v zgornjem (U), srednjem (M) in spodnjem (L) delu poganjka; A - širina celic, B - prostornina celic. * statistično značilna (p<0.05) razlika med vrstama F. japonica in F. ×bohemica. 31Dolenc Koce et al: Cytological analysis of Fallopia shoots Lower shoot region In the lower shoot region cells of fully differ- entiated and mature (10th and 15th) internodes were measured. These cells were collected in the fully developed plants from the 3rd sampling on (Tab. 1). At the 4th and 5th sampling, the cells of F. japonica had significantly (p=0.038) larger vol- ume than earlier in the growth season. The same pattern was observed also in F. ×bohemica but the difference was not significant (p=0.292). On the other hand, the cell width was similar during all mature period (Fig. 3, Suppl. Tab. 4). In the lower region, starch granules were present in cells of both Fallopia species at all sampling times. Discussion Japanese and Bohemian knotweed are im- portant invasive species in Slovenia and Europe. They form large and dense monospecific stands along rivers, railways, roads and other ruderal habitats, and severely decrease native biodiver- sity. In spring they restore the shoot from the underground rhizome and very quickly develop high stem and broad leaves. In our previous study (Strgulc Krajšek and Dolenc Koce 2015) it was shown that only octoploid Japanese knotweed (F. japonica var. japonica) and hexaploid Bohemian knotweed (F. ×bohemica) present highly invasive populations in Slovenia which form monospecific as well as mixed population with both species. Giant knotweed (F. sachalinensis) is less common and at the moment does not present such severe invasive threat. The same observations were reported also for Belgium (Herpigny et al. 2012). Since level of ploidy can affect the cell and organ size as well as plant fitness (Barow and Meister 2003) we aimed to compare the dynamics of cell growth during the season in F. japonica and F. ×bohemica species to determine if cytological traits are correlated to morphological differences of adult plants (F. ×bohemica is taller and has bigger leaves than F. japonica). Cell size gradually increased from the small- est apical cells to the fully developed cells in the lower shoot regions as plants developed during the growth season (Fig. 1). Cell width increased less than cell volume which indicates that cells primarily elongated. At the end of the season both Fallopia species had cells of similar size. The dynamics of cell growth shows that the only significant difference was measured at the 2nd sam- pling when middle size cells of F. ×bohemica had 896% higher volume than of F. japonica (Fig. 2). Nevertheless, when cell data for all shoot regions and all sampling times at are pulled together and compared for both Fallopia species it is revealed that F. ×bohemica has bigger cells than F. japonica and differences are close to significant (p=0.065 for Table 3: Cell size in the middle shoot region of F. japonica and F. ×bohemica. Mean value ± standard error are presented (N=5-10). * statistically significant (p<0.05) difference between F. japonica and F. ×bohemica Tabela 3: Velikost celic v srednjem delu stebla pri F. japonica in F. ×bohemica. Rezultati prikazujejo povprečno vrednost ± standardno napako (N=5-10). * statistično značilna (p<0.05) razlika med vrstama F. japonica in F. ×bohemica. Sampling F. japonica F. ×bohemica Volume (×103 μm3) Width (μm) Volume (×103 μm3) Width (μm) 2 51.96±8.03* 45.97±0.94 465.79±182.65* 49.27±5.50 3 173.04±73.764 57.47±6.13 293.36±42.58 54.04±3.59 4 309.89±110.42 42.13±3.26 305.94±92.37 49.29±3.56 5 256.18±33.90 46.03±5.89 249.69±69.78 59.22±2.64 32 Acta Biologica Slovenica, 58 (1), 2015 cell width and p=0.135 for cell volume). Similar growth kinetics was described for tested Fallopia species in Belgium with F. japonica and F. ×bo- hemica having more comparable kinetics than F. sachalinensis with lower competitive ability for light and nitrogen (Herpigny et al. 2012). In that study it was also shown that F. ×bohemica had more variable growth and functional traits (shoot height, ramification, leaf size, foliar nitrogen concentra- tion) which could be related to its hybrid origin. In our study, the variability of the cell width and cell volume was generally higher in F. japonica (CV range from 6.5 to 95.3%) than of F. ×bohemica (CV range from 8.0 to 78.4%) but only one cell trait was tested which is not enough to generalize the conclusion about cell growth. We also tested correlation between cell size and ploidy level which was not significant therefore we conclude that polyploidy in investigated F. japonica and F. ×bohemica species had no effect on cell size and consequently on the size of the shoot. Additionally, the presence of starch grains in all plant material was observed from the initial developmental phases until the fully developed plants. Cells of F. ×bohemica accumulated starch already at the beginning of the growth season which could contribute to its higher growth rate when compared to F. japonica plants. In the fully devel- oped and mature stems there were no differences in starch accumulation between Fallopia species. Conclusions Fallopia japonica and F. ×bohemica have similarly large stem cells and their size is not correlated to the ploidy level. Cell size of both Fallopia species gradually increased during the growth season from the smallest meristematic cells at the shoot apex to the Figure 4: Stem cells with starch grains in F. japonica (A-C) and F. ×bohemica (D-E). A, D - meristematic cells at the 1st sampling; B, E - meristematic cells at the 3rd sampling; C, F - cells of the middle shoot region at the 4th sampling. Bar presents 100 μm. Slika 4: Celice stebla s škrobnimi zrni pri F. japonica (A-C) in F. ×bohemica (D-E). A, D - meristemske celice pri 1. vzorčenju; B, E - meristemske celice pri 3. vzorčenju; C, F - celice v srednjem delu stebla pri 4. vzorčenju. Merilo predstavlja 100 μm. 33Dolenc Koce et al: Cytological analysis of Fallopia shoots Table 4: Cell size in the lower/mature shoot region of F. japonica and F. ×bohemica. Mean value ± standard error are presented (N=5-10). Tabela 4: Velikost celic v spodnjem/zrelem delu stebla pri F. japonica in F. ×bohemica. Rezultati prikazujejo povprečno vrednost ± standardno napako (N=5-10). Sampling F. japonica F. ×bohemica Volume (×103 μm3) Width (μm) Volume (×103 μm3) Width (μm) 3 292.53±121.31 78.47±6.38 455.54±103.63 71.06±4.49 4 698.03±123.22 57.34±3.14 685.04±115.98 61.07±2.80 5 630.55±102.06 58.18±3.09 577.60±103.38 71.26±4.21 largest mature cells of the 10th and 15th internode Cell volume increased more intensely indicat- ing higher level of cell elongation than expansion of cell width F. ×bohemica accumulated starch earlier in the growth season and in younger tissues than F. japonica. Povzetek Japonski (Fallopia japonica) in češki dresnik (F. ×bohemica) sta v Evropi in Severni Ameriki zelo invazivni tujerodni rastlinski vrsti. Spomladi iz prezimujoče korenike požene poganjek, ki hitro raste do končne višine (2-4 m) in ima široke liste, ki zasenčijo podrast. V raziskavi smo preučevali celično rast in tvorbo škrobnih zrn v treh območjih stebla, da bi ugotovili: (i) Ali ima oktoploidni japonski dresnik večje celice kot heksaploidni češki dresnik? (ii) Ali je velikost celic v meris- temu, mladem in zrelem območju stebla različna in imata vrsti dresnika različno dinamiko celične rasti? (iii) Ali vrsti dresnika različno kopičita škrob? V eni rastni sezoni smo petkrat (dvakrat na začetku rastne sezone, ob vegetativni zrelosti rast- lin, v času cvetenja in ob zaključku rastne sezone) vzorčili celice stebla na vršičku, v srednjem delu stebla, kjer se celice še diferencirajo, in v nižje ležečih zrelih delih stebla. Rastlinski material smo fiksirali v fiksativu in ročno pripravili prečne in vzdolžne prereze za svetlobno mikroskopijo z vidno in UV-svetlobo. Na posnetih slikah smo z računalniškim programom za analizo slike (Im- ageJ) izmerili širino in dolžino celic, iz česar smo izračunali prostornino celic. Prisotnost škroba v tkivu smo dokazali z raztopino jodovice. V povprečju so bile celice češkega dresnika nekoliko večje kot celice japonskega dresnika, vendar razlike niso bile statistično značilne razen v členkih srednjega dela stebla z diferencirajočimi se celicami. Tudi dinamika celične rasti je bila podobna pri obeh vrstah dresnika razen pri 2. vzorčenju, ko so bile celice češkega dresnika bolj podaljšane. Češki dresnik je v primerjavi z japonskim kopičil škrob prej in v mlajših tkivih. Acknowledgements This study was financially supported by the Slovenian Research Agency through research programme Plant Biology P1-0212. References Bailey, J. P., Bímová K., Mandák, B., 2009. Asexual spread versus sexual reproduction and evolution in Japanese knotweed s.l. sets the stage for the “Battle of the clones”. Biological Invasions, 11, 1189–1203. Barow, M., Meister, A., 2003. Endopolyploidy in seed plants is differently corrrelated to systematics, organ, life strategy and genome size. Plant, Cell and Environment, 26, 571-584. 34 Acta Biologica Slovenica, 58 (1), 2015 Herpigny, B., Dassonville, N., Ghysels, P., Mahy, G., Meerts, P., 2012. Variation of growth and functional traits of invasive knotweeds (Fallopia spp.) in Belgium. Plant Ecology, 213, 419-430. John, P.C.L., Qi, R., 2008. Cell division and endoreduplication: doubtful engines of vegetative growth. Trends in Plant Science, 13(3), 121-127. Kondorosi, E., Roudier, F., Gendreau, E., 2000. Plant cell-size control: growing by ploidy? Current Opinion in Plant Biology, 3, 488-492. Krizek, B.A, 2009. Making bigger plants: key regulators of final organ size. Current Opinion in Plant Biology, 12, 17-22. Masterson, J., 1994. Stomatal size in fossil plants: evidence for polyplidy in majority of angiosperms. Science, 264, 421-424. Mizukami, Y., 2001. A matter of size: developmental control of organ size in plants. Current Opinion in Plant Biology, 4, 533-539. Parepa, M., Fischer, M., Krebs, C., Bossford, O., 2014. Hybridization increases invasive knotweed success. Evolutionary Applications, 7, 413–420. Rasband, W.S., 1997-2014. ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/. Sugimoto-Shirasu, K., Roberts, K., 2003. »Big it up«: endoreduplication and cell-size control in plants. Current Opinion in Plant Biology, 6, 544-553. Strgulc Krajšek, S., Jogan, N., 2011. Rod Fallopia Adans. v Sloveniji. Hladnikia, 28, 17-40. Strgulc Krajšek, S., Dolenc Koce, J., 2015. Sexual reproduction of knotweed (Fallopia sect. Reynou- tria) in Slovenia. Preslia, 87, 17-30. Šturm, R., Bačič, T., 2013. Eleocharis palustris group (Eleocharis R. Br. subser. Eleocharis) in Slo- venia: revision in herbarium LJU. Hladnikia, 31, 11-29. te Beest, M., Le Roux, J. J., Richardson, D. M., Brysting, A. K., Suda, J., Kubešová, M., Pyšek, P., 2012. The more the better? The role of polyploidy in facilitating plant invasions. Annals of Botany, 109, 19-45. Vreš, B., 2007. Fallopia – knotweed (Fallopia – dresnik, slakovec). In: Martinčič, A., Wraber, T., Jogan, N., Podobnik, A., Turk, B., Vreš, B. (eds): Mala flora Slovenije: ključ za določanje praprotnic in semenk [Slovenian flora: determination key for Pteridophyta and Spermatophyta], 4th ed. Tehniška založba Slovenije, Ljubljana, p. 211. The effect of different compounds of selenium and iodine on selected biochemical and physiological characteristics in common buckwheat and pumpkin sprouts Vpliv različnih oblik selena in joda na izbrane biokemijske in fiziološke lastnosti pri kalicah navadne ajde in buč Mateja Germa,*, Nina Kacjan Maršića, Janja Turka, Marjetka Pirca, Aleksandra Goloba, Ana Jeršeb,c, Ana Krofličb,c, Helena Šircelja, Vekoslava Stibiljb,c aBiotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia bJožef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia cJožef Stefan International Postgraduate School, Jamova 39, SI-1000 Ljubljana, Slovenia *correspondence: mateja.germ@bf.uni-lj.si Abstract: There is little data about possible interactions between selenium and iodine on plants. Se is essential for I metabolism in the thyroid in mammals. Thus, it is of great importance to carry out the research with simultaneous application of both elements in plant cultivation that are used for human consumption. Seeds of common buckwheat and pumpkins were soaked in solutions: 10 mgSe/L in the form of selenite or selenate, and 1000 mgI/L in the form of iodide or iodate and their combinations. The content of chlorophyll a and b, and carotenoids were measured. Further, the measurements of fluorescence of chlorophyll a were performed. Control buckwheat sprouts and sprouts from seeds soaked in Se(VI) and Se(VI)+I(-1), had the lowest and similar amount of chlorophyll a and carotenoids. There was little effect of different treatments on potential photochemical efficiency of photosystem II (PS II) in common buckwheat sprouts. In pumpkin sprouts neither of treatment affected the amount of photosynthetic pigments, as well as potential photochemical efficiency of (PS II) which was around 0.8. Key words: sprouts, common buckwheat, pumpkins, selenium, iodine Izvleček: Zelo malo podatkov obstaja o interakciji med selenom in jodom pri rastlinah. Selen je bistvenega pomena za delovanje ščitnice. Zato je pomembno, da preučujemo hkraten vpliv obeh elementov na rastline, ki jih uporabljamo za hrano ljudi. Seme navadne ajde in buč smo namakali v različnih raztopinah Se in I; sele- nata in selenita ter jodida oz. jodata ter vseh njunih kombinacij. Merili smo sledeče fiziološke in biokemijske lastnosti kontrolnih in obravnavanih rastlin: fotokemično učinkovitost fotosistema II (FS II) ter vsebnost fotosinteznih barvil (klorofil a, klorofil b in karotenoidi). Kontrolne kalice ajde in kalice, zrastle iz semen, namakanih s Se(VI) in se(VI)+(I-1), so imele najnižjo in podobno vsebnost klorofila a in karotenoidov. Obravnavanja so malo vplivala na potencialno fotokemično učinkovitost fotosistema II pri kalicah navadne ajde (kalice iz semen, namakanih v Se(IV)+I(-1), so imele najnižjo potencialno fotokemično učinkovitost fotosistema II). Nobeno obravnavanje ni vplivalo na vsebnost fotosinteznih barvil in potencialno fotokemično učinkovitost ACTA BIOLOGICA SLOVENICA LJUBLJANA 2015 Vol. 58, [t. 1: 35–44 36 Acta Biologica Slovenica, 58 (1), 2015 fotosistema II pri kalicah buč. Potencialna fotokemična učinkovitost fotosistema II pri kalicah buč je bila okoli 0,8. Ključne besede: kalice, navadna ajda, buče, selen, jod Introduction Several minerals and trace elements e.g., iodine, iron, selenium, and zinc, are essential for normal thyroid hormone metabolism (Zimmermann and Köhrle 2002). Iodine (I) and selenium (Se) are not essential nutrients for plants but both play important roles in human and animal organisms (Smoleń et al. 2014). Plant roots can take up Se as selenate, selenite or organoselenium compounds, such as selenocysteine (SeCys) and selenom- ethionine (SeMet) (White et al. 2004). Selenite is rapidly converted to organoselenium compounds in the root, whereas selenate is delivered to the xylem and transported to the shoot, where it is assimilated into organoselenium compounds and redistributed within the plant (Terry et al. 2000). Role of Se is beneficial in plants capable of ac- cumulating large amounts of this element. It acted as an antioxidant, inhibiting lipid peroxidation in ryegrass and increased yield under ambient radiation conditions in pumpkins (Hartikainen et al. 2000, Germ et al. 2005). There is scarce information about the effect of iodine on plants neither possible interaction with selenium fertiliza- tion. Uptake of iodine from the soil to the plants depend from adsorption–desorption processes in the soil (Zia et al. 2014). Leaf vegetables have higher absorption capacity than fruit vegetable in ten chosen plants in the study from Weng et al. (2013). Plants take up iodine through the root system, preferably as iodide (Fuge 2005, Smoleń et al. 2011). Dai et al. (2006) evidenced that iodide (I(-1)) and iodate (IO3-) added to the soil, do not significantly affect spinach biomass production. Application of high doses of I(-1) to lettuce has a phytotoxic effect on plant physiology. In contrast, IO3- treatments increased the biomass of the plants which showed an elevated photosyn- thetic rate, stomatal conductance, and transpiration comparing to control plants. Blasco et al. (2010) reported about the response of lettuce to iodine biofortification. They found out that application of IO3-, in contrast to I(-1), increased biomass production, stimulated NO3- reduction and NH4+ incorporation and optimised the photorespiratory process. Zhu et al. (2003), who studied the effect of iodine on spinach found out that iodine is not beneficial to the growth of spinach (Spinacia oleracea), but level of iodide above 10 μM was detrimental to yields, while iodate had little effect on the biomass production. Authors suggest that the detrimental effect of iodide on plant growth is probably due to its excessive accumulation in plant tissues. Voogt et al. (2010) reported that when they treated lettuce with I(-1) and IO3- no impact on plant biomass or quality was found, and accumulation of I(-1) was more effective than IO3-. Iodine was mainly distributed to the outer leaves in lettuce. Landini et al. (2011) treated tomato (Solanum lycopersicium) with I(-1) and found out that tomato plants were no sensitive to high levels of iodine, stored both in vegetative tissues and fruits. They also reported that iodine was taken up better when supplied to the roots using hydroponically grown plants. However a considerable amount of iodine was also stored after leaf treatment that indicated that iodine is transport also through phloem. Buckwheat is a good source of nutrition- ally important elements (Ikeda et al. 2006). It contains proteins that have a better balance of amino acids compared with cereals (Yoon et al. 2009). Buckwheat sprouts appeared some years ago as a new vegetable (Kim et al. 2004). Seeds of buckwheat and pumpkins can be used as ad- ditives to improve the quality of bread or other products (Stibilj et al. 2004). Pumpkin seeds have been used for centuries in traditional medicine, mainly in cases of problems of the kidney or the urinary tract (Kreft et al. 2002). Despite its importance, studies focused on the effect of Se and I on plant physiological and biochemical characteristics are scarce (Zhu et al. 2004; Smoleń et al. 2014). The aim of the study was to determine the pos- sible simultaneous effect of iodine and selenium on the biochemical and physiological characteristics 37Germ et al.: The effect of Se and I on buckwheat and pumpkins on common buckwheat and pumpkin sprouts from seeds soaked in Se and I solution. Materials and methods Common buckwheat and pumpkin seeds were soaked in solution for 4 h in 200 mL distilled water (MilliQ) (control), or in solutions of sodium selenate (10 mg Se(VI) /L)), sodium selenite (10 mg Se(IV) /L)), potassium iodide (1000 mg(I(-1)) /L)), potassium iodate (1000 mg I(V) /L)) and combinations: 10 mg Se(VI) /L+ 1000 mg I(-1) /L; 10 mg Se(VI) /L+ 1000 mg I(V) /L; 10 mg Se(IV) /L+ 1000 mg I(-1)/L; 10 mg Se(IV) /L+ 1000 mg I(V) /L. After soaking seeds were distributed in plastic bowls, which were covered with filter paper. During germination, seeds were watered with tap water as needed. Sprouts were grown in controlled conditions in the growth chamber with constant temperature 22°C and 65–70% relative air humidity. Measurements were done after 12 and 10 days of growing common buckwheat and pumpkin sprouts, respectively. Contents of chlorophyll a and b and carotenoids were determined using a UV/VIS Spectrometer System (Lambda 12, Perkin-Elmer, Norwalk, CT, USA). The total chlorophyll content was determined as described in Lichtenthaler and Buschmann (2001a, 2001b). Fluorescence measurements were performed on the cotyledons of randomly selected sprouts using the fluorometer (PAM 2500 Portable Chlorophyll Fluorometer, WALZ). Prior to measurements samples were dark adapted for 20 min. The fluo- rescence parameters recorded included minimal (F0) and maximal (Fm) chlorophyll fluorescence that were provided by dark-adaptation clips. The difference between Fm and F0 is called the variable fluorescence Fv/ Fm = Fm – F0/ Fm. Fv/Fm ratio is com- mon parameter used in fluorescence which reflects the capacity to trap electrons by the photosystem (PS) II reaction centre (Schreiber et al. 1995). Statistical analysis The data were evaluated by ANOVA (Stat- graphicsVersion 4) and the differences were tested using the Duncan test with a significance level of 0.05. Results Amount of chlorophyll a in control buckwheat sprouts was lower and statistical significantly dif- ferent from amount of chlorophyll a in sprouts from seeds, soaked in Se(IV), I(V) and Se(IV)+I(V). The lowest amount of chlorophyll a was measured in the sprouts from seeds, soaked in Se(VI) and sta- tistically different from sprouts from seeds, soaked in Se(IV), I(-1), I(V), Se(IV)+I(-1), Se(IV)+I(V) and Se(VI)+I(V) (Fig. 1). The pattern was similar regarding chlorophyll b although not statistically significant (data not shown). Amount of carotenoids in control buckwheat sprouts were statistically different from the amount of carotenoids from seeds, soaked in Se(IV), I(-1), I(V), Se(IV)+I(-1) and Se(IV)+I(V). The lowest amount of carotenoids was measured in the sprouts from seeds, soaked in Se(VI) and was statistically lower form sprouts from seeds, soaked in Se(IV), I(-1), I(V), Se(IV)+I(-1), Se(IV)+I(V) and Se(VI)+I(V) (Fig. 2). Neither of treatment affected the amount of chlorophyll a (Fig. 3) and carotenoids (Fig. 4) in pumpkins sprouts. Similar pattern appeared for chlorophyll b (data not shown). Photochemical efficiency of PS II There was little effect of different treatment on potential photochemical efficiency of photosystem II (PS II) in common buckwheat sprouts. The lowest potential photochemical efficiency of PS II was measured in common buckwheat sprouts from seeds, soaked in Se(IV)+I(-1). In pumpkin sprouts, neither treatment affected potential pho- tochemical efficiency of PS II. Values were similar and around 0.8 (Table 1). 38 Acta Biologica Slovenica, 58 (1), 2015 Figure 1: Concentration of chlorophyll a per DM in common buckwheat sprouts. Mean ± SE, n = 6, C - control. Mean values, marked with the same letter, are not significantly different at p ≤ 0.05. Slika 1: Koncentracija klorofila a na SM v kalicah navadne ajde. Predstavljene so povprečne vrednosti + SE (n = 6). C - kontrolne kalice. Stolpci, označeni z različnimi črkami, se med seboj statistično značilno razlikujejo pri p ≤ 0,05. Figure 2: Concentration of carotenoids per DM in common buckwheat sprouts. Mean ± SE, n = 6, C - control. Mean values, marked with the same letter, are not significantly different at p ≤ 0.05. Slika 2: Koncentracija karotenoidov na SM v kalicah navadne ajde. Predstavljene so povprečne vrednosti + SE (n = 6). C - kontrolne kalice. Stolpci, označeni z različnimi črkami, se med seboj statistično značilno razlikujejo pri p ≤ 0,05. 39Germ et al.: The effect of Se and I on buckwheat and pumpkins Figure 3: Concentration of chlorophyll a per DM in pumpkin sprouts. Mean ± SE, n = 6, C - control. Mean values, marked with the same letter, are not significantly different at p ≤ 0.05. Slika 3: Koncentracija klorofila a na SM v kalicah buč. Predstavljene so povprečne vrednosti + SE (n = 6). C - kontrolne kalice. Stolpci, označeni z različnimi črkami, se med seboj statistično značilno razlikujejo pri p ≤ 0,05. Figure 4: Concentration of carotenoids per DM in pumpkin sprouts. Mean ± SE, n = 6, C - control. Mean values, marked with the same letter, are not significantly different at p ≤ 0.05. Slika 4: Koncentracija karotenoidov na SM v kalicah buč. Predstavljene so povprečne vrednosti + SE (n = 6). C - kontrolne kalice. Stolpci, označeni z različnimi črkami, se med seboj statistično značilno razlikujejo pri p ≤ 0,05. 40 Acta Biologica Slovenica, 58 (1), 2015 Table 1: Potential photochemical efficiency of PS II in common buckwheat and pumpkin sprouts. Mean values, marked with the same letter for each species, are not significantly different at p ≤ 0.05, SE – standard error, n = 8. Tabela 1: Potencialna fotokemična učinkovitost FS II v kalicah navadne ajde in buč. Srednje vrednosti, označene z različnimi črkami, se med seboj statistično značilno razlikujejo pri p ≤ 0,05, SE – standardna napaka, n = 8. Treatment Species Pumpkins Common buckwheat Treatment mean SE mean SE Control 0.83a 0.006 0.72abc 0.02 SeIV 0.83a 0.003 0.75ab 0.02 SeVI 0.82a 0.002 0.70bc 0.02 I(-1) 0.83a 0.004 0.78ab 0.01 I(V) 0.83a 0.002 0.71abc 0.05 Se(IV)+I(-1) 0.83a 0.003 0.43d 0.06 Se(IV)+I(V) 0.83a 0.004 0.73ab 0.04 Se(VI)+I(-1) 0.83a 0.002 0.80a 0.01 Se(VI)+I(V) 0.83a 0.006 0.63c 0.03 Discussion Biochemical response Different forms of Se and I had different effect on the amount of chlorophyll a, b, and carotenoids. Control buckwheat sprouts and sprouts from seeds, soaked in Se(VI) and Se(VI)+I(-1) had the lowest and similar amount of chlorophyll a and carotenoids. There were no differences in the amount of photosynthetic pigments in pumpkin sprouts. Similar results were given by Mechora et al. (2011, 2014) who reported about similar amount of chlorophyll a and b in red cabbage and cabbage, treated with Se(VI). Cabbage was fertilized via the soil with an aqueous solution of Na selenate (33 mL per plant) containing 2 µg Se L-1, every second day for 2 months and second group was foliarly sprayed with 20 mg Se L-1 in the form of Na selenate (~ 0.30 mL per plant) twice in the growing season. Red cabbage was as cabbage fertilized at a rate of 33 ml per plant, in the form of an aqueous solution containing Na selenate at a concentration of Se 2 μg L -1 , every second day for two months. The second group of red cabbage was fertilized with Se 0.5 mg L -1 twice in the growing season. In addition, on the study, performed on hydroponically cultivated lettuce, the addition of Se(IV) (2–30 µM) and Se(VI) (2–60 µM) did not affected the amount of photosynthetic pigments (Hawrylak-Nowak 2013). In sweet basil foliarly treated with (1-50 mg Se·dm-3) selenite, no significant effect on the content of chloroplast pigments was detected (Hawrylak-Nowak 2008a). Similarly, the concentration of Se(VI) < 10 mg/ kg did not affect the amount of chlorophylls in Lollium perenne cultivated in a soil (Hartikainen et al. 2000). Foliarly spraying with higher con- centration of I(-1) and I(V) in the solution as in this study and soil fertilizatioin (15 mg I dm-3) to the radish plants with I(-1) and I(V) did also not affect the amount of photosynthetic pigments (Strzetelski et al. 2010). On the other hand Xue et al. (2001) found out that the addition of Se(VI) in lower and higher concentration (0.1 mg/kg and 1.0 mg/kg soil respectively) induced synthesis of chlorophyll in young lettuce leaves, while in older leaves of lettuce, only higher concentration of Se(VI) induced the amount of chlorophyll. In younger leaves of Lollium perenne the addition of Se(VI) in concentration ≥ 10 mg/kg in the soil lowered the amount of chlorophylls while on the 41Germ et al.: The effect of Se and I on buckwheat and pumpkins other hand enhanced the amount of chlorophyll in older leaves (Hartikainen et al. 2000). In hydroponic experiment barley seedlings were subjected to 2, 4, 8, 16 ppm Se in the form of Se(VI). Chlorophyll content of the seedlings was affected significantly in a dose dependent manner (Akbulut and Çakır 2010). Significant decrease was observed in the chlorophyll content at ≥ 4 ppm Se applications, similar concentrations as used in the present study. Decrease of the total chlorophyll concentration depending on the Se form (selenate or selenomethionine) and dosage (25, 50, and 100 μM Se) were detected in hydroponicaly grown maize (Hawrylak-Nowak 2008b). Similarly in Tartary buckwheat, the foliar addition of Se(VI) (1 g Se/m3) at 10 times lower concentration in comparison to our used concentration of Se(VI), also lowered the amount of chlorophylls (Breznik et al. 2005). Under hydroponic cultivation in let- tuce plants a marked decrease in photosynthetic pigments concentration was found after passing the toxicity threshold, which has been designated at a level of 15 µM for selenite and 20 µM for selenate (Hawrylak-Nowak 2013). In the study from Strzetelski et al. (2010), soil fertilization with iodine was carried out before radish sowing to the level of 15 mg I·dm-3 soil. Foliar application of this element was performed twice using iodine solution in a concentration per pure element of 0.2%, in dose of 0.4 dm3 · m-2. Iodine foliar and soil application in radish, regardless of iodine forms (I(-1), IO3-), dose and application method, had no significant effect on the content of dry matter, as well as on the level of photosynthetic pigments in leaves. The objective of study from Blasco et al. (2011) was to determine the effect of the application of different doses (20, 40 and 80 μM) and forms of iodine (iodate [IO3- ] and iodide [I(-1)]) on photosynthesis and carbohydrate metabolism in lettuce plants. The Chl a content did not differ between I(-1)-treated lettuce plants and controls but was significantly reduced in plants treated with 80 μM IO3-. Physiological response Potential photochemical efficiency in common buckwheat (except sprouts from seeds, soaked in Se(IV)+I(-1)) and pumpkin sprouts was mainly around 0.7 and 0.8 respectively. Values, close to theoretical maximum 0.83 (Schreiber et al. 1995) meant that different forms of Se and I and their combination did not damage photosynthetic apparatus. Similar results were given regarding common buckwheat (Breznik et al. 2005, Tadina et al. 2007) and Tartary buckwheat (Breznik et al. 2005), foliarly treated with Se(VI) (1 g Se/m3). In addition, in the experiment, where Se(VI) was added to red cabbage (Mechora et al. 2011) and chicory (Germ et al. 2007), potential as well as effective photochemical efficiency were similar in treated and control plants. In pumpkins foliar spraying with Na-selenate solution (1.5 mg Se L-1) did not influence the potential photochemical efficiency of PS II (Germ et al. 2005). A positive effect of Se on potential photochemical efficiency was reported for the strawberries, cultivated in soil enriched with Se (0.1 mg Se kg-1soil and 1 mg Se kg-1 soil in the form of H2SeO4), but the same treatment had no positive effect on barley (Valkama et al. 2003). Conclusions There is scarce information about the effect of different forms of I on plants and particularly with the combination with Se. Concentrations, used in the present study, mainly caused no nega- tive effect on the biochemical and physiological characteristics of sprouts. Povzetek Pri nas in po svetu se je močno povečalo zanimanje za ajdo, tudi zaradi izredno skladne sestave hranilnih snovi v njenih zrnih. Vsebuje zelo kakovostne beljakovine. V tradicionalni medicini se uporabljajo bučna semena že stoletja predvsem v primerih težav z ledvicami ali sečili. Kalice vsebujejo veliko beljakovin, vitaminov, ogljikovih hidratov, olj, rudninskih snovi in s tem pripomorejo k biološki polnovredni prehrani. Selen in jod sta elementa, ki sta ključnega pomena za pravilno delovanje ščitnice in sta nujno potrebna pri izgradnji tiroidnega hormona, njegovi aktivaciji in metabolizmu. Ker je Se neobhodno potreben za metabolizem I v ščitnici raziskovalci menijo, da je smiselno gojenim rastlinam sočasno dodajati oba elementa. 42 Acta Biologica Slovenica, 58 (1), 2015 Kalice navadne ajde in buč smo gojili v rastni komori s stalno temperaturo 22°C in 65–70% r.z.v. Seme ajde in buč smo namakali v različnih raztopinah selena in joda. Preučevali smo vpliv naslednjih obravnavanj: selenat (10 mg Se(VI) /L)), selenit (10 mg Se(IV) /L)), jodid (1000 mg I(-1) /L)), jodat (1000 mg I(V) /L)) in kombinacije 10 mg Se(VI) /L+ 1000 mg I(-1) /L; 10 mg Se(VI) /L+ 1000 mg I(V) /L; 10 mg Se(IV) /L+ 1000 mg I(-1)/L; 10 mg Se(IV) /L+ 1000 mg I(V) /L in K (kontrola – destilirana voda brez dodanega selena in/ali joda). Merili smo sledeče fiziološke in biokemijske lastnosti kontrolnih kalic in kalic, zrastlih iz obravnavanih semen: fotokemično učinkovitost fotosistema II (FS II) ter vsebnost fotosinteznih barvil (klorofil a, klorofil b in karotenoidov). Kontrolne kalice in kalice, zrastle iz semen, namakanih v Se(VI) ter Se(VI)+I(-1), so imele najnižjo in podobno vsebnost klorofila a in karo- tenoidov. Obravnavanja so imela majhen vpliv na potencialno fotokemično učinkovitost fotosistema II pri kalicah navadne ajde. Namakanje semen v različnih raztopinah Se in I ni vplivalo na vsebnost fotosinteznih barvil in potencialno fotokemično učinkovitost fotosistema II pri kalicah buč. Z raziskavo smo želeli ugotoviti, ali selen in jod, ki ju dodajamo hkrati, vplivata na kalice navadne ajde in buč. Raziskava je zanimiva zato, ker je malo podatkov o hkratnem delovanju selena in joda na biokemijske in fiziološke parametre rastline in tudi zato, ker sta selen in jod elementa, ki sta zelo pomembna za delovanje ščitnice. Acknowledgments This research was financed by the Ministry of Education, Science and Sport, Republic of Slovenia, through the programmes “Biology of plants” (P1-0212), “Young researchers” (34326) and projects J4-4224 and J4-5524. References Akbulut, M., Çakır, S., 2010. The effects of Se phytotoxicity on the antioxidant systems of leaf tissues in barley (Hordeum vulgare L.) seedlings. Plant Physiol. Bioch., 48, 160–166. Blasco, B., Rios, J.J., Cervilla, L.M., Sanchez-Rodriguez, E., Rubio-Wilhelmi, M.M., Rosales, M.A., Ruiz, J.M., Romero, L., 2010. Photorespiration Process and Nitrogen Metabolism in Lettuce Plants (Lactuca sativa L.): Induced Changes in Response to Iodine Biofortification. J. Plant Growth Regul., 29, 477–486. Blasco, B., Rios, J.J., Leyva, R., Melgarejo, R., Constan-Aguilar, C., Sanchez-Rodriguez, E., Rubio- Wilhelmi, M.M., Romero, L., Ruiz, J. M., 2011. Photosynthesis and metabolism of sugars from lettuce plants (Lactuca sativa L. var. longifolia) subjected to biofortification with iodine. Plant Growth Regul., 65, 137–143. Breznik, B., Germ, M., Gaberščik, A., Kreft, I., 2005. Combined effects of elevated UV-B radiation and addition of selenium on common (Fagopyrum esculentum Moench) and tartary (Fagopyrum tataricum (L.) Gaertn.) buckwheat. Photosynthetica, 43 (4), 583–589. Dai, J., Zhu, Y., Huang, Y., Zhang, M., Song, J., 2006. Availability of iodide and iodate to spinach (Spinacia oleracea L.) in relation to total iodine in soil solution. Plant Soil, 289, 301–308. Fuge, R., 2005. Soils and iodine deficiency. Essentials of Medical Geology, 417–433. Germ, M., Kreft, I., Osvald, J., 2005. Influence of UV-B exclusion and selenium treatment on pho- tochemical efficiency of photosystem II, yield and respiratory potential in pumpkins (Cucurbita pepo L.). Plant Physiol. Bioch. (Paris), 43, 445–448. Germ, M., Stibilj, V., Osvald, J., Kreft, I., 2007. Effect of selenium foliar application on chicory (Cichorium intybus L.). J. Agric. Food Chem., 55 (3), 795–798. Hartikainen, H., Xue, T., Piironen, V., 2000. Selenium as an anti-oxidant and pro-oxidant in ryegrass. Kluwer Academic Publishers. Printed in the Netherlands, Plant Soil 225, 193–200. 43Germ et al.: The effect of Se and I on buckwheat and pumpkins Hawrylak-Nowak, B., 2008a. Enhanced selenium content in sweet basil (Ocimum basilicum L.) by foliar fertilization. Vegetable Crops Research Bulletin, 69, 63–72. Hawrylak-Nowak, B., 2008b. Changes in Anthocyanin Content as Indicator of Maize Sensitivity to Selenium. J. Plant Nutrit., 31, 1232–1242. Hawrylak-Nowak, B., 2013. Comparative effects of selenite and selenate on growth and selenium accumulation in lettuce plants under hydroponic conditions. Plant Growth Regul., 70, 149–157. Ikeda, S., Yamashita, Y., Tomura, K., Kreft, I., 2006. Nutritional comparison in mineral characteristics between buckwheat and cereals. Fagopyrum, 23, 61–65. Kim, S.L., Kim, S.K., Park, C.H., 2004. Introduction and nutritional evaluation of buckwheat sprouts as a new vegetable. Food Res. Int., 37, 319–327. Kreft, I., Stibilj, V., Trkov, Z., 2002. Iodine and selenium contents in pumpkin (Cucurbita pepo L.) oil and oil-cake. European Food Research and Technology A. Zeitschrift für Lebensmittel- Untersuchung und -Forschung, 215, 279–281. Landini, M., Gonzali, S., Perata, P., 2011. Iodine biofortification in tomato. J. Plant Nutr. Soil Sci., 174, 480–486. Lichtenthaler, H.K., Buschmann, C., 2001a. Chlorophylls and carotenoids - Measurement and cha- racterisation by UV-VIS. In: Current Protocols in Food Analytical Chemistry. John Wiley & Sons, Madison, pp. F4.3.1-F4.3.8. Lichtenthaler, H.K., Buschmann, C., 2001b. Extraction of photosynthetic tissues: Chlorophylls and carotenoids. In: Current Protocols in Food Analytical Chemistry. John Wiley & Sons, Madison, pp. F4.2.1-F4.2.6. Mechora, Š., Stibilj, V., Radešček, T., Gaberščik, A., Germ, M., 2011. Impact of Se (VI) fertilization on Se concentration in different parts of red cabbage plants. International Journal of Food, Agri- culture & Environment – JFAE, 9 (2), 357–361. Mechora, Š., Stibilj, V., Kreft, I., Germ, M., 2014.The physiology and biochemical tolerance of cabbage to Se (VI) addition to the soil and by foliar spraying. J. Plant Nutr., 37 (13), 2157–2169. Schreiber, U., Bilger, W., Neubauer, C., 1995. Chlorophyll fluorescence as a nonintrusive Indicator for Rapid Assessment of in Vivo Photosynthesis. In: Schulze, E.D., Caldwell, M.M., (eds.): Ecophysiology of Photosynthesis, Springer-Verlag, Berlin, Heidelberg, New York, pp. 49–69. Smoleń, S., Rożek, S., Ledwożyw-Smoleń, I., Strzetelski, P., 2011. Preliminary evaluation of the influence of soil fertilization and foliar nutrition with iodine on the efficiency of iodine bioforti- fication and chemical composition of lettuce. J. Elem., 613–622. Smoleń, S., Kowalska, I., Sady, W., 2014. Assessment of biofortification with iodine and selenium of lettuce cultivated in the NFT hydroponic system. Sci. Hortic., 166, 9–16. Stibilj, V., Kreft, I., Smrkolj, P., Osvald, J., 2004. Enhanced selenium content in buckwheat (Fagopyrum esculentum Moench) and pumpkin (Cucurbita pepo L.) seeds by foliar fertilisation. European Food Research and Technology A. Zeitschrift für Lebensmittel-Untersuchung und -Forschung, 219, 142–144. Strzetelski, P., Smoleń, S., Rożek, S., Sady, W., 2010. The effect of diverse iodine fertilization on nitrate accumulation and content of selected compounds in radish plants (Raphanus sativus L.). Acta Sci. Pol., Hortorum Cultus 9 (2), 65–73. Tadina, N., Germ, M., Kreft, I., Breznik, B., Gaberščik, A., 2007. Effects of water deficit and selenium on common buckwheat (Fagopyrum esculentum Moench.) plants. Photosynthetica, 45 (3), 472–476. Terry, N., Zayed, A.M., De Souza, M.P., Tarun, A.S., 2000. Selenium in higher plants. Ann. Rev. Plant Physiol. Plant Mol. Biol., 51, 401–432. Valkama, E., Kivimäenpää, H., Hartikainen, H., Wulff, A., 2003. The combined effects of enhanced UV-B radiation and selenium on growth, chlorophyll fluorescence and ultrastructure in strawberry (Fragaria x ananassa) and barley (Hordeum vulgare) treated in the field. Agr. Forest Meteorol., 120, 267–278. 44 Acta Biologica Slovenica, 58 (1), 2015 Voogt, W., Holwerda, H.T., Khodabaks, R., 2010. Biofortification of lettuce (Lactuca sativa L.) with iodine: the effect of iodine form and concentration in the nutrient solution on growth, development and iodine uptake of lettuce grown in water culture. J. Sci. Food Agric, 90, 906–913. Weng, H., Hong, C., Xia, T., Bao, L., Liu, H., Li, D., 2013. Iodine biofortification of vegetable plants – an innovative method for iodine supplementation. Chinese Science Bulletin, 58, 17, 2066–2072. White, P.J., Bowen, H.C., Parmaguru, P., Fritz, M., Spracklen, W.P., Spiby, R.E., Meacham, M.C., Mead, A., Harriman, M., Trueman, L.J., Smith, B. M., Thomas, B., Broadley, M.R., 2004. Inte- ractions between selenium and sulphur nutrition in Arabidopsis thaliana. J. Exp. Bot., 55, Sulphur Metabolism in Plants Special Issue, pp. 1927–1937. Xue, T., Hartikainen, H., Piironen, V., 2001. Antioxidative and growth-promoting effect of selenium in senescing lettuce. Plant Soil, 237, 55–61. Yoon, Y.-H., Lee, J-G, Jeong, J.-C., Jang, D.-C., Park, C.-S., 2009. The effect of temperature and light conditions on growth and antioxidantcontents of Tartary buckwheat sprout. In: Development and utilization of buckwheat sprouts as medicinal natural products, Park, C.H., Kreft, I., (eds.): Pro- ceedings of the International Symposium on buckwheat Sprouts. Bongpyoung, Korea, pp. 54–59. Zhu, Y.-G., Huang, Y.-Z., Hu, Y., Liu, Y.-X., 2003. Iodine uptake by spinach (Spinacia oleracea L.) plants grown in solution culture: effects of iodine species and solution concentrations. Environ. Int., 29, 33–37. Zhu, Y.-G., Huang, Y.-Z., Hu, Y., Liu, Y., Christie, P., 2004. Interactions between selenium and iodine uptake by spinach (Spinacia oleracea L.) in solution culture. Plant Soil, 261, 99–105. Zia, H.M., Watts, J.M., Gardner, A., Chenery, R.S., 2014. Iodine status of soils, grain crops, and irrigation waters in Pakistan. Environ. Earth Sci., 73 (12), 7995–8008. Zimmermann, M.B., Köhrle, J., 2002.The impact of iron and selenium deficiencies on iodine and thyroid metabolism: biochemistry and relevance to public health. Thyroid, 12 (10), 867–78. Biodiversity, the present ecological state of the Aral Sea and its impact on future development Vrstna pestrost, sedanje ekološko stanje Aralskega jezera in njegov vpliv na prihodnji razvoj Mihael J. Tomana*, Igor Plotnikovb, Nikolai Aladinb, Philip Micklinc, Zaualkhan Ermakhanovd aUniversity of Ljubljana, Biotechnical Faculty, Jamnikarjeva 101, SI-Ljubljana, Slovenia b Laboratory of Brackish Water Hydrology, Zoological Institute of RAS, Universitetskaya nab. 1, 191934 St. Petersburg, Russian Federation c Western Michigan University, Dept. of Geography, Kalamazoo, Michigan 49008, USA d Aral branch of Kazakh Research Institute of Fishery, Baktybay batyr str. 2, Aralsk, Kazakhstan *correspondence: mihael.toman@bf.uni-lj.si Abstract : The Aral sea used to be the fourth largest lake in the world. Its catchment area is huge, two main rivers (Amu Darya and Syr Darya) feed the lake. The balance of hydrological regime changed drastically after 1960 due to regulation of both main rivers and diversion of water for agricultural irrigation and intense cotton production. Salinity increased and most of invertebrate and fish species disappeared. A significant drop of water level has been recorded in the past 20 years and Aral Lake is presently divided into a small northern lake basin and a larger south basin. Kokaral dam con- struction resulted in increased water level and decreased salinity. Many invertebrate species reappeared in Small Aral and fish returned from Syr Darya river. Ecological situation in Large Aral is different, eastern part of this basin is completely dried out. The data on salinity levels, some chemical characteristics and above all the data about zooplankton, zoobenthos and fish in Small Aral have been recorded and presented in the article. Salinity ranges between 1 and 8 g/L, the lowest is near the river inlet. Five species of zooplankton (Keratella quadrata, Brachionus plicatilis, Evadne anonyx, Calanipeda aquaedulcis, Cyclops vicinus) and rotifers from the genus Synchaeta are very abundant, ten species are less numerous and seven summer species very rare. Different zoobenthos species are present, but only four abundant (Hediste diversicolor, Chironomus plumosus, Syndosmya segmentum and Cyprideis torosa). Zoobenthos mainly consist of Polychaeta, Mollusca, Crustacea and Diptera. The highest diversity was found near the Kokaral dam. Many fish species are commercially important: 14 of them are abundant, including endemic bream Abramis brama orientalis, Chalcalburnus chalcoides aralensis, carp Cyprinus carpio aralensis, and Aral roach Rutilus rutilus aralensis. White-eye bream Abramis sapa aralensis, silver carp Hypophtalmichthys molitrix, orfe Leuciscus idus oxianus, and snakehead Channa argus warpachowskii are less numerous. Aral barbel Barbus brachycephalus brachycephalus and Turke- stan barbel Barbus capito conocephalus remain very rare. It can be concluded that significant positive changes occurred after Kokaral dam construction. Particularly, biocenoses and the Aral lake environment have been improved and fisheries returned. Today Kazakhstan Government is discussing an idea to improve this dam and dike ACTA BIOLOGICA SLOVENICA LJUBLJANA 2015 Vol. 58, [t. 1: 45–59 46 Acta Biologica Slovenica, 58 (1), 2015 and we support this discussion and advise to make it higher. All can lead to improve ecological state of the Small Aral. Keywords: Aral Sea, biodiversity, ecological state, zooplankton, zoobenthos, fish Izvleček: Aralsko jezero je bilo četrto največje jezero na svetu. Njegovo pri- spevno območje je zelo veliko, dve glavni reki sta pritekali v jezero, Amu Darja in Sir Darja. Hidrološko stanje jezera se je drastično spremenilo po letu 1960 po regulaciji in preusmeritvi obeh glavnih rek za namakanje bombažnih nasadov. Povečala se je slanost, številne vrste nevretenčarjev in rib so izginile. V 20 letih se je gladina vode v jezeru opazno znižala in jezero se je razdelilo na dva dela, manjši severni bazen in večji južni bazen. Po izgradnji jezu Kokaral se je gladina vode zvišala in slanost znižala. Mnoge nevretenčarske vrste so se vrnile v Mali Aral, iz Sir Darje so prišle tudi ribe. Ekološko stanje v Velikem Aralu je drugačno, vzhodni del tega bazena je popolnoma suh. V članku so zbrani podatki o slanosti, nekateri kemijski parametri in predvsem združbe zooplanktona, zoobentosa in rib v Malem Aralu. Slanost variira med 1 g/L in 8g/L, najnižja pri rečnem vtoku. Zelo pogostih je pet zooplanktonskih vrst (Keratella quadrata, Brachionus plicatilis, Evadne anonyx, Calanipeda aquaedulcis, Cyclops vicinus), ena nedoločena vrsta kotačnika Synchaeta. Deset vrst je manj pogostih, zelo redkih pa je šest vrst pomladnih zooplantontov. Prisotnih je tudi več različnih vrst zoobentosa, le štiri vrste pa so pogoste (Hediste diversicolor, Chironomus plumosus, Syndosmya segmentum, and Cyprideis torosa). Zoobentos sestavljajo Polychaeta, Mollusca, Crustacea in Diptera. Največja pestrost je bila ugotovljena ob jezu Kok- aral. Mnoge ribje vrste so gospodarsko pomembne, 14 od njih je pogostih, vključno z endemnimi taksoni Abramis brama orientalis, Chalcalburnus chalcoides aralensis, Cyprinus carpio aralensis, Rutilus rutilus aralensis. Manj številčne so Abramis sapa aralensis, Hypophtalmichthys molitrix, Leuciscus idus oxianus, Channa argus war- pachowskii. Zelo redki sta dve vrsti mrene Barbus brachycephalus brachycephalus in Barbus capito conocephalus. Ugotavljamo, da so se opazne in pozitivne spremembe zgodile po izgradnji jezu Kokaral. Zlasti se je izboljšala vrstna pestrost združb in je- zersko okolje nasploh, zato se je vrnilo ribištvo. Danes Kazahstanska vlada razmišlja o izboljšanju jezu in nasipa. To razmišljanje podpiramo in obenem svetujemo povišanje jezu, kar bi prineslo izboljšanje ekološkega stanja Malega Arala. Ključne besede: Aralsko jezero, vrstna pestrost, ekološko stanje, zooplankton, zoobentos, ribe Introduction The Aral Sea is a terminal lake, lying amidst the vast deserts of Central Asia. From the 1600’s to the 1960’s, the hydrological regime of the Aral Sea was in reasonable balance. This lake is Trans- boundary Lake and 7 countries (Afghanistan, Iran, Kazakhstan, Kyrgyzstan, Tajikistan, Turkmenistan, Uzbekistan) are contributing water into it. Since 1960 the anthropogenic regression and saliniza- tion of the Aral Sea has begun. This has resulted in the disappearance of most of invertebrates and fish species. Some of them have become extinct. At the end of the 1980’s due to the fall of water level, the Aral Sea was divided into northern Small Aral and the southern Large Aral having different hydrological regimes. Regression and salinization of Large Aral continues. After the construction of the Kokaral dam (Aladin, 2014) Small Aral Sea level has increased and a gradual decline in its salinity has began. Because of this dam lake restoration is possible. To date, salinity of the Small Aral Sea has become lower than it was before the 1960’s. 47Toman et al.: Biodiversity and ecological state of the Aral Sea There is a process of restoration of former biodi- versity. Many invertebrate species are reappearing due to salinity decrease. Commercial freshwater fish species returned into the Small Aral from Syr Darya River and lakes in its lower reaches where they survived. Their populations are in good state. Fisheries are restoring and catches are growing (Aladin and Plotnikov, 2012; Aladin et al., 2012; Plotnikov et al., 2012). At present Aral Sea is divided to the number residual parts (lotic and lentic). Large Aral Sea is currently the most suffering part of the lake. In the beginning of 21st century it was divided in 3 parts: Western Large Aral Sea, Eastern Large Aral Sea and Tsche-Bas Bay. Since last few years ap- peared a new fourth part of Large Aral Sea – New Central Aral Sea. Summer 2014 marked another milestone for the Large Aral Sea. For the first time in modern history, the Eastern Large Aral Sea has completely dried. So we have again currently only 3 parts Western Large Aral Sea, Tsche-Bas Bay and New Central Aral Sea. In autumn 2009 some people without any calculations and without direct observations reported that Eastern Large Aral Sea dry up com- pletely (http://earthobservatory.nasa.gov/Features/ WorldOfChange/aral_sea.php). Next year 2010 Eastern Large Aral Sea came back because it was a very wet year and a lot of water came from Amu Darya River delta. Figure 1. Map of sampling places in Aral Sea. 1 – Saryshiganak Bay; 2 – Butakov Bay; 3 – Shevchenko Bay; 4 – Kokaral island; 5 – Berg Strait; 6 – Auzy-Kokaral Strait. Standard stations: u – ichthyological, l – hydrobiological, n – hydrochemical. Slika 1. Označena vzorčna mesta v Aralskem jezeru. 1 – zaliv Saryshiganak, 2 – zaliv Butakov, 3 – zaliv Shevchenko, 4 – otok Kokaral, 5 – ožina Berg, 6 – ožina Auzy_Kokaral. Standardne postaje: u – ihtiološka, l – hidrobiološka, n – hidrokemijska. 48 Acta Biologica Slovenica, 58 (1), 2015 Material and Methods Number of field trips around Aral Sea were made. We studied the following places: Small Aral Sea, Tsche-Bas Bay; Western Large Aral Sea (Only Chernyshov Bay); New Central Aral Sea. Small Aral Sea level is 42.0 m a.s.l. with a volume of 27.1 km3. Water covers an area of 3288 km2. Basin has maximal depth of 15.5 m (average depth of 8.2 m). In our studies of Aral Sea we are using IL2BM platform (Integrated Lotic/Lentic Basin Manage- ment) (http://www.ilec.or.jp/en/). Salinity was measured using YSI-85 and conductometer LF-330. Other chemical param- eters were determined using standard methods. Oxygen concentration and pH were measured at the sampling points. Parameters such as COD (permanganate method), NH4+, NO2-, NO3- and PO43- were analysed in the laboratory. Plankton samples were collected according to standard methodology using plankton net (mesh size 60µm). Invertebrates (zoobenthos) were sampled using Petersen bottom sampler (0.025 m2) and sediments were washed through the sieve No 36. All samples were fixed with formalin (4%) and investigated using stereo microscope (MBS- 10). All collected animals were taken to the Aral branch of Kazakh Research Institute of Fishery and Zoological institute of RAS. For species identifica- tion Atlas of the Aral Sea invertebrates was used. Fish were sampled several years. They were caught with fixed fishing nets (mesh size 18-65 mm). Results and discussion Chemical data Oxygen concentrations, COD, concentrations of NH4+, NO2-, NO3-, PO43- and pH were measured in different years and very little changes were found. More changes were detected in salinity (Table 1). Salinity in different parts of Small Aral Sea was from 1 g/L up to 8 g/L. The lowest level of salinity was observed in May 2014 near the Kokaral dam (near Syr Darya delta) and the highest in August in Butakov Bay near Akespe village. The level of Small Aral in investigated period was from 42.1 to 42.6 m a.s.l. The lowest was in August, the highest in May (unpublished data by Ermakhanov from institutional report). Much higher salinity was measured in Tsche- Bas Bay, ranging from 78 g/L up to 89 g/L. The lowest level of salinity was observed in May near connection to New Central Aral Sea and the highest – in August near the northern coast of Tsche-Bas Bay. The level of Tsche-Bas Bay was from 28.7 to 29.1 m a.s.l. The lowest was in August, the highest was in May. In Western Large Aral Sea (Only Chernyshov Bay) salinity was very different compared to Small Table 1: Selected chemical data from Small Aral Sea in the years 2007 – 2013. Tabela 1: Izbrani kemijski podatki o malem Aralu v letih 2007 – 2013. Year pH O2, mg/dm3 COD mg/dm3 Salinity, ‰ Biogenes, mg/dm3 NH+4 NO-2 NO-3 PO3-4 2007 7.90-8.30 6.71-13.63 4.3-12.0 6.3 0.65-1.25 0.003-0.055 0.00-0.60 0.005-0.068 2008 6.85-7.20 5.32-11.42 1.5-12.2 12.1 0.05-1.23 0.002-0.135 0.02-0.59 0.008-0.050 2009 6.93-7.32 6.20-12.51 1.4-11.7 12.9 0.38-0.83 0.012-0.101 0.05-0.43 0.010-0.025 2010 7.20-7.30 7.84-12.30 5.8-7.0 11.0 0.23-0.37 0.003-0.039 4.17-5.94 0.000-0.040 2011 7.20-7.45 7.62-12.74 2.4-4.6 9.9 0.36-0.39 0.028-0.032 4.35-5.30 0.022-0.036 2012 7.20-7.25 7.74-9.28 3.2-3.9 5.7 0.37-0.39 0.022-0.029 3.99-4.97 0.017-0.025 2013 7.10-8.15 7.70-9.20 3.1-3.5 5.3 0.12-0.24 0.009-0.027 3.22-3.33 0.015-0.019 49Toman et al.: Biodiversity and ecological state of the Aral Sea Aral with very high values, from 143 g/L up to 169 g/L. The lowest level of salinity was observed in May 2014 near sampling camp of Mangistau Bioresource Company and the highest in August near the northern coast of Chernyshov Bay. The level of Western Large Aral Sea in investigated period was from 24.9 to 25.5 m a.s.l. The lowest was in August, the highest was in May. Salinity in New Central Aral Sea varied from 6 g/L up to 77 g/L. The lowest level was observed in May near the Kokaral dam and the highest in August near the connection to Tsche-Bas Bay. The level of New Central Aral Sea at the place of planned southern dike was from 28.5 to 28.9 m a.s.l. and at Kokaral dam from 29.5 to 31.6 m a.s.l. The lowest level was in August, the highest was in May. Zooplankton Zooplankton biocenoses in Small Aral Sea mainly consist of three Rotifera, Cladocera and Copepoda (Table 2). Only few species of freshwater and brackish planktonic Protozoa were detected (Plotnikov et al., 2014; Smurov, 1995). Five species of zooplankton were numerous: Keratella quadrata (Müller), Brachionus plicatilis Müller, Evadne anonyx Sars, Calanipeda aquae- dulcis Kritchagin, Cyclops vicinus Uljanin and one undeterminated species of rotifers from the genus Synchaeta (unpublished data by Ermakhanov from institutional report). Less numerous were ten species: Brachionus quadridentatus Hermann, Brachionus calyciflorus Pallas, Hexarthra oxyuris (Zernov), Bosmina longirostris Müller, Chydorus sphaericus Müller, Ceriodaphnia reticulata (Jurine), Podonevadne camptonyx (Sars), Phyllodiaptomus blanci (Guerne et Richard), Mesocyclops leuckarti (Claus), Acanthocyclops viridis (Jurine). Copepod taxa from order Harpacticoida were also less numer- ous (unpublished data by Ermakhanov from institutional report). Seven species of zooplankton mainly from rotifers group were very rare and all of them were observed only in summer time: Asplanchna priodonta Gosse, Keratella cochlearis (Gosse), Notholca acuminata (Ehrenberg), Filinia longiseta (Ehrenberg), Moina mongolica Daday, Diaphano- soma brachyurum Lievin, Podonevadne angusta groups: (Sars) (unpublished data by Ermakhanov from institutional report). In Tsche-Bas Bay only two species of zoo- plankton were numerous: brine shrimp Artemia parthenogenetica Bowen et Sterling from May till September, and halophilic ciliate Fabrea salina Henneguy during all summer months from June till August (Plotnikov et al., 2014). Zooplankton species Moina mongolica was very rare and only few individuals (parthenogenetic females) were observed only in summer time. In Western Large Aral Sea (Only Chernyshov Bay) only one species of zooplankton brine shrimp Artemia parthenogenetica was numerous due to high salinity. It occurs from May till September. One species of halophilic ciliate Fabrea salina was observed only at the end of summer in August and was very rare (Plotnikov et al. 2014). Near the Kokaral dam the biodiversity of zooplankton is the highest. Three species were nu- merous: Keratella quadrata, Brachionus plicatilis, Calanipeda aquaedulcis, seven species were less numerous: Brachionus quadridentatus, Bosmina longirostris, Chydorus sphaericus, Ceriodaphnia reticulata, Phyllodiaptomus blanci, Mesocyclops leuckarti, Acanthocyclops viridis. Near connection to Tsche-Bas Bay the biodi- versity of zooplankton are the lowest. Only three species of zooplankton were numerous: brine shrimp Artemia parthenogenetica Plotnikov et al., 2014, euryhaline Moina mongolica from May till September, and halophilic ciliate Fabrea salina during all summer months from June till August. Zoobenthos Zoobentic biocenosis in Small Aral Sea mainly consists of groups: Polychaeta, Mollusca, Crusta- cea, Insecta/Diptera (Table 3). Only few species of Foraminifera and Nematoda were found in samples (Filippov et al. 1993; Plotnikov et al. 2014). Four species of zoobenthos were numerous: Hediste diversicolor (Müller), Chironomus plumo- sus (L.), Syndosmya segmentum Récluz, Cyprideis torosa (Jones) (Plotnikov et al., 2014; unpublished data by Ermakhanov from institutional report). Six species of zoobenthos were less numer- ous: Chironomus behningi Goetghebuer, Glyp- totendipes gripekoveni Kieffer, Limnocythere aralensis Schornikov, Limnocythere inopinata 50 Acta Biologica Slovenica, 58 (1), 2015 Table 2: Taxonomic composition of zooplankton in the Small Aral Sea in in the years 2011 – 2013 (species only found in the samples are indicated). Tabela 2: Taksonomska sestava zooplanktona v Malem Aralu v letih 2011 – 2013 (naštete so le vrste, ki so bile najdene v vzorcih). Taxon Occurrence, % 2011 2012 2013 Rotifera Asplanchna priodonta Gosse - 5 5 Synchaeta spp. 50 60 74 Keratella quadrata (Gosse) 68 77 74 Keratella cochlearis (Gosse) - - 5 Brachionus quadridentatus Hermann 41 40 26 B. calyciflorus Pallas - 9 26 B. plicatilis Müller 23 40 68 Notholca acuminata Ehrenberg 5 5 5 Filinia longiseta (Ehrenberg) 23 27 - Hexarthra oxyuris (Zernov) 5 36 37 Cladocera Bosmina longirostris (Müller) 10 18 21 Chydorus sphaericus (Müller) 15 23 26 Moina mongolica Daday 10 9 - Ceriodaphnia reticulata (Jurine) 15 36 21 Diaphanosoma brachyurum (Lievin) - - 5 Podonevadne angusta (Sars) 5 5 - P. camptonyx (Sars) 10 5 37 Evadne anonyx Sars 73 73 79 Copepoda Phyllodiaptomus blanci (Guerne et Richard) 14 14 11 Calanipeda aquaedulcis Kritschagin 100 100 100 Cyclops vicinus Uljanin 60 86 79 Mesocyclops leuckarti (Claus) 20 23 26 Acanthocyclops viridis (Jurine) 28 36 32 Harpacticoida gen. sp. 25 14 26 51Toman et al.: Biodiversity and ecological state of the Aral Sea (Baird), Tyrrenocythere amnicola donetziensis (Dubowsky), Amnicythere cymbula (Livental), unspecified species from genera Chironomus, Candona and family Ceratopogonidae were also less numer- ous (Plotnikov et al. 2014; unpublished data by Ermakhanov from institutional report). Four species of zoobenthos were very rare and all of them were observed from May till Septem- ber: Dreissena polymorpha aralensis (Andrusov), Cerastoderma isthmicum Issel, Paramysis inter- media (Czerniavsky), Palaemon elegans Rathke. Unspecified species from genus Chironomus and family Ceratopogonidae were also less numer- ous (Plotnikov et al., 2014; unpublished data by Ermakhanov from institutional report). Unspecified species from genera Cryptochi- ronomus, Cladotanytarsus, Tanytarsus, Cas- piohydrobia were very rare and all of them were observed from May till September (unpublished data by Ermakhanov from institutional report). Table 3: Taxonomic composition of zoobenthos in Small Aral Sea in in the years 2012 – 2013 (species only found in the samples are indicated). Tablela 3: Taksonomska sestava zoobentosa v Malem Aralu v letih 2012 – 2013 (naštete so le vrste, ki so bile najdene v vzorcih). Taxa Occurrence, % 2012 2013 Polychaeta Hediste diversicolor (Müller) 86 80 Insecta: Diptera Chironomus behningi (Goetghebuer) 25 18 Chironomus sp. - 36 Ch. plumosus (Linne) 50 50 Glyptotendipes gripekoveni (Kieffer) - 10 Cryptochironomus sp. 30 5 Cladotanytarsus sp. - 8 Tanypus villipennis (Kieffer) - 8 Tanytarsus sp. 17 8 Ceratopogonidae gen. sp. - 18 Mollusca: Bivalvia Syndosmya segmentum Récluz 60 46 Dreissena polymorpha aralensis (Andrusov) 5 4 Cerastoderma isthmicum Issel 25 4 Mollusca: Gastropoda Caspiohydrobia spp. 5 - Crustacea Paramysis intermedia (Czerniavsky) - 4 Palaemon elegans Rathke 5 - 52 Acta Biologica Slovenica, 58 (1), 2015 In Tsche-Bas Bay three species of zoobenthos were numerous from May till September: salt tol- erant halophilic ostracod Eucypris inflata (Sars), euryhaline ostracod Cyprideis torosa (Jones) and halophilic larvae of Chironomus salinarius Kieffer (Plotnikov et al. 2014). Two species of zoobenthos were very rare: euryhaline Turbellaria Mecynostomum agile (Beklemischev) and large ciliate Frontonia ma- rina Fabre-Domergue. Unspecified species of foraminifers and nematodes were also were very rare (Plotnikov et al. 2014). In Western Large Aral Sea (Only Chernyshov Bay) only one species of zoobenthos halophilic larvae of Chironomus salinarius were numerous and present from May till September. One rare species of zoobenthos was found: large ciliate Frontonia marina. Very rare were unspecified species of foraminifers and nematodes (Plotnikov et al. 2014). Near the Kokaral dam the biodiversity of zoo- benthos is the highest. Four species were numer- ous: Hediste diversicolor (Müller), Chironomus plumosus (L.), Syndosmya segmentum, Cyprideis torosa. The following four species such as Chi- ronomus behningi Goetghebuer, Limnocythere aralensis, Tyrrenocythere amnicola donetziensis, Amnicythere cymbula were less numerous, while species Cerastoderma isthmicum and Palaemon elegans were very rare. Near the connection to Tsche-Bas Bay the biodiversity of zoobenthos was the lowest. Three species of zoobenthos from May till September were numerous and occurred from May till Sep- tember: salt tolerant halophilic ostracod Eucypris inflata (Sars), euryhaline ostracod Cyprideis torosa and halophilic larvae of Chironomus salinarius. Euryhaline large ciliate species Frontonia marina was very rare. Unspecified species of foraminifers and nematodes were also were very rare (Plotnikov et al. 2014). Fish In Small Aral Sea 14 species of commercial fish were numerous: pike Esox lucius Linnaeus, bream Abramis brama orientalis Berg, asp (zherekh) Aspius aspius iblioides (Kessler), crucian carp Carassius carassius gibelio Bloch, Aral shemaya Chalcalburnus chalcoides aralensis (Berg), carp Cyprinus carpio aralensis Spitshakow, grass carp Ctenopharyngodon idella (Valenciennes), sabrefish Pelecus cultratus (Linnaeus), Aral roach Rutilus rutilus aralensis Berg, rudd Scardinius erythropthalmus (Linnaeus), wels Silurus glanis Linnaeus, perch Perca fluviatilis (Linnaeus), pike perch or zander Stizostedion lucioperca (Linnaeus), Black Sea flounder Platichthys flesus (Linnaeus) (Ermakhanov et al. 2012) (Table 4). Less numerous were 4 species: white-eye bream Abramis sapa aralensis Tjapkin, silver carp Hypophtalmichthys molitrix (Valenciennes), orfe Leuciscus idus oxianus (Kessler), snakehead Channa argus warpachowskii Berg (Ermakhanov et al. 2012). Only 5 species of commercial fish were very rare: Baltic herring Clupea harengus membras (Linnaeus), spotted silver carp Aristichtys nobilis (Richardson), black carp Mylopharyngodon piceus (Richardson), Aral barbel Barbus brachycephalus brachycephalus Kessler, Turkestan barbel Barbus capito conocephalus Kessler (Ermakhanov et al. 2012). Fishery is under control by authorities. Nine fish species in the lake are not commercial. Six of them are numerous: ruff Gymnocephalus cer- nuus (Linnaeus), nine-spined stickleback Pungitius platygaster aralensis (Kessler), Caspian atherine Atherina boyeri caspia Eichwald, bubyr goby, transcaucasian goby Pomatoschistus caucasicus Berg [= Knipowitschia caucasica (Berg)], sand goby Neogobius fluviatilis pallasi (Berg), round goby Neogobius melanostomus affinis (Eichwald) (Ermakhanov et al., 2012). Three species of not commercial fish are rare: syrman goby Neogobius syrman eurystomus (Kessler), tubenose goby Pro- terorchinus marmoratus (Pallas), bighead goby Neogobius kessleri gorlap Iljin (Ermakhanov et al. 2012). In Tsche-Bas Bay and in Western Large Aral Sea (Only Chernyshov Bay) fish are not living now due to high salinity (Ermakhanov et al. 2012). The highest biodiversity of fish was found near the Kokaral dam. Nine species of commercial fish were numerous: pike Esox lucius Linnaeus, bream Abramis brama orientalis Berg, Aral shemaya Chalcalburnus chalcoides aralensis (Berg), carp Cyprinus carpio aralensis Spitshakow, Aral roach Rutilus rutilus aralensis Berg, wels Silurus glanis Linnaeus, perch Perca fluviatilis (Linnaeus), pike perch or zander Stizostedion lucioperca (Linnaeus), Black Sea flounder Platichthys flesus (Linnaeus). 53Toman et al.: Biodiversity and ecological state of the Aral Sea Two species of commercial fish were less nu- merous: silver carp Hypophtalmichthys molitrix (Valenciennes) and snakehead Channa argus warpachowskii Berg. Near connection to Tsche-Bas Bay the biodi- versity of fish is the lowest. Only one species of commercial fish was very rare in this part of the New Central Aral Sea. Black Sea flounder Platichthys flesus was caught several times from May to June (Ermakhanov et al. 2012). Table 4: Species composition of ichthyofauna in the Small Aral Sea. Tabela 4: Vrstna sestava ihtiofavne v Malem Aralu. Taxa Status Esocidae Esox lucius Linnaeus (Pike) A, C- Cyprinidae Rutilus rutilus aralensis Berg (Aral roach) A, C Leuciscus idus oxianus (Kessler) (Orfe) A, C- Aspius aspius iblioides (Kessler) (Asp, zherekh) A, C Scardinius erythropthalmus (Linnaeus) (Rudd) A, C- Barbus capito conocephalus Kessler (Turkestan barbell) A, C-, RB Barbus brachycephalus brachycephalus Kessler (Aral barbell) A, C-, RB Abramis brama orientalis Berg (Bream) A, C Abramis sapa aralensis Tjapkin (White-eye bream) A, C- Chalcalburnus chalcoides aralensis (Berg) (Aral shemaya) A, C- Pelecus cultratus (Linnaeus) (Sabrefish) A, C- Carassius carassius gibelio Bloch (Crucian carp) A, C- Cyprinus carpio aralensis Spitshakow (Carp) A, C Ctenopharyngodon idella (Valenciennes) (Grass carp) I, C- Hypophtalmichthys molitrix (Valenciennes) (Silver carp) I, C- Aristichtys nobilis (Richardson) (Spotted silver carp) I, C- Mylopharyngodon piceus (Richardson) (Black carp) I, C- Siluridae Silurus glanis Linnaeus (Wels) A, C- Gasterostidae Pungitius platygaster aralensis (Kessler) (Nine-spined stickleback) A, NC Percidae 54 Acta Biologica Slovenica, 58 (1), 2015 Stizostedion lucioperca (Linnaeus) (Pike perch, zander) A, C Perca fluviatilis Linnaeus (Perch) A, C- Gymnocephalus cernuus (Linnaeus) (Ruff) A, NC Clupeidae Clupea harengus membras (Linnaeus) (Baltic herring) I, C-- Atherinidae Atherina boyeri caspia Eichwald (Caspian atherine) I, NC Gobiidae Pomatoschistus caucasicus Berg (Bubyr goby, transcaucasian goby) [= Knipowitschia caucasica (Berg)] I, NC Neogobius fluviatilis pallasi (Berg) (Sand goby) I, NC Neogobius melanostomus affinis (Eichwald) (Round goby) I, NC Neogobius syrman eurystomus (Kessler) (Syrman goby) I, NC Proterorchinus marmoratus (Pallas) (Tubenose goby) I, NC Neogobius kessleri gorlap Iljin (Bighead goby) I, NC Channidae Channa argus warpachowskii (Berg) (Snakehead) I, C Pleuronectidae Platichthys flesus (Linnaeus) (Black Sea flounder) I, C Abbreviations: A – aboriginal; I – introduced; C – commercial; C- – commercial but low stocks C-- – while commercial but stocks very low for fishery; NC – not commercial; RB – in Red Book. All above mentioned data collected from May till September is the evidence of great practical and commercial importance of all four Aral Sea Areas in Republic of Kazakhstan: Small Aral Sea, Tsche-Bas Bay, Western Large Aral Sea (Only Chernyshov Bay), New Central Aral Sea. As it is said in the introduction to this paper big positive changes in Aral Sea environment and in Aral Sea fisheries happened immediately after construction of a Kokaral dam in the Berg strait. Today local people and Kazakhstan Government are discussing an idea to improve this dike. We are supporting this discussion and we advise to make it higher from 42-43 meters above ocean level up to 46 - 48 meters as it was advised by us in 1992. We also propose to build two more dams in addition to this Central dam: 1) The Northern dam could be build in the entrance to the Bolshoy Sarychaganak Bay near Trekhgorka place (three-headed mountain place in English). The canal from Kamyslibash Lake to Bolshoy Sarychaganak Bay should be build too. The dam should be as high as 49-50 meters a.s.l. Geographical coordinates of future dike could be N 49°29’16”, E 61°15’51”. 2) The Southern dam could be build at the southern edge of New Central Aral Sea where it unites with Tsche-Bas Bay. This dam will en- able keeping the water that is running away from Kokaral dam in Republic of Kazakhstan via its spillway. Geographical coordinates of future dike could be N 45°55’37”, E 59°40’15”. New Central Aral Sea which appeared in 2005 - 2006 after new Kokaral dam was built in 55Toman et al.: Biodiversity and ecological state of the Aral Sea comparison to the other three parts of the Aral Sea described in the article is studied very poor. Fauna is not studied yet so more studies should be done as soon as possible. Povzetek Usoda četrtega največjega jezera na svetu do leta 1980, je sicer poznana tudi svetovni javnosti, veliko manj pa je bilo objav o spremembah ekološkega stanja, življenjskih združb in posebno gospodarsko pomembnih rib. Članek govori o kemizmu in slanosti, biodiverziteti plankton- skih, bentoških in ribjih združb v različnih letih, ekološkem stanju in hidrološkem režimu ter možnosti ohranjanja in izboljšanja trenutnega stanja predvsem v severnem delu nekdanjega jezera, danes imenovanega Mali Aral. Hidrološki režim jezera se je drastično začel spreminjati že kmalu po letu 1960, ko so Sovjeti z regulacijami preusmerili dve veliki reki, Syr Darjo in Amu Darjo, ki sta sicer polnili veliko Aralsko jezero. Razlog preusmeritve rek je bilo namakanje velikih površin posajenih z bombažem v nekdanjih sovjetskih republikah Uzbekistanu in Kazahstanu. Vode je jezeru je pričelo primanjko- vati, gladina se je hitro zmanjševala in že v 80. letih prejšnjega stoletja se je veliko jezero razdelilo v dva bazena, severni Mali Aral in južni Veliki Aral s povsem drugim hidrološkim režimom. Slanost v teh bazenih se je izjemno povečala, marsikje je dosegla vrednost prek 100 g/L soli. To je bil začetek vrstnega siromašenja življenjskih združb, izumiranja nekaterih vrst, med njimi tudi gosp- odarsko pomembnih rib. Jezersko dno je postalo puščavsko območje, ki je vsebovalo tudi različne toksične snovi, posledice kemijskih in bioloških poskusov v času hladne vojne. Biotsko izjemno diverzitetni otok Barsakelmes je postal puščavski. Raziskovalci so skušali rešiti preostanek severnega dela z izgradnjo večjega nasipa in jezu imenovanega Kokaral v bližini delte Syr Darje, ki je preprečeval odtekanje vode v puščavo. Nivo vode se je nekoliko zvišal, predvsem pa se ni več zmanjševal. Največji učinki so se pokazali v slanosti, ta se je zmanjšala pod 10 g/L, zato so se vrnili mnogi nevretenčarji v planktonu in bentosu in z njimi tudi nekatere ribje vrste predvsem iz porečja in manjših jezer prispevnega območja. Ribje populacije v Malem Aralu so danes v do- brem stanju in omogočajo tudi kontroliran in za nekatere vasi gospodarsko pomemben ribolov. Usoda južnega Velikega Arala pa je še naprej negotova, v suhem letu 2014 je v del te kotanje povsem presušil. Planktonska združba je danes v Malem Aralu zmerno pestra, prevladujejo vrste iz skupin Rotif- era, Cladocera in Copepoda, nekaj je protozojskih vrst. Pet vrst je zelo pogostih, med njimi rotatorija Keratella quadrata in Brachiounus plicatilis, vodna bolha Evadne anonyx in dve vrsti kopepo- dov Calanipeda aquaedulcis in Cyclops vicinus. Poleg teh je manj pogostih še deset vrst in zelo redkih, ter še vedno ogroženih sedem vrst, med njimi nekatere, v drugih jezerih sicer zelo pogoste vrste, npr. Keratella cochlearis, Filinia longistea ter Moina mongolica. Podobno pestra je tudi združba nevretenčarjev v bentosu, sestavljena predvsem iz skupin Poly- chaeta, Mollusca, Crustacea in Diptera. Zelo pogosta vrsta je Chironomus plumosus, ki je značilen predstavnik občasno anoksičnih jezerskih sedimentov in obenem dobro prilagojen na večjo slanost. Zelo pogost je tudi polihet Hediste diversi- color, ki kaže na slan tip celinskega vodnega telesa. Marsikje invazivna vrsta Dreissena polymorpha aralensis se v Malem Aralu sporadično pojavlja in je zelo redka. Posebna pozornost je bila v naših raziskavah dana ribjim združbam. Kar 14 vrst gospodarsko pomembnih rib je danes v Malem Aralu, med njimi bi izpostavili ščuko Esox lucius, krapa Car- assius carassius gibelio, aralsko vrsto rdečeoke Rutilus rutilus aralensis in Platichthys flesus, ki velja za eno najbolj okusnih rib. Manj pogosta je vrsta aralskega ploščiča Abramis sapa aralensis. Izlov dveh redih, sicer gospodarsko pomembnih vrst poher, aralske pohre Barbus brachycephalus brachycepahlus in turkestanske pohre B. capito conecephalus, je strogo kontroliran. V jezeru je kar devet vrst gospodarsko nepomembnih rib, med njimi tudi endemne. Njihov obstoj ni vezan na ribištvo, ampak na ekološke razmere v malem Aralu. Kazahstanska vlada si močno prizadeva vzdrževati pridobljeno ekološke stanje v Malem Aralu in ga celo izboljšati z nadgradnjo in obnovo jezu Kokaral ter gradnjo dveh novih pregrad. Ve- liko bolj negotova je usoda Velikega Arala, kjer 56 Acta Biologica Slovenica, 58 (1), 2015 nižanje vodostaja in slanostne razmere omogočajo preživetje le nekaterim zelo specializiranim nevretenčarskim vrstam. Acknowledgements This paper is dedicated to the memory of Dr. Sandeep Joshi, Director of SERI (Shristi Eco- Research Institute) who passed away on 23rd September 2014 in Delhi. References Aladin, N., 2014. The dam of life or dam lifelong. The Aral Sea and the construction of the dam in Berg Strait. Part one (1988-1992) El Alfoli - IPAISAL’s biyearly journal No , Issue 15, 2014, 1-3. Aladin, N., Plotnikov, I., 2012. Restoration of the Northern Aral Sea with the help of Kokaral dike. In: International conference of Urmia Lake. Challenges and solutions. Urmia. Aladin N., Plotnikov I., Micklin P., 2012. 20 years anniversary (1992-2012) of the contraction of the first earthen dike in Berg‘s strait of the Aral Sea (Kazakhstan). SIL news, 61, 5-7. Ermakhanov, Z. K., Plotnikov, I. S., Aladin, N. V., Micklin, P., 2012. Changes in the Aral Sea ich- thyofauna and fishery during the period of ecological crisis. Lakes & Reservoirs: Research and Management, 17, 3–9. Filippov, A. A., Petukhov, Komendantov, V. A., Yu., A., 1993. Zoobenthos of Berg strait (Aral Sea) in May 1992. Proceedings of Zoological Institute RAS, 250, 72–80. [in Russian] http://www.ilec.or.jp/en/ The International Lake Environment Committee Foundation (ILEC). http://earthobservatory.nasa.gov/Features/WorldOfChange/aral_sea.php Earth Observatory. Plotnikov, I. S., Aladin, N.V., Keyser, D., Ermakhanov, Z. K., 2012. Transformation of aquatic animal biodiversity in the Aral Sea. It is not dying, but transforming in accordance with water availability and its salinity. In: Towards a Sustainable Society in Central Asia: An Historical Perspective on the Future. Plotnikov, I. S., Aladin, N. V., Ermakhanov, Z. K., Zhakova, L. V., 2014. The New Aquatic Biology of the Aral Sea, Part II, Chapter 6: The Aral Sea. The Devastation and Partial Rehabilitation of a Great Lake Series, Springer Earth System Sciences, Vol. 10178. Eds. P. Micklin, N.V., Aladin, I. Plotnikov, pp. 137-170. Smurov, A. O., 1995. Materials for fauna of Tintinnina infusorians (Polymeromorpha, Oligotricha) of modern Aral (Small Sea). Proceedings of Zoological Institute RAS, 262, 189-194 [in Russian]. Photobiology, The Science of Light and Life Fotobiologija, Znanost o svetlobi in življenju Björn, L.O., 2015. Photobiology - The Science of Light and Life, 3rd ed. Springer, New York, Heidelberg, Dordrecht, London. ACTA BIOLOGICA SLOVENICA LJUBLJANA 2015 Vol. 58, [t. 1: 57–59 Svetloba, ki prihaja od Sonca, je ključnega pomena za življenje. Je gonilna sila procesov v biosferi, služi kot vir energije za organizme in jim zagotovlja informacije o okolju, v katerem živijo. Za rastline je svetloba pomembna iz več vidikov; svetlobo vežejo v procesu fotosinteze, svetloba tudi usmerja rast in razvoj rastlin od kalitve do cvetenja. Svetloba paima tudi toplotne in škodljive učinke na rastlinsko tkivo. Rastline zaznavajo ka- kovost, jakost, trajanje in smer svetlobe s pomočjo različnih molekul pigmenta, medtem ko pri živalih, svetloba večinoma omogoča informiranje preko vida. Različne valovne dolžine svetlobe imajo različne učinke na organizme. Knjigo Fotobiologija: Znanost o svetlobi in življenju je uredil Lars Olof Björn, profesor na Oddelku za biologijo Univerze v Lundu, in na Fakulteti za znanosti o življenju (South China Normal University). Lars Olof Björn ima dolgo- letne izkušnje na področju fotobioloških raziskav. Njegovo raziskovalno delo je usmerjeno v foto- biologijo rastlin, zanima pa ga tudi vid živali, fotobiologija kože in bioluminiscenca. Profesor Björn ni samo urednik knjige, je tudi avtor in 58 Acta Biologica Slovenica, 58 (1), 2015 soavtor 22 od 29 skupno poglavij. Ta, že tretja izdaja te knjige, se bistveno razlikuje od prvih dveh izdaj, predvsem glede slikovnega gradiva in obsega vsebine. Slikovni material je bogatejši kot v prejšnjih izdajah in vključuje tudi barvne fotografije in risbe. Dodanih je tudi nekaj novih poglavij: »Fotoaktivne beljakovine«, »Proteinski fotoreceptorji in njihov razvoj«, »Privzem svetlobe v procesu fotosinteze«, »Okužbe, odvisne od svetlobe«, in »Pomen ultravijoličnega sevanja v evoluciji«. Knjiga celovito obravnava naravo svetlobe, strukturne in funkcionalne prilagoditve organizmov ter njihove interakcije s svetlobo. Uvodna poglavja knjige (od 1 do 7) povzemajo splošne vidike svetlobe: interakcije svetlobe s snovjo, merjenje svetlobe, svetloba kot orodje za znanstvene raziskave, ter lastnosti svetlobe v kopenskih in vodnih okoljih. Drugi del knjige se ukvarja s strukturnimi in funkcionalnimi lastnostmi živih organizmov v odnosu do svetlobe. Vključuje različne ravni organizacije, od molekul do organizmov. Naslov osmega poglavja je »Akcijska spektroskopija v biologiji«. Akcijska spektroskopija je metoda, ki služi za identifikacijo vrste molekul, ki absorbirajo aktivno svetlobo. Akcijski spekter nam pove sto- pnjo fiziološke aktivnosti v odvisnosti od valovne dolžine svetlobe in je uporaben za določanje učinkov določenih valovnih dolžin na fiziološko aktivnost. To znanje je še posebej pomembno pri načrtovanju poskusov s svetlobnimi viri pod nadzorovanimi razmerami, vključno z raziskavami UV sevanja. Deveto in deseto poglavje podajata izsledke na področju bioluminiscene ter vsebujeta vsebine o pomenu svetlobnih spektrov v biologiji, predvsem za fotosintezo, za vid živali ter za in- terakcije med rastlinami in njhovimi opraševalci. Ti dve poglavji odgovarjata na vprašanja, zakaj so rastline zelene, kaj določa spektre pigmentov, kako so usklajeni pigmenti in vid in kako različne biotske strukture odbijajo in pršijo svetlobo, kot tudi kako nastane strukturna obarvanost, bele lise in presevnost pri listih. Enajsto poglavje se osredotoča na fotoak- tivne proteine, ki so odgovorni za vrsto različnih procesov v rastlinah kot je uravnavanje encimske aktivnosti, svetlobno regulirano delovanje ionske črpalke in ionskih kanalov, privzem svetlobe pri fotosintezi, fotorecepcija in bioluminiscenca. Dvanajsto in trinajsto poglavje govorita o zaz- navanju svetlobne pri organizmih. V zoologiji se izraz fotoreceptor nanaša na celice, ki se odzivajo na svetlobo (na primer čepki v očeh), medtem ko se pri rastlinah beseda fotoreceptor nanaša na molekule pigmenta, ki absorbirajo svetlobo in povzročajo zaporedje različnih reakcij in s tem povezanih »informacij«. Posebna pozornost je namenjena reševanju posebnih »težav«, ki so se pojavljale v evoluciji živali, kot so oči v vodi, kromatična aberacija, oči dvoživk, oči insektov, oči z zrcalno optiko ter oči, ki imajo sposobnost skeniranja. Šestnajsto in sedemnajsto poglavje povzemata pomembne informacije o fotosintezi, procesu, v katerem rastline privzemajo sončno energijo. Šesnajsto poglavje podaja izsledke o razvoju fotosinteze in njenem vplivom na okolje, kar je pomembno z vidika človekovega vpliva na okolje ter z vidika rastlinske pridelave. Meha- nizem privzema svetlobe v procesu fotosinteze je predstavljen v sedemnajstem poglavju. Naslov osemnajstega poglavja je: »Kako svetloba spremeni cirkadiani ritem?« To poglavje vsebuje pregled cirkadianih ritmov različnih organizmov, od gliv, cianobakterij, alg, semen rastlin, živali in ljudi in razlaga, kako uporabiti dnevne in letne cikle v svojo korist. Posebna pozornost je namenjena reševanju praktičnih težav, kot so izmensko delo, »jet lag«, in motnje spanja in njhovim vplivom na cirkadiani ritem pri človeku. V tem poglavju avtorji navajajo več kot 700 referenc. Osemnajsto poglavje ponuja vpogled v procese, povezane s fotomorfogenzo in fotoperiodizmom pri rastli- nah. Rastline, kot sesilni organizmi, uporabljajo informacije o svojem okolju, ki temeljijo tudi na svetlobnih razmerah. To je zelo pomembno, saj svetloba ne vpliva le na fotosintezo, ampak tudi na kalitev, apikalno rast, rast internodijev in listov, proizvodnjo barvil, delovanje listnih rež, dormanco popkov, razvejanost in cvetenje. Svetloba služi tudi kot pomembna informacija v kombinaciji z magnetnim poljem. Dvajseto poglavje z naslovom »Svetlobno odvisni magnetnni kompas«, razpravlja o magnetorecepciji, ki je odvisna od svetlobe z vedenjskega, fiziološkega, nevrobiološkega in biofizikalnega vidika. Enaindvajseto poglavje povzema mehanizme fototoksičnosti v povezavi s svetlobo. Fototoksičnost označuje spremembo lastnosti snovi, ki prvotno niso strupene, ampak postanejo strupene po izpostavljenosti svetlobi. To poglavje vključuje različna podpodročja, ki 59Book review razpravljajo o fototoksičnosti v rastlinski obrambi, o fototoksičnih zdravilih in kozmetiki ter o metabol- nih procesih, ki lahko pripeljejo do fototoksičnih učinkov. Pomemben del tega poglavja je namenjen policikličnim aromatskim ogljikovodikom kot fototoksičnim snovem v vodnih okoljih, saj nji- hova strupenost nastopi po izpostavljenosti UV-B sevanju. Čeprav številna poglavja omenjajo tudi različne vidike UV-B sevanja, se dvainvajseto poglavje osredotoča na posledice tanjšanja ozonske plasti za življenje, medtem ko je sedemindvajseto poglavje zanimivo branje o vlogi UV sevanja pri razvoju življenja. Trindvajseto in štiriindvajseto poglavje sta tudi povezani z učinki UV-B sevanja. Triindvajseto poglavje poroča o fotobioloških in ekoloških vidikih vitamina D, medtem ko štiriindvajseto obravnava fotobiologijo človeške kože, vključno z imunosupresijo in nekaterimi fotosenzitivnostnimi motnjami. Splošno je znano, da lahko UV sevanje uničuje mikroorganizme, vendar je manj dokazov o njegovih pozitivnih učinkih na življenje. Kratko poglavje (poglavje številka 25) z naslovom »Svetloba poveča možnost okužbe« navaja primere pri različnih organizmih. Šestindvajseto poglavje govori o bioluminiscenci, ki se pojavlja pri različnih skupinah organizmov, predvsem tistih, ki živijo v morju. Bioluminis- cenca ima različne vloge kot so razmnoževanje, zaščita plena pred plenilci, pridobivanje hrane, zaščita pred reaktivnimi kisikovimi spojinami in popravljanje poškodb DNA. Poleg bioluminiscence to poglavje obravnava mehanizme nastajanja svetlobe, nadzora oddajanja svetlobe in načine človekovega izkoriščanja bioluminiscence. Zad- nje poglavje povzema nekatere praktične vidike delovanja svetlobe, ki jih lahko uporabimo v procesu izobraževanja. Ta obsežna knjiga je edinstven nabor znanja o svetlobi in življenju in prinaša obilico novosti na področju fotobiologije. Vsebuje številne primere z različnih ravni biološke organizacije. Knjiga je nepogrešljiv pripomoček za različne bralce, od študentov in univerzitetnih učiteljev, do znan- stvenikov s področja biologije pa tudi iz drugih znanstvenih področij. Alenka Gaberščik Oddelek za biologijo, Univerza v Ljubljani, Ljubljana, Slovenija 1. slovensko posvetovanje mikroskopistov Piran 2015 ACTA BIOLOGICA SLOVENICA LJUBLJANA 2015 Vol. 58, [t. 1: 61 V organizaciji Slovenskega društva za mik- roskopijo (SDM) je 18. in 19. maja 2015 v Piranu potekalo 1. slovensko posvetovanje mikrosko- pistov. Srečanja se je udeležilo 107 udeležencev, predavateljev, gostov in razstavljalcev, večina iz Slovenije in tudi nekaj iz drugih držav. V otvorit- venem nagovoru je predsednik SDM, prof. dr. Sašo Šturm, izpostavil ključna namena posveta - pred- stavitev različnih mikroskopskih metod in njihove uporabe na področju naravoslovnih znanosti, znanosti o materialih in v industriji ter oblikovanje priložnosti za boljše povezovanje mikroskopistov v Sloveniji. V plenarnem predavanju sta prof. dr. Jasna Štrus z Univerze v Ljubljani in prof. dr. Miran Čeh z Inštituta Jožef Stefan predstavila pregled razvoja mikroskopije v Sloveniji, zlasti elektronske mikroskopije. Tega so zaznamovali dosežki različnih slovenskih strokovnjakov, ki so delovali na področju razvoja opreme in tehnik priprave vzorcev za mikroskopske analize. Med ključnimi mejniki sta izpostavila pionirsko delo prof. dr. Aleša Strojnika, ki je leta 1955 v Ljubljani skonstruiral presevni elektronski mikroskop. Za raziskave materialov je bil l. 1954 v Sloveniji nameščen prvi elektronski mikroskop, ki mu je l. 1965 sledila postavitev presevnega elektronskega mikroskopa za biološke in medicinske raziskave. V dvodnevnem programu smo v nadaljevanju spremljali 5 vabljenih predstavitev, ki so odražala izrazito interdisciplinarnost srečanja. V okviru vabljenih predavanj so bile predstavljene nasled- nje vsebine: (i) vrstična tunelska mikroskopija in uporaba te metode za vizualizacijo površin z atomsko ločljivostjo ter za kontrolirano manipu- lacijo osnovnih gradnikov v raziskavah materialov in v nanotehnologiji; (ii) kombinirana uporaba fluorescenčne in elektronske mikroskopije za lokalizacijo membranskih proteinov in vivo ter na ultrastrukturnem nivoju, s poudarkom na študiju urotelijskih celic; (iii) avtomatska analiza neko- vinskih vključkov v jeklu z vrstično elektronsko mikroskopijo, ki je bila razvita za sistematično sledenje proizvodnih procesov; (iv) napredna kvan- titativna mikroanaliza z valovnodolžinsko disper- zijsko spektroskopijo, prikazana na primeru študija kemijske sestave kristalov in (v) superločljivostna fluorescenčna mikroskopija, s poudarkom na mikroskopiji z vzbujenim praznjenjem emisije (mikroskopija STED: STimulated Emission Depletion), pri razvoju katere so sodelovali tudi raziskovalci iz Slovenije in jo uspešno uporabili v različnih raziskavah v celični fiziologiji. Novosti na področju razvoja mikroskopije, raznolikost mikroskopskih metod in možnosti njihove uporabe v raziskavah, diagnostiki in industriji so predstavili udeleženci v 24 krajših predavanjih, ki so jim sledile zanimive razprave. Širok spekter predavateljev iz vrst študentov, uveljavljenih raziskovalcev in strokovnjakov je omogočal izmenjavo različnih izkušenj in znanj. Poleg tega je bilo na srečanje uvrščenih 31 post- erskih predstavitev, ki so jih pripravili avtorji iz različnih univerz, inštitutov in drugih organizacij, pogosto v okviru medinstitucionalnih sodelovanj. Vsi prispevki so objavljeni v zborniku Slovensko posvetovanje mikroskopistov - Knjiga povzet- kov (COBISS.SI-ID 3463247). K celovitejšemu pregledu tehnoloških novosti so prispevali tudi strokovni predstavniki proizvajalcev mikroskopov ter instrumentov za pripravo vzorcev, ki so organ- izirali informativne predstavitve in demonstracijo opreme na razstavnem prostoru. Udeleženci smo se strinjali, da je tovrstno srečanje dobra priložnost za izmenjavo znanj med različnimi področji znanosti in dobrodošla spodbuda za interdisciplinarno sodelovanje na področju razvoja in uporabe mikroskopije v raziskavah, storitvenih dejavnostih, industriji in izobraževanju. Nada Žnidaršič Oddelek za biologijo, Biotehniška fakulteta, Univerza v Ljubljani 63 INSTRUCTIONS FOR AUTHORS 1. Types of Articles SCIENTIFIC ARTICLES are comprehensive descriptions of original research and include a theo- retical survey of the topic, a detailed presentation of results with discussion and conclusion, and a bibliography according to the IMRAD outline (Introduction, Methods, Results, and Discussion). In this category ABS also publishes methodological articles, in so far as they present an original method, which was not previously published elsewhere, or they present a new and original usage of an estab- lished method. The originality is judged by the editorial board if necessary after a consultation with the referees. The recommended length of an article including tables, graphs, and illustrations is up to fifteen (15) pages; lines must be double-spaced. Scientific articles shall be subject to peer review by two experts in the field. REVIEW ARTICLES will be published in the journal after consultation between the editorial board and the author. Review articles may be longer than fifteen (15) pages. BRIEF NOTES are original articles from various biological fields (systematics, biochemistry, genetics, physiology, 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. CONGRESS NEWS acquaints readers with the content and conclusions of important congresses and seminars at home and abroad. ASSOCIATION NEWS reports on the work of Slovene biology associations. 2. Originality of Articles Manuscripts submitted for publication in Acta Biologica Slovenica should not contain previously 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 Slovene if the topic is very local. As a rule, congress and association news will appear in Slovene. 4. Titles of Articles Title must be short, informative, and understandable. It must be written in English and in Slovene language. The title should be followed by the name and full address of the authors (and if possible, fax number and/or e-mail address). The affiliation and address of each author should be clearly marked as well as who is the corresponding author. 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 up to 250 words, and for brief note articles, 100 words. Article must have an abstract in both English and Slovene. 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. Running title This is a shorter version of the title that should contain no more than 60 characters with spaces. 64 Acta Biologica Slovenica, 58 (1), 2015 8. Introduction The introduction must refer only to topics presented in the article or brief note. 9. Illustrations and Tables Articles should not contain more than ten (10) illustrations (graphs, dendrograms, pictures, photos etc.) and tables, and their positions in the article should be clearly indicated. All illustrative material should be provided in electronic form. Tables should be submitted on separate pages (only horizontal lines should be used in tables). Titles of tables and illustrations and their legends should be in both Slovene and English. Tables and illustrations should be cited shortly in the text (Tab. 1 or Tabs. 1-2, Fig. 1 or Figs. 1-2; Tab. 1 and Sl. 1). A full name is used in the legend title (e.g. Figure 1, Table 2 etc.), written bold, followed by a short title of the figure or table, also in bold. Subpanels of a figure have to be unambiguously indicated with capital letters (A, B, …). Explanations associated with subpanels are given alphabetically, each starting with bold capital letter (A), a hyphen and followed by the text. 10. The quality of graphic material All the figures have to be submitted in the electronic form. The ABS publishes figures either in pure black and white or in halftones. Authors are kindly asked to prepare their figures in the correct form to avoid unnecessary delays in preparation for print, especially due to problems with insufficient contrast and resolution. Clarity and resolution of the information presented in graphical form is the responsibility of the author. Editors reserve the right to reject unclear and poorly readable pictures and graphical depictions. The resolution should be 300 d.p.i. minimum for halftones and 600 d.p.i. for pure black and white. The smallest numbers and lettering on the figure should not be smaller than 8 points (2 mm height). The thickness of lines should not be smaller than 0.5 points. The permitted font families are Times, Times New Roman, Helvetica and Arial, whereby all figures in the same article should have the same font type. The figures should be prepared in TIFF, EPS or PDF format, whereby TIFF (ending *.tif) is the preferred type. When saving figures in TIFF format we recommend the use of LZW or ZIP compression in order to reduce the file sizes. The photographs can be submitted in JPEG format (ending *.jpg) with low compression ratio. Editors reserve the right to reject the photos of poor quality. Before submitting a figure in EPS format make sure first, that all the characters are rendered correctly (e.g. by opening the file first in the programs Ghostview or GSview – depending on the operation system or in Adobe Photoshop). With PDF format make sure that lossless compression (LZW or ZIP) was used in the creation of the *.pdf file (JPEG, the default setting, is not suitable). Figures created in Microsoft Word, Excel, PowerPoint etc. will not be accepted without the conver- sion into one of the before mentioned formats. The same goes for graphics from other graphical programs (CorelDraw, Adobe Illustrator, etc.). The figures should be prepared in final size, published in the magazine. The dimensions are 12.5 cm maximum width and 19 cm maximum height (width and height of the text on a page). 11. Conclusions Articles shall end with a summary of the main findings which may be written in point form. 12. Summary Articles written in Slovene must contain a more extensive English summary. The reverse also applie s. 13. 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, (Trinajstić and Franjić 1994); if a work by three or more authors is cited, (Pullin et al. 1995); and if the reference appears in several works, (Honsig- Erlenburg et al. 1992, Ward 1994a, Allan 1995, Pullin et al. 1995). If several works by the same author 65 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 included: Toman (1992: 5) or (Toman 1992: 5–6).The bibliography shall be arranged in alphabetical order beginning with the surname of the first author, comma, the initials of the name(s) and continued in the same way with the rest of the authors, separated by commas. The names are followed by the year of publication, the title of the article, the international abbreviation for the journal (periodical), the volume, the number in parenthesis (optional), and the pages. Example: Mielke, M.S., Almeida, A.A.F., Gomes, F.P., Aguilar, M.A.G., Mangabeira, P.A.O., 2003. Leaf gas exchange, chlorophyll fluorescence and growth responses of Genipa americana seedlings to soil flood- ing. Experimental Botany, 50 (1), 221–231. 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., McLean, I.F.G., Webb, M.R., 1995. Ecology and Conservation of Lycaena dispar: Britis h 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 posve- tovanja DZVS, Gozd Martuljek, pp. 1-7. 14. Format and Form of Articles The manuscripts should be sent exclusively in electronic form. The format should be Microsoft Word (*.doc) or Rich text format (*.rtf) using Times New Roman 12 font with double spacing, align left only and margins of 3 cm on all sides on A4 pages. Paragraphs should be separated by an empty line. The title and chapters should be written bold in font size 14, also Times New Roman. Possible sub-chapter titles should be written in italic. All scientific names must be properly italicized. Used nomenclature source should be cited in the Methods section. The text and graphic material should be sent to the editor-in-chief as an e-mail attachment. For the purpose of review the main *.doc or *.rtf file should contain figures and tables included (each on its own page). However, when submitting the manuscript the figures also have to be sent as separate attached files in the form described under paragraph 10. All the pages (including tables and figures) have to be numbered. All articles must be proofread for professional and language errors before submission. A manuscript element checklist (For a manuscript in Slovene language the same checklist is appro- priately applied with a mirroring sequence of Slovene and English parts): English title – (Times New Roman 14, bold) Slovene title – (Times New Roman 14, bold) Names of authors with clearly indicated addresses, affiliations and the name of the corresponding author – (Times New Roman 12) Author(s) address(es) / institutional addresses – (Times New Roman 12) Fax and/or e-mail of the corresponding author – (Times New Roman 12) Keywords in English – (Times New Roman 12) Keywords in Slovene – (Times New Roman 12) Running title – (Times New Roman 12) Abstract in English (Times New Roman 12, title – Times New Roman 14 bold) 66 Acta Biologica Slovenica, 58 (1), 2015 Abstract in Slovene – (Times New Roman 12, title – Times New Roman 14 bold) Introduction – (Times New Roman 12, title – Times New Roman 14 bold) Material and methods – (Times New Roman 12, title – Times New Roman 14 bold) Results – (Times New Roman 12, title – Times New Roman 14 bold) Discussion – (Times New Roman 12, title – Times New Roman 14 bold) Summary in Slovene – (Times New Roman 12, title – Times New Roman 14 bold) Figure legends; each in English and in Slovene – (Times New Roman 12, title – Times New Roman 14 bold, figure designation and figure title – Times New Roman 12 bold) Table legends; each in English and in Slovene – (Times New Roman 12, title – Times New Roman 14 bold, table designation and table title – Times New Roman 12 bold) Acknowledgements – (Times New Roman 12, title – Times New Roman 14 bold) Literature – (Times New Roman 12, title – Times New Roman 14 bold) Figures, one per page; figure designation indicated top left – (Times New Roman 12 bold) Tables, one per page; table designation indicated top left – (Times New Roman 12 bold) Page numbering – bottom right – (Times New Roman 12) 15. 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. With articles written in Slovene and dealing with a very local topic, both reviewers will be Slovene. In the compulsory accompanying letter to the editor the authors must nominate one foreign and one Slovene reviewer. However, the final choice of referees is at the discretion of the Editorial Board. The referees will remain anonymous to the author. The possible outcomes of the review are: 1. Fully acceptable in its present form, 2. Basically acceptable, but requires minor revision, 3. Basically acceptable, but requires important revision, 4. May be acceptable, but only after major revision, 5. Unacceptable in anything like its present form. In the case of marks 3 and 4 the reviewers that have requested revisions have to accept the suitability of the corrections made. In case of rejection the corresponding author will receive a written negative decision of the editor-in-chief. The original material will be erased from the ABS archives and can be returned to the submitting author on special request. After publication the corresponding author will receive the *.pdf version of the paper.