ELECTRICAL CHARGE OF BASIDIOSPORES OF HYMENOMYCETES (FUNGI) AND ITS BIOLOGICAL SIGNIFICANCE

EOSLAVASEENTE KANDEOSTE ELEKTRILAENG JA SELLE BIOLOOGILINE TÄHENDUS

MARET SAAR

A thesis for applying for the degree of Doctor of Philosophy in Applied Biology

9lLWHNLULÀORVRRÀDGRNWRULNUDDGLWDRWOHPLVHNVUDNHQGXVELRORRJLDHULDODO

7DUWX

Eesti Maaülikooli doktoritööd

Doctoral Theses of the Estonian University of Life Sciences

ELECTRICAL CHARGE OF BASIDIOSPORES OF HYMENOMYCETES (FUNGI) AND ITS BIOLOGICAL SIGNIFICANCE

EOSLAVASEENTE KANDEOSTE ELEKTRILAENG JA SELLE BIOLOOGILINE TÄHENDUS

MARET SAAR

A thesis for applying for the degree of Doctor of Philosophy in Applied Biology

Väitekiri fi losoofi adoktori kraadi taotlemiseks rakendusbioloogia erialal

Tartu 2015 Institute of Agricultural and Environmental Sciences Estonian University of Life Sciences

According to the verdict No 6-14/14-7 of July 9, 2015 the Doctoral Committee of Agricultural and Natural Sciences of the Estonian University of Life Sciences has accepted the thesis for the defence of the degree of Doctor of Philosophy in Applied Biology.

Opponent: Prof. Roy Kennedy Institute of Science and the Environment, University of Worcester, UK

Supervisors: D. Sc. Erast Parmasto Institute of Agricultural and Environmental Sciences, Estonian University of Life Sciences

Prof. Tiiu Kull Institute of Agricultural and Environmental Sciences, Estonian University of Life Sciences

Defence of the thesis: Estonian University of Life Sciences, room 2A1, Kreutzwaldi 5, Tartu, on September 17, 2015, at 11:15 a.m.

The English language was edited by Roger Evans, and the Estonian by Piret Kruuspere.

Publication of this thesis is supported by the Estonian University of Life Sciences and by the Doctoral School of Earth Sciences and Ecology created under the auspices of European Social Fund.

© Maret Saar, 2015

ISSN 2382-7076 ISBN 978-9949-536-94-8 (trükis) ISBN 978-9949-536-95-5 (pdf) CONTENTS

1. LIST OF ORIGINAL PUBLICATIONS ...... 7 2. INTRODUCTION ...... 8 2.1. Background ...... 8 2.2. Review of the literature ...... 14 2.3. Research aims ...... 21 3. MATERIALS AND METHODS ...... 23 3.1. Sampling, samples, charge type, mean charge-to-mass quotient, mean charge ...... 23 3.2. Hymenial emission rate, territorial emission rate ...... 26 3.3. Data analysis ...... 27 3.4. Relaxation time, washout of spores ...... 29 4. RESULTS ...... 30 4.1. Charge type of a spore sample ...... 30 4.2. Spore charge-to-mass quotient and spore charge ...... 32 4.3. Intraspecimen and intraspecifi c variabilities of spore charge- to-mass quotient ...... 33 4.4. Hymenial emission rate of charged spores ...... 33 4.5. Upper limit of the territorial emission rate of spores and their charges ...... 34 4.6. Charge relaxation time ...... 34 4.7. Role of charges in washout of spores ...... 35 5. DISCUSSION ...... 37 5.1. Factors infl uencing the charge type of a spore population .....37 5.2. Mean absolute value of the spore charge-to-mass quotient and the spore charge, their variability ...... 38 5.3. Hymenial emission rate of charged spores, upper limit of the territorial emission rate of charged spores and charges ...... 39 5.4. Relaxation time of spore charges in the atmosphere ...... 40 5.5. Role of charges in washout of spores from the atmosphere....40 6. CONCLUSIONS ...... 42 7. REFERENCES ...... 44 8. GLOSSARY ...... 50

5 9. SUMMARY IN ESTONIAN ...... 51 10. ACKNOWLEDGEMENTS ...... 58 PUBLICATIONS ...... 59 CURRICULUM VITAE...... 113

6 1. LIST OF ORIGINAL PUBLICATIONS

This thesis is based on the following publications, which are referred in the further text by their Roman numeral.

I. Saar, Maret (2013). Investigation of the electrostatic charge of basidiospores of the Phellinus igniarius group. Central European Journal of Biology, 8 (5), 410 - 422.

II. Saar, Maret & Salm, Jaan (2014). Emission rate of charged spores in basidiomycetous fungi and the relaxation time of their electric charges. Aerobiologia, 30 (1), 71 - 89.

III. Saar, Maret & Parmasto, Erast (2014). Primary basidiospore charge and taxonomy of Agaricomycetes. Central European Journal of Biology, 9 (9), 874-887.

Table 1. Authors’s contribution to each paper (%) IIIIII Concept 100 100 60 Design 100 90 100 Data collection 100 100 100 Data analyses 100 85 50 Manuscript preparation 100 80 80

7 2. INTRODUCTION

2.1. Background

Fungi spread mostly by windborne spores (Ingold 1971). Wind-dispersed spores are violently or passively discharged into the atmosphere (Ingold 1971). When spores liberate from spore-bearing structures, both the violently ejected spores and the passively released spores can carry electric charges (Buller 1909, Gregory 1957, Swinbank et al. 1964, Garrett 1972, Webster et al. 1988, McCartney et al. 1982). Charges are present also on freshly discharged pollen grains of anemophilous plants (Bowker & Crenshaw 2003, 2007).

The knowledge of primary charges of wind-dispersed pollen grains is precise. The investigation by Bowker & Crenshaw (2007) of seven species covered deciduous, coniferous and herbaceous plants. This showed that nearly all pollen grains are electrically charged. Each species had a bipolar distribution of pollen charges. Often the distribution of spore charges was roughly centered about zero. In some species, the distributions were tightly centered, while in others there was a wide spread of charges. Seldom the distributions were skewed toward the positive. The magnitude of charge (the average of the absolute value) carried by a typical pollen grain was 5,250 e (referred to as 0.84 fC in Bowker & Crenshaw 2007). Some pollen grains carried charges up to 250,000 e (40 fC).

Little appears to be known about primary electric charges carried by fungal spores. Primary charges of passively released conidia were investigated in Blumeria graminis (DC.) Speer. McCartney et al. (1982) sought to determine whether barley mildew spores carry electric charges as large as the minimum charge (3 × 104 e per spore) that, by a theory, would be needed to infl uence spore deposition to a crop. Field measurements showed a bipolar distribution of spore charges and the charge magnitude smaller than 104 e per spore. The conclusion that conidia of Setosphaeria turcica (Luttr.) K.J. Leonard & Suggs carry electric charges when liberating from conidiophores was made by Leach from his experiments with sporulating maize leaf lesions (1976). This conclusion should be revised because Leach had omitted alternative interpretations of his experimental results (Aylor & Paw U 1980). Primary charges of

8 ascospores were examined only in the fungal component of lichens. To determine the existence and nature of charges on freshly ejected ascospores, Garrett (1972) performed tests in four lichen species, using fi ve different ascocarps for each. Spores of three species generally bear positive electric charges whereas spores of one species bear negative. Fifteen fungal species were involved in the works on the primary charges of violently ejected basidiospores (Buller 1909, Gregory 1957, Swinbank et al. 1964, Webster et al. 1988). However, these works provide fragmentary information insuffi cient for understanding the phenomenon. Therefore, the opinion of Gregory (1973) has remained valid up to now: little is known about the phenomenon of the electrostatic charges on freshly liberated basidiospores.

These works show us that electrically charged spores are emitted by various ballistospore-bearing structures: fruitbodies with basidia entirely exposed to the air, fruitbodies exposing only outgrowths of basidia, and single-celled fungi producing aerial ballistoconidia. Basidiomata of the Agaricomycetes (Buller 1909, Gregory 1957, Swinbank et al. 1964, Webster et al. 1988) and Tremellomycetes (Webster et al. 1988), and yeast-like anamorphic stages of the Tremellomycetes (Webster et al. 1988) were involved in these works.

Few species, which were studied and in which the primary charges were found, belong to different basidiomycetous groups (Table 1). This fact points to the possibility of the presence of primary electrostatic charges in all ballistosporic basidiomycetes. Ballistospores may be formed as the result of sexual or asexual reproduction. These spores may be produced by basidia, hyphae, yeast cells, or even other ballistospores (Swann & Hibbett 2007). Ballistosporic species are present not only in the Agaricomycotina, but also in other subphylums of the Basidiomycota, in the Ustilaginomycotina and Pucciniomycotina (Swann and Hibbett 2007). Phylogenetic studies of the relationships between the species in the Basidiomycota, based on multiple analyses of ribosomal RNA gene sequences, showed that the ballistic type of spore discharge must have evolved very early in the evolutionary history of the Basidiomycota as it is found in members of the earliest diverging lineages within the group (Swann and Hibbett 2007).

9 The present doctoral dissertation is focused on primary charges characterising the basidiospores of hymenomycetous species of the Agaricomycetes. Here the term “Hymenomycetes” is used in the sense of the traditional concept, formulated by C.H. Persoon more than two centuries ago (Clemencon 2012). Hymenomycetes are those basidiomycetes that form their basidia in a more or less continuous layer exposed to the open air at full maturity; their basidia are unicellular and entirely exposed to the air (this preposition excludes “jelly fungi”). The Hymenomycetes is a polyphyletic assemblage of basidiomycetes (Hibbett 2007a). This polyphyletic group embraces the crust fungi, chanterelles, spine fungi (hydnums), bracket fungi (polypores), gilled fungi (agarics) and boletes (Clemencon 2012). The hymenomycetes embraces about 11% of all fungi described (Clemencon 2012), about 8,000 to 11,000 species worldwide (according to the number of described species given in Hawksworth & Rossman 1997). About 1500 species represent this group in a territory of 45 000 km2 with favourable growth conditions for both boreal coniferous forest fungus species and nemoral deciduous forest species, in Estonia (Eesti seenestik 2000). Due to the species richness and the diversity, this fungal group is appropriate for a study of phylogenetic signals in the primary charges. Such aspect of the primary charges has not been studied or discussed previously and is one of main subjects of this dissertation.

The hypothesis of phylogenetic signals in the primary charges of the hymenomycetous basidiospores was supported by some pre-existing experimental data indicating the endogenous origin of spore charges. Ballistic basidiospores are unicellular wind-dispersal units of asymmetric form, having mostly an ovoid, ellipsoid, spherical or elongated general view and the diameter of 2-16 µm of a sphere of equivalent volume (Ingold 2001, Clemencon 2012). Aerosol particles with the size of these basidiospores, being in the atmosphere for a long time—suffi cient to reach a steady state with atmospheric ions—, carry electrostatic charges which have an average absolute value in the range of about 3-15 e and are distributed symmetrically in sign (Seinfeld & Pandis 2006, Roos 1996). Contrary to the charges of old atmospheric aerosol particles, the freshly liberated ballistospores carried on average tens or hundreds of elementary charges (Swinbank et al. 1964; Webster et al. 1988) and were in some spore populations only positive (Gregory 1957) or only negative (Swinbank et al. 1964). These fundamental distinctions between the

10 freshly liberated spores and the old aerosol particles in the polarity and in the absolute value of charges exclude the exogenous origin of spore charges, i.e. charging the spores after liberation by the atmospheric ions (a hypothesis proposed in Webster et al. 1988). These distinctions point to the endogenous origin of spore charges, i.e. charging due to processes occurring in the fungus (a hypothesis proposed in Swinbank et al. 1964 and Gregory 1979).

The ballistic spore liberation mechanism (ballistosporic discharge) is a complex of processes, with a duration varying in the range of nine magnitude orders, from ten microseconds to ten kiloseconds. The spores on a basidium are formed simultaneously and are liberated successively (Buller 1922). Depending on species, the duration of the basidiospore formation—from the onset of karyogamy to spore formation on budding sterigmata—is about 6-37 h (Clemencon 2012), the interval between the liberation of the fi rst and the fourth spore of the basidium is about 10-300 s (Ingold 1971). Ballistic spore-liberation from a sterigma has an invariable preliminary, the excretion of a drop at the hilum appendix (Buller 1922). The drop initial develops within the spore wall; after about 3 hours the drop initial reaches the diameter of 0.5-0.8 µm (Clemencon 2012). Then it stays that way for several hours (Clemencon 2012). After the dissolution of the thin outer wall layer surrounding the drop, the drop expands during some seconds. In about 20 µs after the drop reaches the maximum size, the spore jumps off from the sterigma (Pringle et al. 2005). Spore discharge distance is controlled by the spore morphology and size, and by the drop size (Stolze-Rybczynski et al. 2009). The micromechanical processes involved in the ballistosporic discharge mechanism are well understood following studies over more than a century (Money 1998, Clemencon 2012) and the theory, based on surface tension forces, is verifi ed (Pringle et al. 2005). However, the biochemical processes involved are known incompletely (Stolze-Rybczynski 2009, Money & Fischer 2009). For example, it is not clear how the osmolytes are delivered to the spore surface in a suffi cient concentration to act as nuclei for the condensation of water (Money & Fischer 2009). In addition to the previously mentioned processes which are directly associated with a basidium and spores, there are processes which—orchestrating the production of basidia on hymenium—are associated with basidia and spores indirectly. Hymenium is organized to produce and liberate spores in different ways (Buller 1922). For

11 example, the gilled species with white or pale spores have a non-mottled hymenium (e.g., Hymenopellis radicata (Relhan) R.H. Petersen, Figure 2 in Ingold 1965), while the gilled species with pigmented spores (e.g., Agaricus campestris L.) have the mottled hymenium. This consists of a mosaic of small, irregular areas (< 2 mm across) in differing stages of development; each small area produces numerous generations of basidia throughout the sporulation period, developmental waves pass over the hymenium in an irregular manner (Buller 1922).

The present dissertation aimed at gaining an understanding of factors infl uencing the polarity and absolute value of charges. A spore population, i.e., a mass of spores liberating simultaneously from a basidioma during minutes or hours, was brought into focus. The polarity distribution of spore charges in a spore sample (called the charge type of spores), and the average absolute values in a spore sample of both the spore charge- to-mass quotient and the spore charge were investigated.

Work on fungal spore discharge impacts the atmospheric science and the science of medicine. In atmospheric science, the interest in the fungal component of atmospheric aerosol particles is primarily related to ice nucleation and cloud droplet activation or atmospheric chemistry (Deguillaume et al. 2008, Despré s et al. 2012). The ice nucleation ability of fungal spores was studied experimentally (Iannone et al. 2011, Haga et al. 2014). Several characteristics of global fungal spore emission were estimated (Elbert et al 2007, Winiwarter et al. 2009, Heald & Spracklen 2009, Sesartic & Dallafi or, 2011). One model for the impact of fungal spore ice nuclei on stratiform clouds and precipitation showed that, at the global level, the role of spores is negligible in comparison with roles of bacteria and dust, but a potential impact on local climate might be expected (Sesartic et al. 2013). Further research should focus on regional and local effects of fungal spores, especially in the tropical and boreal regions. Refi nements of the estimates of fungal spore emission—based on more observational data about fungal spore emission and deposition, and about spore size distribution—are needed (Despré s et al. 2012, Sesartic et al. 2013).

At global scale, the species richness of fungal spores circulating in the continental air is dominated by basidiomycetes (64 %) and most of these species belong to the Agaricomycetes (Fröhlich-Nowoisky et al.

12 2012). There are large variations in the spatiotemporal concentrations of basidiospores. Near the northern forest limit in Fennoscandia (in Kevo, Utsjoki, Finlandia) basidiospores contribute 44-48% of the yearly total number of spores at a height of between 10-20 m above ground level, 22-41% in the northern coniferous zone (Oulu, Finland) (Rantio- Lehtimäki et al. 1985), and 12-50% in the northern mixed forest zone (Turku, Finland; Stockholm and Eskilstuna, Sweden) (Finnish Pollen Bulletin 1993, 1994, 1995, 1996, 1998; Rantio-Lehtimäki et al. 1985; Rubulis 1984). There are regions with autumn peaks in concentration of airborne basidiospores and regions where both late spring and autumn peaks occur (Levetin 1995). In south-southwest Finland, the concentration of basidiospores peaks in July-October, the weekly average concentration reaching values of 15,000-34,000 spores m-3 and the daily average concentration values up to 85,000 spores m-3 (in Turku; Finnish Pollen Bulletin 1993, 1994, 1995, 1996, 1998). The diurnal rhythm of basidiospore concentration varies greatly from month to month; the maximum mostly occuring during the period of 06-09 o’clock and the minimum, 12-18 o’clock (in Turku; Mäkinen & Rantio-Lehtimäki 1979).

While it is well known that basidiospores contribute an important fraction to the airborne fungal spora in general, the contribution of basidiospores to spore plumes—the short-term fl uctuations (lasting about an hour or shorter) of the airborne spore concentration over the value of 100,000 spores m-3 (Levetin & Horner 2002)—is not well known. The upper limit (the maximum possible value) of the territorial emission rate of hymenomycetous basidiospores is also unknown.

Freshly emitted basidiospores and pollen grains can carry high electric charges, which considerably exceed the steady state charges of atmospheric aerosol particles. However, atmospheric air is continuously ionized, containing a steady level of both negative and positive air ions. The highly charged basidiospores and pollen grains attract oppositely charged air ions, and thus their charge diminishes or relaxes during time. We estimated the time of such charge relaxation.

13 2.2. Review of the literature

Our understanding of primary electrostatic charges of ballistic basidiospores derives from the studies of Arthur Henry Reginald Buller (1874-1944), Philip Herries Gregory (1907-1986), and the groups of Peter Swinbank (dates unknown) and of John Webster (1925-2014) carried out between the years 1909-1988.

Discovering the phenomenon. The presence of primary electrostatic charges on the basidiospores of hymenomycetes was discovered by Buller in 1909. At that time he was studying the production, liberation, and dispersion of the spores of hymenomycetes. To answer the question whether or not ballistic basidiospores following their liberation from a hymenophore carry electrostatic charges, he observed the fall of basidiospores between electrically charged and vertically placed parallel plain plates by means of a horizontal microscope (Fig. 1 and Fig. 2). Observing spores of Agaricus campestris L. [referred to as Psalliota campestris in (Buller 1909)], Polyporus squamosus (Huds.) Fr. and some other unmentioned species, he found that “in all cases…the majority of spores receive positive or negative electric charges of different strengths, whilst a certain number do not become charged at all”. This statement of results shows us that the bipolar spore charges were present in the all basidiomata of the all species examined by Buller. Since the question about the charges was just one among his many others, Buller abandoned this, saying that “a further investigation…was thought unnecessary for my present purpose”.

Figure 1. Apparatus used by Buller for detecting the electrical charges on falling spores (shown by two vertical sections). Lamp was used to detect any accidental fl ow of current. Brass plates were 1.2 cm wide and 2 cm high, 1.5 mm apart. (Fig. 67 in Buller 1909, modifi ed).

14 Figure 2. The paths of spores falling between two brass plates as seen by Buller. (Fig. 68 in Buller 1909, modifi ed).

Verifi cation of the discovery. Buller’s laboratory based discovery, using pieces of basidiomata, was later verifi ed by fi eld observations (Gregory 1957) with basidiomata growing under natural conditions. Gregory, observing spores (Fig. 3) of seven species, confi rmed the presence of charges on the majority of spores, but not the occurrence of bipolar distribution of spore charges in all species as did Buller. Both positively and negatively charged spores were simultaneously released from a basidioma in Agaricus campestris L., Coprinellus hiascens (Fr.) Redhead, Vilgalys & Moncalvo [referred to as Coprinus hiascens in (Gregory 1957)], C. micaceus (Bull.) Vilgalys, Hopple & Jacq. Johnson [Coprinus micaceus], Pholiota highlandensis (Peck) A.H. Sm. & Hesler [Flammula carbonaria], P. squarrosa (Vahl) P. Kumm., and Polyporus squamosus (Huds.) Fr. Only positively charged spores were released from basidiomata in Ganoderma applanatum (Pers.) Pat. With this species, (in contrast to others where only one spore sample per species was observed) a series of observations involving a dozen spore samples from several basidiomata in different locations was carried out over a period of two years. In the species with spores of bipolar charge type, Gregory also noticed that in some of these species, the positively charged spores prevailed, as opposed to the negatively charged spores in others.

15 Figure 3. Apparatus used by Gregory for detecting the electrical charges on falling spores (Figure 1 in Gregory 1957, modifi ed). The box had the form of a cube with 2.5 cm sides, and with a slit 0.5 cm wide. The vertical charged metal plates (parallel to each other) were 1 cm apart. After expose for an hour, observations were made on the density of deposit of the spores adhering to the charged plates.

Measurement of the polarity and magnitude of primary charges in a species. To consider the effect of the charges on spore movement in confi ned spaces of hymenophore (within tubes, between gills, etc), it was necessary to assess the order of magnitude (absolute value) of the spore charges. The magnitude of the basidiospore primary charges was for the fi rst time measured by Swinbank et al. in 1964. They determined experimentally the quotient of spore charge-to-mass (Fig. 4) and the mean spore mass (by direct weighing) in Serpula lacrymans (Wulfen) J. Schröt. Using these data, they calculated the mean charge on a spore. Mean values were following: -8.17×10-5 C kg-1 [referred to as -245 esu g-1 in (Swinbank et al.1964)] for the quotient of spore charge- to-mass, (5.52±0.48)×10-11 g for a spore mass, -28 e (-25…-31 e) [(- 1.35±0.12)×10-8 esu] for a spore charge. A majority of spores carried a negative charge in a spore sample: the proportion of the non-charged spores was several times lower than that of the charged spores. It was found that the primary electric charges of spores do not play a role in the moving between gills or within tubes.

16 Figure 4. Apparatus used by Swinbank and colleagues for the determination of the spore charge-to-mass quotient. (Fig. 1 and 2 in Swinbank et al. 1964, modifi ed). Vertical parallel metal plates were 2.6 cm wide, 7.5 cm high, and 1.5 cm apart. The potential difference between plates was 84 volts throughout the experiments with fi eld applied. Slit was 0.7 mm wide. After expose for a day or more, observations were made on the density of deposit of the spores adhering to the bottom glass plate.

Estimation of magnitude of primary charges in ten species. In the early 1980s, Webster’s group together with Terence Ingold, one of the champions of innovative mycological research (Money 1998), proposed that the mechanism of ballistospore discharge in basidiomycetes may involve two processes. These processes were following: jump off of the spore driven by the surface energy of liquid present on the spore, and electrostatic repulsion of the charged spore from its sterigma. The relative effect of the surface tension force and the electrostatic force was later reconsidered by Webster and his group (1988). They found that the electric charges of spores do not play a role in the escape of spores from sterigmata.

For the assessment of the role of spore charges, a range of variation of absolute values of the basidiospore primary charges was found in a group of both morphologically and phylogenetically different species (Table 1). To calculate the spore charge, the quotient of spore charge-to-

17 mass was determined experimentally (Fig. 5 and 6) and the spore mass was estimated using literature data on spore sizes and density. Absolute values of the quotient of spore charge-to-mass varied from 0.09×10-4 C kg-1 (standard error of measurement ±0.04×10-4 C kg-1) for Agaricus bisporus to 12.8×10-4 C kg-1 (±12.9×10-4 C kg-1) for Itersonilia perplexans. Absolute values of the spore charge varied from 7 e (minimum of 2 e, maximum of 142 e) [originally 0.12×10-17 C (0.04×10-17 C, 2.27×10-17 C)] for A. bisporus to 6,710 e (minimum of 893 e, maximum of 54,806 e) [originally 107.5×10-17 C (14.3×10-17 C, 878.0×10-17 C)] for I. perplexans.

Figure 5. Chamber used for observation of falling spores by the Webster’s group. Spores fell between the charged plates. The spores were illuminated by a stroboscopic fl ash lamp, their trajectories were photographed by a microscope. (Fig. 2 in Webster et al. 1988, modifi ed).

18 Figure 6. Photographs of spore trajectories, made by stroboscopic illumination at 10 fl ashes per second. At the beginning, for about 0.3 s, the spores fell freely under gravity and subsequently, when an electrostatic fi eld was applied, under the infl uence of a horizontal electrostatic fi eld. (Fig. 3 in Webster et al. 1988, modifi ed).

19 Table 1. Species and amounts of spores involved in the studies of primary charges of basidiomycetous spores in 1909-1988. Marks: A – absolute value of spore charges was studied, P – polarity of spore charges was studied, 1 – Buller 1909, 2 – Gregory 1957, 3 – Swinbank et al. 1964, 4 – Webster et al. 1988.

AGARICOMYCETES AGARICALES AGARICACEAE Agaricus bisporus (J.E. Lange) Imbach A4 (38 spores) Agaricus campestris L. A4 (80 spores) P1 (number of spores unknown) P2 (a spore sample) MARASMIACEAE Marasmius oreades (Bolton) Fr. A4 (22 spores) PHYSALACRIACEAE Flammulina velutipes (Curtis) Singer A4 (13 spores) PSATHYRELLACEAE Coprinellus hiascens (Fr.) Redhead, Vilgalys & Moncalvo P2 (a spore sample) Coprinellus micaceus (Bull.) Vilgalys, Hopple & Jacq. Johnson A4 (25 spores) P2 (a spore sample) SCHIZOPHYLLACEAE Schizophyllum commune Fr. A4 (52 spores) STROPHARIACEAE Pholiota highlandensis (Peck) A.H. Sm. & Hesler P2 (a spore sample) Pholiota squarrosa (Vahl) P. Kumm. P2 (a spore sample) AURICULARIALES AURICULARIACEAE Auricularia mesenterica (Dick.) Pers. A4 (13 spores) BOLETALES SERPULACEAE Serpula lacrymans (Wulfen) J. Schröt. A3 (several spore samples) A4 (26 spores) P3 (several spore samples) SUILLACEAE Suillus luteus (L.) Roussel A4 (12 spores) POLYPORALES GANODERMATACEAE Ganoderma applanatum (Pers.) Pat. P2 (1-3 spore samples from 12 basidiomes) POLYPORACEAE Polyporus squamosus (Huds.) Fr. P1 (number of spores unknown) P2 (a spore sample) TREMELLOMYCETES CYSTOFILOBASIDIALES CYSTOFILOBASIDIACEAE Itersonilia perplexans Derx (a ballistosporic yeast, an asexual morph) A4 (75 spores)

20 Despite the fact that the studies include data on electrostatic charges in 15 species, information on both the polarity and the magnitude of charges exists only in three species (Table 1). In most species, either the magnitude (7 species) or the polarity (5 species) was recorded. The majority of the data were found using very small samples of spores (< 100 spores per species), which were realised from a piece of basidiome. These four studies (Buller 1909, Gregory 1957, Swibank et al. 1964, Webster et al. 1988) are single sporadic attempts to understand an electrostatic phenomenon in basidiomycetes.

2.3. Research aims

The overall objective of my dissertation research was to gain a better understanding of the electrostatic charges of freshly liberated ballistic basidiospores and to elucidate the biological importance of these charges. Experiments for this dissertation were performed with the objective to characterise the polarity distribution of spore charges in spore populations and the mean spore charge-to-mass quotient in spore populations. Based on the experimental data obtained on spore charges, the effect of charges on the washout of spores from the air and the quantity of charges carried into the air by spores per unit time per unit area of forested territory were theoretically determined.

Aim 1. Identifying the polarity distributions of spore charges, measuring the spore charge-to-mass quotient, and estimating the spore charge in the hymenomycetous fungi growing in natural conditions.

Our hypotheses were based on the assumption that the spore charge is created within the basidioma. We hypothesized that the charge type of spores releasing from a particular basidioma is determined by phylogenetical factors, rather than physiological, environmental or spatio- temporal factors. Among those we considered: substrata, the major states of full-grown basidioma, seasonal periods, circadian periods, different years, air temperature and relative humidity, geographical locations, and fungal phylogenetic affi nities.

We hypothesized that the absolute values of the mean spore charge-to- mass quotient and of the mean spore charge are independent of the

21 sporulation intensity of the basidioma. We also hypothesized that the intraspecifi c variability of these characteristics could be explained by the natural variability of the mean size of spores.

Aim 2. Estimating the relaxation time of the primary spore charges in the atmosphere.

This research would help to answer the question about the time during which the initial primary charge on an airborne basidiospore diminishes in real atmospheric conditions.

Aim 3. Studying the effect of the primary charges on the washout of hymenomycetous basidiospores.

We hypothesized that the primary spore charges increase the effi ciency of collisions between spores and the water droplets of fogs, clouds and drizzles when the droplets fall through spore laden air.

Aim 4. Calculating the upper limit of the territorial emission rate of spores and their charges for some fungal species in some forests.

We hypothesized that basidiomata of the same species growing in a neighbourhood have the same sporulation dynamics and release spores synchronously, and that the circadian dynamics of the hymenial emission rate of spores and their charges is regular. Based on these hypotheses, we assumed that in a fungal species the upper limit of quantity of spores emitted into the air per unit time by basidiomata growing per unit area of forest is proportional to the number of the basidiomata on the area and to the upper limit of hymenial emission rate of spores.

The present era of mycological research is characterized by a predominance of genetic approaches and models to investigate the diversity of fungi, and the relationships between fungi and other organisms. Few researchers are investigating the processes of active discharge of spores from spore-bearing structures currently. Nevertheless, it is hoped that this doctoral research will open a new line of enquiry from which some real understanding of the development of the basidium as well as of the acting of basidiospores as cloud droplet nuclei and ice nuclei will come.

22 3. MATERIALS AND METHODS

3.1. Sampling, samples, charge type, mean charge-to-mass quotient, mean charge

Spore samples were taken in natural conditions by self-designed and -built portable devices placing the collecting chambers adjacent to the undersurface of hymenophores (Figure 7). In our experimental design, the operating principle—falling of spores in the homogeneous horizontal electric fi eld—was similar to those used in previous studies (Buller 1909, Gregory 1957, Swinbank et al. 1964, Webster et al. 1988). We determined the vertical component of the trajectories of spores according to the spore deposition sites on the electrodes (vertical plane parallel metal plates). A half of the distance between the electrodes refl ected the horizontal component of the trajectories of spores. Our way of getting information about spore trajectories differed from the ways used in the previous studies (I).

Figure 7. Device for measurement of the spore charge-to-mass quotient in natural forest conditions: a general view of the chamber located on the bottom plate; b cross- section through the middle of chamber, the electrical scheme, and spore trajectories (non-charged spore, spore with the charge too small to detect, and spore with the charge bigger than the detection limit). Vertical parallel metal plates were 45 mm wide, 34 mm high, and 11, 14, 17, 20, 22.5 or 25 mm apart. Strength of a homogeneous horizontal electrostatic fi eld between the plates was 30, 24, 19, 17, 15 or 13 kV m-1.

23 A collecting chamber was exposed under a hymenophore for 15-1500 min depending on the spore emission rate of the basidioma and on the purpose of the experiment. After exposure, electrodes and glass slides, which were lying under them, were taken to a lab and examined by a light microscope. Occurrence of spores on one electrode—and not on the other electrode—indicated a spore sample of a unipolar (either positive or negative) charge type. In the case of the bipolar charge type, the spores were found on both electrodes. (I).

The electrodes were scanned under a light microscope (×370) using 510 microscopic fi elds per electrode. A vertical stripe, 11 mm wide, was scanned in the middle of electrode. Microscopic fi elds were evenly situated over the scanning area, forming 15 vertical and 34 horisontal series. The spores were counted in every fi eld. The amount of spores on the scanning area N was computed as follows:

2 where Sscanning is the area of 34 x 11 mm scanned on the electrode, Smicrofi eld the area of the microscopic fi eld of 0.096 mm2, A the integer equal to the height of the electrode in units of millimetre, h the integer equal to the distance (in units of millimetre) from the electrode’s upper edge to the centre of the microscopic fi eld, and xh the mean number of spores per microscopic fi eld located at the distance h. The amount of the spores on a glass slide, in the middle transversal part being 11 mm wide, was found in a similar way.

The quotient of the spore charge q and the spore mass m could be found by the equation

where qm is the spore charge-to-mass quotient (q/m), g the acceleration of gravity, L the distance between the electrodes, U the difference of potential on the electrodes, and h the distance of the spore from the upper edge of the electrode (Swinbank et al. 1964, Webster et al. 1988).

In a spore sample, the mean values of spore charge-to-mass quotient were found out for both the spores with positive charges and the spores with 24 negative charges. We computed the mean value of the spore charge-to- mass quotient using the equation

where the marks are as above. The measurement error of the mean spore charge-to-mass quotient was evaluated as a sum of both the instrumental and the random errors (I).

Estimates of the mean spore charge values Q were based on the calculation Q = m × Qm , where the value of the mean spore charge-to- mass quotient Qm was identifi ed from the experiment and the spore mass m was found approximately. A spore mass was found by the dimensions of spores given in the literature (II, Table 4) and by the density of 103 kg m-3. To estimate the 95% confi dence interval of the mean value of the spore charge, limits were calculated using the lower and the upper limits of the 95% confi dence interval of the mean value of the spore charge- to-mass quotient. Infl uence of the assumptions made for the mass of spore was discussed (I).

Samples consisted mostly of 103-106 spores. If a spore deposit on an electrode was too dense for the spore counting, only the charge type of spore sample was identifi ed. Data from samples consisting of less than about 500 spores were not included, because then the random error was not negligible in the measurement error of the mean value of spore charge-to-mass quotient. (I).

Samples were collected in 1972-1974, 1975, 1978, 1980 and 1986 from fungi growing in Estonia, Azerbaijan and Adygea (Russia) (III, Table 1). Spatially and temporally the most-studied species were Fomes fomentarius (L.) J. Kickx f., Fomitopsis pinicola (Sw.) P. Karst. and Ganoderma applanatum (Pers.) Pat. The quantity of spore samples and basidiomes examined was the greatest in F. pinicola, followed by F. fomentarius, G. applanatum, and Lactarius turpis (Weinm.) Fr. The largest number of genera and families examined was in the order Agaricales. In 51 basidiom, covering 21 species, spore samples were collected repeatedly (III, Table 2). In several species,

25 spore samples were collected in different seasonal (vernal, aestival and autumnal), circadian (nocturnal, morning, diurnal and evening) periods and in different air conditions (III, Fig. 1a, 1b, 1c), and from basidiomata growing on different tree substrata.

To examine the infl uence of physiological conditions of basidiomata on the charge type of spore population, in some species spore samples were collected from turgescent and non-turgescent (ceasing, drying, collapsing) basidiomata (III, Table 3). When exploring the phylogenetic signal in the charge types of hymenomycetous basidiospores, one of the most important steps in experiment was a choice of appropriate species. These species should represent both different lineages and closely related groups of species, genera, and families in the Agaricomycetes (I, III).

3.2. Hymenial emission rate, territorial emission rate

Hymenial emission rates of charged spores were calculated using the spore amounts on electrodes, the area of the chamber ceiling slot and the sampling durations. In bipolar samples, the hymenial emission rates were measured separately for spores with positive charges and spores with negative charges.

To estimate the upper limit of the hymenial emission rate for spores and their charges in species with poroid hymenophore, we used the data for spore production in Fomes fomentarius and Phellinus alni (Bondartsev) Parmasto (Parmasto 1981). These polypores with narrow pores emitted spores at the maximum rate of 914 and 1,100 spores cm-2 s-1, respectively. In basidiomata with poroid hymenophore with narrow pores, the ratio area of hymenium : area of basidioma undersurface is 3-fold greater than in basidiomata with lamellate hymenophore, and 45-fold greater than in basidiomata with fl at, unstructured hymenophore according to the data of Fischer and Money (2010). Using these ratioships, we calculated the upper limit of the hymenial emission rate of spores as 305-367 spores cm-2 s-1 in the group of basidiomata with lamellate hymenophore, and 20-24 spores cm-2 s-1 in the group of basidiomata with fl at hymenophore. (II).

To estimate the upper limit of the territorial emission rate of spores in a particular species in a particular type of forest, we had, fi rst of

26 all, to calculate the total hymenial area (i.e., the total amount of all hymenophore undersurfaces) per hectare of territory. To calculate this total area, the basidioma number per hectare was multiplied by the mean area of the hymenophore surface. In Phellinus tremulae (Bondartsev) Bondartsev & P.N. Borisov, the total hymenial area per hectare of territory was calculated using literature data on fruitbody numbers per tree (Tamm 2000) and tree numbers per hectare (Krigul 1969), and expert data on the mean area of hymenophore surface (Ülo Tamm, personal communication). In Paxillus involutus (Batsch) Fr. and Cantharellus cibarius Fr., this total hymenial area was found using fruitbody numbers per plot (Lõugas 1980) and the mean areas of hymenophore surface calculated by the dimensions of fruitbodies given in the literature (II). Then the upper limit of the territorial emission rate of spores was found by multiplication of the total hymenial area per hectare by the upper limit of hymenial emission rate of spores.

We used the upper limit of the territorial emission rate of spores also for the charged spores. We proposed that all spores or most of spores are charged in a population of freshly released spores.

To estimate the upper limit of the hymenial emission rate of charges, the upper limit of the territorial emission rate of spores was multiplied by the mean spore charge.

3.3. Data analysis

Testing the difference between the means of samples. To answer the question whether two spore samples differed from each other in terms of mean spore charge-to-mass quotient or by mean charge, a two independent samples t-test for samples of unequal sizes and unequal variances (Howell 2010) was used (I). The degree of freedom in t test was taken equal to the infi nity, because more than 100 spores were involved in the calculation. At the 0.05 level of signifi cance for a two tailed test, the critical value of t was equal to 1.96. If t > 1.96, then a difference between the means was concluded.

Estimating the variation of the means of samples in a set. For a small sized dataset, standard deviation and coeffi cient of variation were calculated by the “unbiased” method (Haldane 1955). Most intraspecimen

27 (i.e., originating from a particular basidiom a) and intraspecifi c (i.e., belonging to a particular species) datasets of the mean spore charge- to-mass quotients and of the mean charges consisted of fewer than ten entities.

Testing the correlation. The Pearson’s correlation test (Howell 2010) was used to identify whether the magnitude of the spore charge-to- mass quotient and the charge magnitude correlated with the hymenial emission rate or with the spore volume. The correlation coeffi cient, its signifi cance value and 95% confi dence level were calculated for each pair of characteristics.

Testing the synchrony of hymenial emission rates between basidiomata. To identify, whether emission rates of charged spores performed by basidiomata were synchronised with each other, repeated spore collection was undertaken from a group of basidiomata in two species (Fomes fomentarius, Ganoderma applanatum). Spearman’s rank-order correlation test (Howell 2010) was used to analyse the data and the Spearman’s rank-order correlation coeffi cient and its signifi cance value were calculated for all pairs of basidiomata in the group.

Testing the difference in hymenial emission rates between basidiomata and between time periods. In Fomes fomentarius and Ganoderma applanatum for sets of simultaneously repeated samples covering several periods and several basidiomata, the Friedman rank test (Howell 2010) was used to estimate differences between basidiomata (in the periods-mean rate of hymenial emission) and between periods (in the basidiomata -mean rate of hymenial emission). The Friedman rank test statistic chi-square and its signifi cance value were calculated for periods and basidiomata. In Fomitopsis pinicola for sets of simultaneously collected samples covering several periods and groups of different basidiomata, a matched t-test (Howell 2010) was used to test whether periods differed by the basidiomata-mean rate of hymenial emission. Test characteristic t and its signifi cance value were calculated for all pairs of periods.

Searching for the phylogenetic signal in charge types of hymenomycetous basidiospores. Earlier, in III, the Hibbett’s phylogenetic overview of the Agaricomycotina (Hibbett 2007b) and the

28 phylogenetic overview of the Agaricales given by Matheney et al. (2007) were used to interpret phylogenetic relationships between the species. In this thesis, we used the classifi cations given in the Index Fungorum database (www.indexfungorum.org, 16 June 2015).

3.4. Relaxation time, washout of spores

We developed a theoretical model for the relaxation of charges of airborne particles and made an estimation using data on air ions in a rural environment in Estonia (II).

The washout is a process based on the collection of spores by water droplets falling through spore-loaded air. The effect of electric charges on collisions between water droplets and basidiospores was estimated based on the study of Tripathi and Harrison (2002). Their study considered aerosol particles with diameters, densities and charges representing basidiospores and their primary charges. We evaluated the effect of charges for basidiospores of Phellinus tremulae and P. nigricans. The spores are subglobose, smooth, with a density of 1 g cm-3 or a bit more (1.2 g cm-3). In P. tremulae, the mean equivalent diameter is 4.4 µm (99.7% of the variation of mean spore sizes is within the range of 3.9-4.7 µm) and in P. nigricans, 6.1 µm (5.6-6.5 µm) (Sell 2008). The mean primary electric charge is 117 e (46-222 e) in P. tremulae, and 204 e (107-496 e) in P. nigricans (I). These spores represent the smallest and the biggest spores in the P. igniarius group, one of the most important wood-rotting fungi of many deciduous trees.

29 4. RESULTS

4.1. Charge type of a spore sample

Distribution of charges according to the polarity was measured in 235 spore samples of 135 basidiomata from 50 species, 38 genera, 23 families, and eight orders of Agaricomycetes (Tables 3 and 5 in III).

Three charge types occurred: unipolar-positive, unipolar-negative, and bipolar. The results in turgescent basidiomata (Table 5 in III) contrasted to the results in the basidiomata, which were collapsing or drying up or ceasing to sporulate in the collecting day or the day after (non-turgescent basidiomata, Table 3 in III). The non-turgescent basidiomata showed different charge types in the intraspecimen spore samples or the absence of charges in a spore sample. The turgescent basidiomata showed that the charged spores occurred in all spore samples, and the charge type of spore samples was the same within both a basidioma and a species.

Table 3. The polarity of primary charges of basidiospores in hymenomycetes. Position of the species in classifi cation accords with the classifi cations given in the Index Fungorum database (www.indexfungorum.org, 16 June 2015). The numbers of turgescent basidiomata and spore samples studied in the doctoral research are given in the brackets. Samples consisted of 103-106 spores.

CANTHARELLALES CANTHARELLACEAE Cantharellus cibarius Fr. – negative charges only (1 basidioma and 1 sample studied) HYMENOCHAETALES HYMENOCHAETACEAE s.l. Coltricia perennis (L.) Murrill – positive charges only (1, 1) Mensularia radiata (Sowerby) Lázaro Ibiza – positive charges only (1, 1) Phellinus alni (Bondartsev) Parmasto – positive charges only (2, 4) P. nigricans (Fr.) P. Karst. – positive charges only (1, 1) P. populicola Niemelä – positive charges only (4, 18) P. tremulae (Bondartsev) Bondartsev & P.N. Borisov – positive charges only (6, 10) Porodaedalea pini (Brot.) Murrill – positive charges only (1, 2) THELEPHORALES BANKERACEAE Sarcodon imbricatus (K.) P. Karst. – negative charges only (1, 1) THELEPHORACEAE Thelephora terrestris Ehrh. – negative charges only (1, 1) POLYPORALES FOMITOPSIDACEAE Climacocystis borealis (Fr.) Kotl. & Pouzar – positive charges only (1, 2) Fomitopsis pinicola (Sw.) P. Karst. – negative charges only (40, 60) F. rosea (Alb. & Schwein.) P. Karst. – negative charges only (4, 7) Piptoporus betulinus (Bull.) P. Karst. – negative charges only (2, 2) Postia leucomallella (Murrill) Jülich – positive charges only (1, 1)

30 MERULIACEAE Junghuhnia nitida (Pers.) Ryvarden – bipolar, negative charges more numerous (1, 1) POLYPORACEAE Fomes fomentarius (L.) J. Kickx f. – negative charges only (9, 34) Polyporus squamosus (Huds.) Fr. – bipolar, negative charges more numerous (1,1) Royoporus badius (Pers.) A.B. De [syn. Polyporus badius (Pers.) Schwein.] – bipolar, negative charges more numerous (1, 1) GANODERMATACEAE Ganoderma applanatum (Pers.) Pat. – positive charges only (6, 18) G. lucidum (Curtis) P. Karst. – positive charges only (1, 1) CORTICIALES CORTICIACEAE Dendrothele griseocana Bres.) Bourdot & Galzin – bipolar, negative charges more numerous (1, 1) RUSSULALES RUSSULACEAE Lactarius helvus (Fr.) Fr. – negative charges only (1, 1) L. torminosus (Schaeff.) Gray – negative charges only (1, 1) L. trivialis (Fr.) Fr. – negative charges only (1, 2) L. turpis (Weinm.) Fr. – negative charges only (9, 12) Russula vinosa Lindblad – negative charges only (1, 1) STEREACEAE Gloeocystidiellum leucoxanthum (Bres.) Boidin [syn. Megalocystidium leucoxanthum (Bres.) Jülich] – negative charges only (1, 1) Stereum rugosum Pers. – bipolar, negative charges more numerous (1, 1) PENIOPHORACEAE Gloiothele citrina (Pers.) Ginns & G.W. Freeman [syn. Vesiculomyces citrinus (Pers.) E. Hagstr.] – bipolar, negative charges more numerous (1, 2) AGARICALES AMANITACEAE Amanita muscaria (L.) Lam. – negative charges only (1, 1) A. vaginata (Bull.) Lam. – negative charges only (1, 1) PLEUROTACEAE Pleurotus ostreatus (Jacq.) P. Kumm. – bipolar, positive charges more numerous (2, 2) PHYSALACRIACEAE Cylindrobasidium evolvens (Fr.) Jülich – negative charges only (1, 1) Flammulina velutipes (Curtis) Singer – negative charges only (1, 1) TRICHOLOMATACEAE Clitocybe sp. – negative charges only (2, 2) Lepista nuda (Bull.) Cooke – negative charges only (1, 1) Tricholoma equestre (L.) P. Kumm. – bipolar, negative charges more numerous (1, 1) T. imbricatum (Fr.) P. Kumm. – bipolar, negative charges more numerous (1, 2) CORTINARIACEAE Cortinarius sp. – bipolar, negative charges more numerous (2, 3) STROPHARIACEAE Pholiota sp. – bipolar, positive charges more numerous (1, 2) Stropharia hornemannii (Fr.) S. Lundell & Nannf. – bipolar, negative charges more numerous (2, 3) BOLETALES CONIOPHORACEAE Coniophora puteana (Schumach.) P. Karst. – negative charges only (1, 2) SERPULACEAE Serpula lacrymans (Wulfen) J. Schröt.– negative charges only (2, 2) BOLETACEAE Xerocomus sp. – bipolar, positive charges more numerous (1, 1) SUILLACEAE Suillus luteus (L.) Roussel – bipolar, positive charges more numerous (1, 1) PAXILLACEAE Paxillus involutus (Batsch) Fr. – bipolar, positive charges more numerous (4, 7)

31 The following are the results shown by the spore samples from the turgescent basidiomata from 47 species, 37 genera, 23 families, and eight orders (Table 3, classifi cation of the Agaricomycetes is modifi ed according to the latest phylogeny researches). Of the six genera with two or more species studied, the all had the same charge type in species of the same genus. Unipolar-positive distribution of spore charges occurred in Phellinus Quél. and Ganoderma P. Karst. Unipolar-negative distribution occurred in Fomitopsis P. Karst., Lactarius Pers. and Amanita Dill. ex Boehm. Bipolar distribution (in which the negative charges are more numerous) occurred in Tricholoma (Fr.) Staude. Of 37 genera studied, seven had unipolar-positive, 16 had unipolar-negative, and 14 genera had bipolar distribution of spore charges.

Of the eight families with two or more genera studied, four had the same charge type in genera and species of the same family (Table 3). Unipolar- positive distribution of spore charges occurred in Hymenochaetaceae (4 genera, 7 species). Unipolar-negative distribution of spore charges occurred in Physalacriaceae (2 / 2) and Russulaceae (2 / 5). Bipolar distribution of spore charges occurred in Strophariaceae (2 genera, 2 species). The families with different charge types in genera of the same family are as follows: Fomitopsidaceae, Polyporaceae, Stereaceae and Tricholomataceae. Three families were of the related type: they had spores with either negative charges only, or bipolar, with more numerous negative charges—Polyporaceae (3 genera, 3 species), Stereaceae (2 genera, 2 species), and Tricholomataceae (3 genera, 4 species). In Fomitopsidaceae (4 genera, 5 species), opposite types of spore charges, unipolar-positive and unipolar-negative, occurred.

Of the fi ve orders with two or more families, one had the same charge type in genera and species of the same order, while four orders had different charged types (Table 3).

4.2. Spore charge-to-mass quotient and spore charge

Mean absolute value of spore charge-to-mass quotient was measured in 128 spore samples of 81 basidiomata, covering 31 species, 26 genera, 19 families, and eight orders (Agaricales, Boletales, Cantharellales, Corticiales, Hymenochaetales, Polyporales, Russulales, and Thelephorales) (II, Table 1). These values varied from 0.74 × 10-4 C kg-1 to 7.32 × 10-4 C kg-1,

32 while the mean absolute values of spore charges varied in the interval of 21-981 e (II, Table 2). Correlation between the spore charge-to-mass quotient and the spore volume was absent (r=-0.07; set of 49 pairs, df=47, p=0.63), but it was signifi cant [r=0.50 (0.26-0.69); set of 49 pairs, df=47, p=0.0002] between the spore charge and the spore volume (II).

4.3. Intraspecimen and intraspecifi c variabilities of spore charge- to-mass quotient

The spore charge-to-mass quotient in the spore samples of Phellinus populicola showed as follows. The mean value of the spore charge- to-mass quotient of a sample of spores varied between samples, its variation coeffi cient was 35% within the species (I, Table 3). The range of intraspecimen variation coincided with the range of intraspecifi c variation (I, Figure 3). Both insignifi cant and signifi cant differences occurred between the intraspecimen samples as well as between the interspecimen samples; the t test for samples of unequal sizes and variances used for all pairs of samples indicated this (I, Table 4). The intraspecifi c variability of the spore charge-to-mass quotient and the spore charge could depend on the natural variability of the spore size (I).

4.4. Hymenial emission rate of charged spores

Hymenial emission rates of charged spores from the 81 basidiomata were in the range of orders of magnitude of 0.1-100 charged spores cm-2 s-1 (data of 128 spore samples; Fig 1a, 1b, 1c and Table 3 in II). The maximum was 343 spores cm-2 s-1 for positive spores and 715 spores cm-2 s-1 for negative spores. The spore samples of Fomes fomentarius, Fomitopsis pinicola, Ganoderma applanatum and Lactarius turpis showed that variability of spore charge-to-mass quotient and spore charges was 4-13 times lower than that of the hymenial emission rates. We did not observe correlation between the hymenial emission rate and the spore charge-to- mass quotient and spore charge. (II).

The series of 4-8 repeated sampling in F. fomentarius and G. applanatum (II, Fig 1a, 1b) showed the following. i) The emission rate was fl uctuating. We observed up to a 3-fold increase and a 35-fold decrease per 80 minutes. ii) In the simultaneously sporulating basiodiomata, the fl uctuation ranges

33 were unequal and the dynamics of hymenial emission rate was mostly asynchronous. Depending on the basidioma, the fl uctuation range extended over three or two orders of magnitude, or remained within one order of magnitude. (II).

4.5. Upper limit of the territorial emission rate of spores and their charges

Total hymenial area per hectare of territory in Phellinus tremulae in aspen stands of 75-year in Estonia was about 10 m2 ha -1. In a year of a quite poor mushroom yield, at the seasonal peak time of fruitbody production—in Paxillus involutus in the paludifi ed Calluna pine-birch forest in Lahemaa (North-Estonia), it was 2 m2 ha-1 and, in Cantharellus cibarius in the lichen pine-birch forest in the same region, it was 5 m2 ha-1. The hypothetical upper limit of the hymenial emission rate of spores was 305-367 spores cm-2 s-1 calculated in basidiomata with lamellate hymenophore, and 914-1100 spores cm-2 s-1 in basidiomata with poroid hymenophore. We used these values for the upper limits of the hymenial rate of charged spores. So, in the case of these forests and these fungal species, the upper limits of the territorial emission rate of charged spores should be the following: about 108 positive spores s-1 ha-1 in P. tremulae in old aspen stands, 105 positive and 105 negative spores s-1 ha-1 in P. involutus in the paludifi ed Calluna pine-birch forest, and 107 negative spores s-1 ha-1 in C. cibarius in the lichen pine-birch forest. Corresponding amounts of charges should be as follows: 1010 e ha-1 s-1 for P. tremulae, 108 e and -108 e s-1 ha-1 for P. involutus, and -109 e s-1 ha-1 for C. cibarius. (II).

4.6. Charge relaxation time

An equation was deduced for the rate of relaxation of the charge on an aerosol particle. This equation showed that the rate of charge relaxation does not depend on the size and charge of particles. Calculations showed that the relaxation time of basidiospore primary charges in real atmospheric conditions near the ground level is 1430 s. Thus the initial charge number diminishes about 2.7 times during this period. (II).

34 4.7. Role of charges in washout of spores

The effect of electric charges on collisions between water droplets and basidiospores was estimated based on the study of Tripathi and Harrison (2002). The collision effi ciency between droplets and aerosol particles 2 2 has been defi ned as E = rc /( A+a) where rc is the initial horizontal displacement of the particle from the central axis of the falling drop for the particle critical trajectory; A is the radius of a droplet and a is the radius of an aerosol particle. In the study, the trajectories of particles of density 1 and 2 g cm-3 were obtained by numerically integrating the electric image charge effects in the reference frame of the drop with diameter 84 µm. Then the collision effi ciency was calculated from the trajectory (Figure 7). The results for particles with a diameter between 4.0-5.6 µm showed that, in the case of neutral particles of density 1 g cm-3, the collision effi ciency is very low. In the case of charged particles with charges greater than 50 e, the collision effi ciency is enhanced.

Figure 7. Collision effi ciencies E plotted as function of radius for aerosol particles carrying charges, 5 e to 500 e, and having density of 2 g cm-3 (a) and 1 g cm-3 (b). The droplet is constant at radius of 42 µm and is electrically neutral. The charged collision effi ciency is independent from the charge carried by the water drop. (Figure 3 from Tripathi & Harrison 2002, modifi ed).

Approximately a twofold increase occurs in collision effi ciency between the drops with diameter 84 µm and the particles with diameter 4-5 µm,

35 when electrically neutral particles are replaced by the charged particles with 100 e per particle (Table 4). Electrical effects on collision effi ciency diminish as the particle size increases. So for a particle with diameter 8 µm or larger and with 100 elementary charges, electrical effects are negligible.

Table 4. Quotient of the collision effi ciency E for charged particles and the collision effi ciency for neutral particles in the case of a water droplet of diameter 84 µm and particles of density 1 g cm-3. (Our calculations. For E, we used values found by Tripathi and Harrison (2002), given here on the Figure 1.)

Particle Particle diameter (µm) charge (e) 4568 10 1 1 1 1 20 1 1 1 1 50 1.4 1.2 1 1 100 2.4 1.5 1.1 1 500 28.6 11.5 5.0 1.1

This shows, that in both P. nigricans and P. tremulae the primary charge enhances collision effi ciency between basidiospores and droplets of diameter 84 µm.

36 5. DISCUSSION

5.1. Factors infl uencing the charge type of a spore population

The results showed that the charge type of freshly released hymenomycetous basidiospores depends on physiological factors. The signifi cance of physiological factors was only observable when comparing the turgescent state with the senescent state of basidiomata or with the deteriorative state (occurring due to the destructive infl uence of some factor) of the mature but not yet of non-old basidiomata. In basidiomata experiencing fatal physiological changes, the charge type varies or non-charged spores are released. In turgescent basidiomata, the charge type remains constant. In a turgescent basidioma, the charge type does not depend on different seasons, different days, different times of day, and on various weather conditions.

The results showed that the charge type is the same in turgescent basidiomata of the same species. In a species, the charge type of spore populations released from turgescent basidiomata does not depend on different years, different geographic places, and on different substrata.

The results showed that the charge type of the spore populations from turgescent basidiomata in a species has a strong phylogenetic signal. It is possible that all taxa from species to monophyletic families are characterized by a one charge type. These different types are obviously correlated with currently unknown phylogenetically differentiated types of bioelectric activity in spore development, which were determined in the ancestor of each of these groups and remained constant during following evolution and radiation of the group.

Existence of species with bipolar charges indicates that the primary electrostatic charge of basidiospores originates from two or more processes (and not from one process) occurring in the fungus. Processes responsible for the formation of the spore primary charge are unknown. These processes could occur in different stages of the development of basidium and spores and in the spore jump off. The most probable stages are the following: the formation of the sterigmata initials and their development, the formation of the Buller’s drop initials and their development between the spore wall layers, the rapid expansion of

37 Buller’s drop, the formation of initials of the adaxial drop and their development on the spore wall, and mechanical separation of the spore from the sterigma.

The situation in pollen grains differs from that in basidiospores. A study by Bowker & Crenshow 2007 on the electrostatic charges on pollen grains immediately upon their release in seven species of wind-pollinated plants shows that each species had a bipolar distribution of pollen charges, often roughly centered around zero. Two possible mechanisms have been postulated for creating the charges on the pollen grains: in the fi rst, pollen acquires a charge by triboelectric charging when separating from the anther and other pollen contained within the anther. While in the second a negative charge is induced on the plant by the fair weather electric fi eld which is then transferred to the pollen. The actual charging mechanism remains unclear.

5.2. Mean absolute value of the spore charge-to-mass quotient and the spore charge, their variability

Our results in the spore charge-to-mass quotients and in the spore charges accord with previous measurements made in basidiospores (Swinbank et al. 1964, Webster et al. 1988). The ranges of absolute values of these two characteristics found earlier are wider and include our results. The primary charges of hymenomycetous basidiospores are far smaller than that of pollen grains of anemophilous plants. In pollen grains of seven plant species, the average of the absolute value of all charges was 5000 e (originally 0.84 fC in Bowker & Crenshow 2007), which is 5-fold greater than our maximum result [the mean charge of 981e (913-1049 e) in Fomes fomentarius] and about 100-fold greater than our minimum result (Junghuhnia nitida) and the near-minimum results (Piptoporus betulinus, Polyporus badius, Suillus luteus, Vesiculomyces citrinum, Table 2 in II).

Factors responsible for the magnitude of charge-to-mass quotient and spore charge are not known (Webster et al. 1988). Our results showed that the intraspecimen and intraspecifi c variability of the mean spore charge-to-mass quotient and the mean spore charge could depend on the natural variability of the mean spore size (I), and they do not depend on the sporulation intensity of the basidioma (II). The interspecifi c variability of the mean spore charge could partially (25%) be explained

38 by spore size (volume) or by some other factor, which correlates with it (II). The greater charges tend to be on larger spores, and the smaller charges – on smaller spores (II). Correlation between the mean spore charge-to-mass quotient and the mean spore volume was absent (II).

5.3. Hymenial emission rate of charged spores, upper limit of the territorial emission rate of charged spores and charges

The upper limits of hymenial emission rates of spores and their charges were calculated on the assumption that, in all species, the spore production potential of the hymenium is similar to that in Fomes fomentarius and Phellinus alni. Consequently, and because of other simplifi cations (II), our estimates should be considered as very broad. With reservation they may, however, be applicable in studies of the contribution of basidiospores and their primary electric charges to the physicochemical processes in the atmosphere.

In three case studies of a dominant species in the local fungal community, the upper limits of territorial emission rates of charged spores were in the range of 106-108 spore s-1 ha-1 and of their charges in the range of 108-1010 e s-1 ha-1.

For a whole fungal community, the upper limits of territorial emission rates of spores and their charges could be found by summing the upper limits of dominant species. Composition of dominant species and amount of basidiomata depend on the type of the forest and on the year (Krastina 2000). In some types of forest, there are tens of dominant hymenomycetous species.

Results of repeated spore samplings in Fomes fomentarius and Ganoderma applanatum showed that a short-term (tens of minutes) mean of the hymenial emission rate of charged spores fl uctuated across large ranges, and that synchrony in the hymenial emission rate was absent between basidiomata. These results were in agreement with earlier observations (Haard and Kramer 1970, Rocket and Kramer 1974, Votintseva 2005, Kadowaki et al. 2010). (II).

Basidiomata release spores in a fl uctuating mode and independently from each other. Hymenial emission rates vary greatly throughout the

39 day. It occasionally happens, that most of the basidiomata within a neighbourhood release spores at the maximum intensity or near it (II). So our results indicated that peaks approaching to the upper limit occur infrequently and irregularly in the circadian dynamics of the territorial emission rates of spores and their charges.

5.4. Relaxation time of spore charges in the atmosphere

Relaxation time found in our study was about threefold higher than that given in Bowker & Crenshaw 2003. The origin of such a short period, 440 s, was due to an overestimation of the value of air conductivity. In addition, they considered total conductivity instead of polar conductivity. (II).

The relaxation time of 1430 s means that highly charged spores can spread over a considerable area depending on the wind velocity, for example, over 3-4 km by the wind of 2-3 m s-1. By upward moving air fl ows occurring above forests during the fungal sporulation seasons, the highly charged spores can reach the atmospheric layers where aerosol particles acts as clod droplet nuclei or ice nuclei.

5.5. Role of charges in washout of spores from the atmosphere

The mechanics of collision and coalescence of falling water drops with aerosol particles of the surrounding air are largely governed by aerodynamic and viscous forces, and electrical charges could play a signifi cant role. Role of electrical charges became insignifi cant and inertial effects become dominant for larger particles interacting with larger droplets (Fletcer 2013). For the particles of density 2 g cm-3, role of electrical charges is insignifi cant when diameters of droplets and particles exceed values of 120 μm and 6 μm, respectively (Tinsley et al. 2001). For the particle of density 1 g cm-3, having diameter 8 µm or larger and 100 elementary charges, electrical effects are negligible in interaction with droplet of diameter 84 μm (Tripathi & Harrison 2002). These data show that primary charges of spores do not play a role in washout of basidiospores of any dimensions by rain. Primary charges do have role in washout of basidiopores of diameter 4-5 µm or less by droplets of diameter 80 or less. Thus the primary charges enhance

40 the deposition of freshly emitted basidiospores in species with small basidiospores by fog, cloud and drizzle. So in areas with frequent fogs or drizzles and in mountain cloud-forests, the primary charges enhance, in comparison with electrically neutral spores, the spore deposition in species with small basidiospores.

41 6. CONCLUSIONS

Conclusion 1. Freshly released hymenomycetous basidiospores are electrically charged. In one spore population spores are either negatively or positively charged or of both types. The charge type of spores depends on physiological factors. Physiological changes occurring in a collapsing or ceasing to sporulate or drying up basidioma have an effect on spore charges, but not the physiological changes occurring in a turgescent basidioma. In turgescent basidiomata, within each basidioma and fungal species charge type is invariable. It is likely that spore charge is a phylogenetically conserved feature, with only one type occurring in each monophyletic group, corresponding to currently recognised taxa at the species, genus and family level. The mean absolute value of spore charge-to-mass quotient is in the range of (0.7-7) × 10-4 C kg-1. The mean absolute value of spore charge is in the range of 20-1000 e. The range of intraspecifi c variability of both these variables is of the same magnitude with the range of intraspecimen variability. The intraspecifi c variability of the mean spore charge-to-mass quotient and the mean spore charge might depend on the natural variation of the mean spore size. The intraspecifi c variability of these characteristics does not depend on the sporulation intensity of the basidioma. We did not detect a correlation between the mean spore charge-to-mass quotient and the mean spore volume.

Conclusion 2. The initial primary charge on an airborne basidiospore diminishes about 2.7 times during a time interval of 23 min and 7.3 times during 46 min. The rate of charge relaxation does not depend on the size and charge of the spores.

Conclusion 3. In hymenomycetous species with small basidiospores (diameter of 4-5 µm or less) and primary charges of 50 e or more, the freshly released spores are washed out from the air by fogs, clouds and drizzles better than the spores remaining airborne for hours. In areas with frequent fogs or drizzles and in mountain cloud-forests, the primary charges enhance the spore deposition in species with small basidiospores.

Conclusion 4. In old pure aspen stands, the upper limits of the territorial emission rate of charged spores and charges are about 108 positive spores and 1010 e ha-1 s-1 for Phellinus tremulae, respectively. In

42 a lichen pine-birch forest in a poor year for mushroom yields, these upper limits for Cantharellus cibarius are 107 negative spores and -109 e s-1 ha-1, respectively. In a paludifi ed Calluna pine-birch forest in a poor year for mushroom yields, these upper limits for Paxillus involutus are 105 positive and 105 negative spores, and 108 e and -108 e s-1 ha-1, respectively. Approaches to the upper limit of the territorial emission rates of spores and their charges occur infrequently and irregularly.

43 7. REFERENCES

Aylor D E, Paw U K T. 1980. The role of electrostatics in spore liberation by Drechslera turcica. Mycologia, 72 (6), 1213-1219. Bowker G E, Crenshaw H C. 2003. The infl uence of fair weather electricity on the charging of wind-dispersed pollen. Proc. of 12th International Conference on Atmospheric Electricity, 9-13 June 2003, Versailles, France, Vol. I, 361-364. Bowker G E, Crenshaw H C. 2007. Electrostatic forces in wind- pollination—Part 1: Measurement of the electrostatic charge on pollen. Atmospheric Environment, 41 (8), 1587-1595. Buller A H R. 1909. Researches on fungi, I, An account of the production, liberation, and dispersion of the spores of hymenomycetes treated botanically and physically, Also some observations upon the discharge and dispersion of the spores of ascomycetes and of Pilobolus. Longmans, Green & Co., London. Buller A H R. 1922. Researches on fungi, II, Further investigations upon the production and liberation of spores in hymenomycetes. Longmans, Green & Co., London. Clémençon H. 2012. Cytology and plectology of the Hymenomycetes. 2nd revised ed., J. Cramer, Stuttgart. Deguillaume L, Leriche M, Amato P, Ariya P A, Delort A N, Pöschl U, Chaumerliac N, Bauer H, Flossmann A I, Morris C E. 2008. Microbiology and atmospheric processes: chemical interactions of primary biological aerosols. Biogeosciences, 5, 1073-1084. Després V R , Huffman J A, Burrows S M, Hoose C, Safatov A S, Buryak G, Fröhlich-Nowoisky J, Elbert W, Andreae M O, Pöschl U, Jaenicke R. 2012. Primary biological aerosol particles in the atmosphere: a review. Tellus B 2012, 64, 015598, DOI: 10.3402/tellusb.v64i0.15598, 58 p. Eesti seenestik. Mycobiota of Estonia. 2000. [Elektrooniline teavik CD- ROM]. Toimetaja K. Kalamees. Eesti Põllumajandusülikooli Zooloogia ja Botaanika Instituut, Tartu. Elbert W, Taylor P E, Andreae M O, Pöschl U. 2007. Contribution of fungi to primary biogenic aerosols in the atmosphere: wet and dry discharged spores, carbohydrates and inorganic ions. Atmospheric Chemistry and Physics, 7 (17), 4569-4588.

44 Finnish Pollen Bulletin 1993. University of Turku, Turku, Finland. Finnish Pollen Bulletin 1994. University of Turku, Turku, Finland. Finnish Pollen Bulletin 1995. University of Turku, Turku, Finland. Finnish Pollen Bulletin 1996. University of Turku, Turku, Finland. Finnish Pollen Bulletin 1998. University of Turku, Turku, Finland. Fischer M W F, Money N P. 2010. Why mushrooms form gills: effi ciency of the lamellate morphology. Fungal Biology, 114 (1), 57-63. Fletcher N H. 2013. Effect of electric charge on collisions between cloud droplets. Journal of Applied Meteorology and Climatology, 52 (2), 517–520. Fröhlich-Nowoisky J, Burrows S M, Xie Z, Engling G, Solomon P A, Fraser M P, Mayol-Bracero O L, Artaxo P, Begerow D, Conrad R, Andreae M O, Després V R, Pöschl U. 2012. Biogeography in the air: fungal diversity over land and oceans. Biogeosciences, 9, 1125-1136, doi:10.5194/bg-9-1125-2012. Garrett R M. 1972. Electrostatic charges on freshly discharged lichen ascospores. Lichenologist, 5, 311-313. Gregory P H. 1957. Electrostatic charges on spores of fungi. Nature, 130, 330-330. Gregory P H. 1973. Microbiology of the atmosphere, 2nd ed., John Wiley & Sons, New York–Toronto. Gregory P H. 1979. Speculations on basidiospore liberation. Bulletin of the British Mycological Society, 13 (2), 128-131. Haard R T, Kramer C L. 1970. Periocity of spore discharge in the Hymenomycetes. Mycologia 62 (6), 1145-1169. Haga D I, Burrows S M, Iannone R, Wheeler M J, Mason R H, Chen J, Polishchuk E A, Pöschl U, Bertram A K. 2014. Ice nucleation by fungal spores from the classes Agaricomycetes, Ustilaginomycetes, and Eurotiomycetes, and the effect on the atmospheric transport of these spores. Atmospheric Chemistry and Physics, 14, 8611-8630, doi:10.5194/acp-14-8611-2014. Haldane J B S. 1955. The measurement of variation. Evolution, 18, 484. Hawksworth D L, Rossman A Y. 1997. Where are all the undescribed fungi? Phytopathology, 87, 888-891. Heald C L, Spracklen D V. 2009. Atmospheric budget of primary biological aerosol particles from fungal spores. Geophysical Research Letters, Vol. 36, L09806, doi:10.1029/2009GL037493.

45 Hibbett D S. 2007a. Agaricomycotina. Jelly Fungi, Yeasts, and Mushrooms. Version 20 April 2007. http://tolweb.org/ Agaricomycotina/20531/2007.04.20 in The Tree of Life Web Project, http://tolweb.org/ Hibbett D S. 2007b. A phylogenetic overview of the Agaricomycotina. Mycologia, 98 (6), 917–925. Howell D C. 2010. Statistical methods for psychology, 7th ed., Wadsworth Cengage Learning, Belmont. Iannone R, Chernoff D I, Pringle A, Martin S T, Bertram A K. 2011. The ice nucleation ability of one of the most abundant types of fungal spores found in the atmosphere. Atmospheric Chemistry and Physics, 11 (3), 1191-1201, doi:10.5194/acp-11-1191-2011. Ingold C T. 1965. Spore Liberation. Oxford University Press. Ingold C T. 1971. Fungal spores: their liberation and dispersal. Clarendon Press, Oxford. Ingold C T. 2001. Range in size and form of basidiospores and ascospores. Mycologist, 15 (4), 165-166. Kadowaki K, Leschen R A B, Beggs J R. 2010. Periodicity of spore release from individual Ganoderma fruiting bodies in a natural forest. Australasian Mycologist, 29, 17-23. Krastina I. 2000. Species composition and dynamics of sporocarp production of macrofungi in pine forests with Va c c i n i u m m y r t il l u s in northern Latvia. Folia Cryptogamica Estonica, 37, 39–50. Krigul T. 1969. Forest surveyor’s reference book. Eesti Põllumajanduse Akadeemia, Tartu. (In Estonian.) Leach C M. 1976. An electrostatic theory to explain a violent spore liberation by Dreschlera turcica and other fungi. Mycologia, 68 (1), 63-86. Levetin E, Horner W E. 2002. Fungal aerobiology: exposure and measurement. In: Chemical Immnunology, vol. 81. Breitenbach M, Crameri R, Lehrer S B (eds.), Fungal allergy and pathogenecity, S. Karger AG, Basel (Switzerland), pp. 10-26. Levetin E. 1995. Fungi. In: Burge H A (ed), Bioaerosols. (Indoor air research series), Lewish Publishers, USA, pp. 87-120. Lõugas T. 1980. About the resources of edible fungi in some forest site types of Lahemaa National Park. In: Year-book of the Estonian Naturalists’ Society, vol. 68, Eesti Loodusuurijate Selts, Tartu, pp. 32-49. (In Estonian, with summary in English.) 46 McCartney H A, Bainbridge A, Legg B J, Jones C D. 1982. Electric charge and the deposition spores of barley mildew Erysiphe graminis. Atmospheric Environment, 16 (5), 1133-1143. Mäkinen Y, Rantio-Lehtimäki A. 1979. Diurnal variations of airborne fungal spores in Turku. Reports from the Aerobiology Laboratory, University of Turku, 1, Turku, Finland. Matheny P B, Curtis J M, Hofstetter V. et al. 2007. Major clades of Agaricales: a multilocus phylogenetic overview. Mycologia, 98 (6), 982–995. Money N P, Fischer M W F. 2009. Biomechanics of spore release in phytopathogens. In: Deising H. (ed.), Plant relationships, 2nd ed., The Mycota, V, Springer-Verlag, Berlin. Money N P. 1998. More g’s than the Space Shuttle: ballistospore discharge. Mycologia, 90 (4), 547-558. Parmasto E. 1981. Quantitative characteristics of sporulation in wood- destroying polyporous fungi. Vtoraja vsesojuznaja konferencija po biopovreždenij. Tezisy dokladov. Čast II (pp. 138-139). Gor’’kij (Russia). (In Russian.) Pringle A, Patek S N, Fisher M, Stolze J, Money N P. 2005. The captured launch of a ballistospore. Mycologia, 97 (4), 866-871. Rantio-Lehtimäki A, Mäkinen Y, Pohjala A. 1985. Seasonal variations in airborne fungus spore frequencies in Finland. Reports from the Aerobiology Laboratory, University of Turku, 7, Turun Yliopiston offsetpaino. Rockett T R, Kramer C L. 1974. Periodicity and total spore production by lignicolous basidiomycetes. Mycologia, 66 (5), 817–829. Roos R A. 1996. The forgotten pollution. Kluwer Academic Publishers, Dordrecht. Rubulis J. 1984. Airborne fungal spores in Stockholm and Eskilstuna, Central Sweden. In: Nilsson S, Rai B (eds.). Nordic aerobiology. [Proc.] 5th Nordic Symposium on Aerobiology, Abisko, 1983, Almqvist & Wiksell Int., pp. 85-93. Seinfeld J H, Pandis S N. 2006. Atmospheric chemistry and physics: from air pollution to climate change. 2nd ed. John Wiley & Sons, Hoboken (USA). Sell I. 2008. Taxonomy of the species in the Phellinus igniarius group. Mycotaxon, 104, 337–347.

47 Sesartic A, Dallafi or T N. 2011. Global fungal spore emissions, review and synthesis of literature data. Biogeosciences, 8 (5), 1181-1192. Sesartic A, Lohmann U, Storelvmo T. 2013. Modelling the impact of fungal spore ice nuclei on clouds and precipitation. Environmental Research Letters, 8 (1), 014029 (8 p.) Stolze-Rybczynski J L, Cui Y, Stevens M H H, Davis D J, Fischer M W F, Money N P. 2009. Adaptation of the spore discharge mechanism in the basidiomycota. Plos ONE, 2009, 4, e4163, doi:10.1371/journal. pone.0004163 Stolze-Rybczynski J L. 2009. Biomechanics of spore discharge in the Basidiomycota. PhD thesis, Miami University, USA. Swann E, Hibbett D S. 2007. Basidiomycota. The Club Fungi. Version 20 April 2007. http://tolweb.org/Basidiomycota/20520/2007.04.20 in The Tree of Life Web Project, http://tolweb.org/ (1 June 2015). Swinbank P, Taggart J, Hutchinson S A. 1964. The measurement of the electrostatic charges on spores of Merulius lacrymans (Wulf.) Fr. Annals of Botany, 28 (2), 239-249. Tamm Ü. 2000. Aspen in Estonia. Eesti Loodusfoto, Tartu. (In Estonian, with summary in English.) Tinsley B A, Rohrbaugs R P, Hei M. 2001. Electroscavenging in clouds with broad size distributions and weak electrifi cation. Atmospheric Research, 59-60, 115-135. The Index Fungorum database. www.indexfungorum.org (16 June 2015). Tripathi S N, Harrison R G. 2002. Enhancement of contact nucleation by scavening of charged aerosol particles. Atmospheric Research, 62, 57-70. Votintseva A A. 2005. Ecological characteristics and biotic linkages in mono- and dicaryotic polyporous fungi. 03.00.16 – Ecology. Dissertation for the Candidate degree in Biology. Scientifi c supervisor V. A. Muhin, PhD. The Ural State University named after A. M. Gorky, Ekaterinburg, Russia. Manuscript. UDK 574:581.524.1:582.284.5 (In Russian.) Webster J, Procton M C F, Davey R A, Duller D A. 1988. Measurement of the electrical charge on some basidiospores and an assessment of two possible mechanisms of ballistospore propulsion. Transactions of the British Mycological Society, 91, 193–203.

48 Winiwarter W, Bauer H, Caseiro A, Puxbaum H. 2009. Quantifying emissions of primary biological aerosol particle mass in Europe. Atmospheric Environment, 43 (7), 1403-1409.

49 8. GLOSSARY anamorph – an asexual reproductive stage of the life cycles of fungi ballistospore – a spore placed asymmetrically on a sterigma and discharged violently immediately after drop-production at the hilum appendix basidioma (pl. basidiomata) – a fruitbody formed in sexual reproduction in basidiomycetes basidium (pl. basidia) – a cell for sexual reproduction in basidiomycetes C – coulomb. The SI unit of electric charge, equal to the quantity of electricity conveyed in one second by a current of one ampere charge relaxation time – the time required for a highly charged spore to return 0.368 of its initial charge during its exponential charge decay e – elementary charge. The equivalent of the electric charge carried by a single proton. 1 e = 1.6 ×10-19 C hilum appendix – a minute projection on the spore, very near to the junction of the sterigma and the spore hymenial emission rate of spores – a number of spores released per unit time per unit area of hymenophore surface. Here [spores s-1 cm-2]. hymenium – fertile surface of a hymenophore forming spores. In basidiomycetes, the hymenial layer is composed of basidia and sterile cells. hymenophore – the particular part on ascomata or basidiomata where the cells for sexual reproduction are formed. The hymenophore may be lamellate, tubular, spiny, fl at etc. phylogenetic signal – a certain character is distributed among a group of related species. A phylogenetic signal is when the species where the character is present are all grouped together in the same branch or closely related branches of the phylogenetic tree. primary charges of spore – the electrostatic charge carried on a spore immediately following liberation from a spore bearing structure rainout – the growth of cloud drops on aerosol particles and the eventual removal of the aerosol particles to the ground by precipitation sterigma – a curved tapering outgrowth of the spore-bearing cell territorial emission rate of spores – a number of spores released per unit time per unit area of forest. Here [spores s-1 ha-1]. washout – the collection of aerosol particles by precipitation that subsequently falls to the ground 50 9. SUMMARY IN ESTONIAN

Eoslavaseente eoste elektrilaeng ja selle bioloogiline tähendus

Eoslavaseente viljakehadest vabanevatel kandeostel avastati elektrilaengute olemasolu juba sada aastat tagasi, aga teadmisi neist on kogunenud vähe. Avastaja Buller (1909) keskendus vaid laengu olemasolu selgitamisele, nagu ka nähtuse järgmine uurija Gregory (1957). Kahel korral (Swinbank et al. 1964, Webster et al. 1988) mõõdeti eoste erilaenguid, et hinnata laengute suurust ja selgitada, kas see on piisav etendamaks otsustavat osa eose eoskannalt eemalepaiskumises või eose liikumisel eoslavakandja sees. Kuna vastus oli eitav, laengutega enam ei tegeldud. Nende töödega tuvastati laengu polaarsus kaheksa liigi eostel ning erilaengu ja laengu absoluutväärtus kümne liigi eostel (Table 1). Absoluutväärtusi mõõtes kasutati enamasti 12-80 eost liigi kohta (Webster et al. 1988), polaarsust selgitades üht suurt eosekogumit liigi kohta (Gregory 1957). Vaid kahel liigil, Ganoderma applanatum (Pers.) Pat. ja Serpula lacrymans (Wulfen) J. Schröt., uuriti mitmel viljakehal mitut suurt eosekogumit, mis olid kogutud vastavalt ühe tunni (Gregory 1957) ja ühe ööpäeva jooksul (Swinbank et al. 1964). Esindatud olid kolm laengutüüpi. Ainult positiivselt laetud eostega olid Ganoderma applanatum eosekogumid (Gregory 1957). Ainult negatiivselt laetud eostega olid Serpula lacrymans eosekogumid (Swinbank et al. 1964). Erinimeliselt laetud eosed olid Agaricus campestris L., Coprinellus hiascens (Fr.) Redhead, Vilgalys & Moncalvo, C. micaceus (Bull.) Vilgalys et al., Pholiota highlandensis (Peck) A.H. Sm. & Hesler, P. squarrosa (Vahl) P. Kumm. ja Polyporus squamosus (Huds.) Fr. eosekogumikes (Buller 1909, Gregory 1957). Nende, aastatel 1909—1988 tehtud töödega saadud andmestik põhineb paljuski vaid väikemahulistel eosekogumikel, mis vabanesid viljakeha tükikestelt laboratoorsetes tingimustes (Buller 1909, Webster et al. 1988). Tervikpilt, milles nii laengute polaarsus kui ka suurus, oli saadud vaid Agaricus campestris, Coprinellus micaceus ja Serpula lacrymans puhul. Primaarsete elektrilaengute olemasolu on iseloomulik mitte ainult eoslavaseente kandeostele. Primaarsed elektrilaengud esinevad ka mittesugulises staadiumis oleva kandseene pärmi ballistospooridel (Webster et al. 1988) ja kottseene lülieostel (McCartney et al. 1982), samblike seenkomponentide kotteostel (Garrett 1972) ja tuultolmlevate taimede õietolmuteradel (Bowker & Crenshaw 2003, 2007).

51 Minu doktoritöö esimeseks ja põhiliseks eesmärgiks oli luua lavaseente kandeoste primaarsetest elektrilaengutest tervikpilt, kasutades suuremahulisi eosekogumeid looduses kasvavatelt seentelt. Selleks oli vaja määrata eoste laengutüüp, s.o. eoste laengute jaotumine polaarsuse järgi eosekogumis, mõõta keskmine erilaeng ja keskmine laeng eosekogumis ning tuvastada tegurid, millest need laengute omadused sõltuvad. Minu hüpoteesid põhinesid eeldusel, et laengud moodustuvad viljakehas või eoste eoskandadelt vabanemisel, mitte aga pärast eoskandadelt vabanemist, õhus olevate ioonide tõttu. Püstitasin järgmised hüpoeesid. 1) Ühelt viljakehalt pärinevate eosekogumite laengutüübis kajastub fülogeneetiline signaal. Laengutüüp ei sõltu viljakeha füsioloogilistest seisunditest ega ka ajalistest, geograafi listest ja keskkonnateguritest. Nendeks teguriteks olid: substraat, õhu temperatuur, õhu suhteline niiskus, geograafi line asukoht, aasta, aastaaeg, ööpäeva osa. 2) Eoste keskmise erilaengu ja keskmise laengu suurus ei sõltu viljakeha sporulatsiooni intensiivsusest. 3) Eoste keskmise erilaengu ja keskmise laengu suuruse liigisisene varieeruvus sõltub eoste suurusest. (I, II, III).

Teisteks eesmärkideks olid: — Hinnata eose primaarse laengu relaksatsiooniaega – ehk vastata küsimustele, kui kaua säilib õhku sattunud eosel veel suur osa esialgsest laengust ja millise aja järel on eoselt kadunud enamus esialgsest laengust. Eeldasin, et eose laeng muutub õhuioonide toimel. (II). — Uurida, kas primaarsed elektrilaengud mõjutavad eoste õhust väljapesemist. Hüpoteesiks oli, et väiksemate piiskadega väljapesemisel suurendavad eoste laengud piiskade kogumisvõimet. (II). — Leida eoste ja nende laengute territoriaalse emissiooni kiiruse ülempiir. Eeldasin, et see sõltub eoste hümeniaalse emissiooni kiiruse ülempiirist ja viljakehade arvust metsa pinnaühikul. Oletasin, et ümbruskonnas kasvavad üht liiki viljakehad sporuleerivad sünkroonselt ja korrapäraselt ning et seetõttu esinevad neil sporulatsiooni intensiivsuse ööpäevarütmis maksimumid üheaegselt. (II).

Eoseid kogusin looduslikes tingimustes kasvavatelt viljakehadelt, paigutades kogumiskambri eoslavakandja alla (I). Eosekogumi moodustasid eosed, mis vabanesid 4x11 mm2 suuruselt eoslavakandja pinna osalt 15-1500 min kestel. Kogumis oli 103-106 eost. Kasutasin endakonstrueeritud ja -ehitatud seadeldist. Kogumiskambri tööpõhimõte

52 oli samasugune nagu ka varasematel uurijatel (Gregory 1957, Swinbank et al. 1964) – horisontaalses homogeenses elektriväljas vabalt langevate laetud eoste sadenemine elektroodidele ja nende all olevale pinnale. Eoste trajektooride määramise viis oli mul aga erinev. Mina kasutasin trajektoori vertikaalkomponendina eose sadenemiskoha kaugust elektroodi ülemisest äärest. Horisontaalkomponendiks oli pool elektroodide vahelisest kaugusest. Nende komponentide suhte abil saab arvutada eose erilaengu (I). Eosekogumis olevate ühenimeliselt laetud eostele keskmise erilaengu leidmiseks lähtusin eoste hulga vertikaalsuunalisest jaotumisest elektroodil. Iga kõrguse jaoks eraldi leidsin seal asuvate eoste erilaengute summa. Nende osasummade abil arvutasin eosekogumi ühenimeliselt laetud osa jaoks eose keskmise erilaengu (I). Eose keskmise laengu sain teada, korrutades seda eose massiga. Eose massi arvutasin eoste kirjandusest leitud mõõtmete järgi, võttes eose tiheduseks 1 g cm-3. Keskmise erilaengu ja keskmise laengu mõõtmisviga moodustus riistavea ja juhusliku vea summast (I). Riistaviga oli vahemikus 24-45%, sõltudes eksperimenditingimustest ja uuritavast seeneliigist. Mõõtmisviga sõltuski peamiselt riistaveast, sest eoste suure hulga tõttu oli juhuviga väike. Eoste hümeniaalse emisioonikiiruse leidsin, jagades eosekogumis olnud eoste arvu kogumisaja kestusega. Eoste elektroodil jaotumist uurides lugesin mikroskoobi abil (0.096 mm2 vaateväli) eosed 34x11 mm2 suurusel alal. Kasutasin 510 vaatevälja, mis paiknesid sellel elektroodi keskel oleval ribal 34 horisontaalse ja 15 vertikaalse seeriana.

Doktoritöö algandmebaasi moodustasid 235 eosekogumit, mis pärinesid 135 viljakehalt, hõlmates 50 liiki, 38 perekonda, 23 sugukonda ja 8 seltsi. Igas kogumis määrasin eoste laengutüübi. Neist 235-st 128-s eosekogumis, mis pärinesid 81-lt viljakehalt 31-st liigist, määrasin ka eoste erilaengu ja laengu ning laetud eoste hümeniaalse emissiooni kiiruse.

Eosekogumikest enamiku kogusin aastatel 1972-1974. Aastatel 1975, 1978, 1980 ja 1986 kogusin lisa, et hõlmata rohkem perekondi ja sugukondi. Välitööd tegin Eestis (Järvseljas, Lahemaal, Valgemetsas, Vooremaal, Tartus, Tartu ümbruses), Adõgees (Guzeriplis, Kaukaasia mäestiku loodeosa, 1974) ja Azerbaidžaanis (Zakatalas, Kaukaasia mäestiku kaguosa, 1974) (III, Table 1). Ajaliselt ja ruumiliselt olid kõige paremini uuritud Fomes fomentarius (L.) J. Kickx f., Fomitopsis pinicola (Sw.) P. K a r s t . j a Ganoderma applanatum. Eosekogumite ja viljakehade arvud oli suurimad liigil F. pinicola, järgnesid F. fomentarius, G. applanatum, ja

53 Lactarius turpis (Weinm.) Fr. Hõlmatud perekondade ja sugukondate arv oli suurim seltsis Agaricales. Mitme eosekogumiga oli esindatud 51 viljakeha 21-st liigist (III, Table 2). Aastaajad, ööpäeva osad, õhu temperatuur, õhu suhteline niiskus ning substraadid olid kõige paremini esindatud liikide F. fomentarius, F. pinicola, G. applanatum, Phellinus tremulae (Bondartsev) Bondartsev & P.N. Borisov ja rühma Phellinus igniarius [koosnes liikidest P. alni (Bondartsev) Parmasto, P. nigricans (Fr.) P. Karst. ja P. populicola Niemelä ] puhul (III, Fig. 1a, 1b, 1c). Viljakeha füsioloogiliste seisundite mõju uurimiseks oli eosekogumeid ka viljakehadelt, mis kogumispäeval või päev hiljem ära kuivasid, kokku varisesid või lõplikult sporuleerimise lõpetasid (III, Table 3). Need elujõuetud viljakehad olid liikidest Lactarius helvus (Fr.) Fr., L. turpis, Hydnellum ferrugineum (Fr.) P. Karst., Leccinum auriantiacum (Bull.) Gray, Tapinella atrotomentosa (Batsch) Š utara ja Stropharia hornemannii (Fr.) S. Lundell & Nannf. Kui käsitlesime fülogeneetilise signaali olemasolu eoste laengutüübis, ei võtnud me elujõuetutelt viljakehadelt pärit eosekogumeid arvesse. Eoste laengutüübis fülogeneetilise signaali olemasolu selgitamisel kasutasin seente klassifi katsiooni nii, nagu see oli kajastatud andmebaasis The Index Fungorum juunis 2015.

Eose primaarse laengu relaksatsiooniaja leidmiseks tuletas artikli II kaasautor valemi. Valem näitas, et relaksatsiooniaeg ei sõltu eose mõõtmetest ega eose laengu suurusest, küll aga sõltub õhu juhtivusest, s.o. õhus olevate ioonide hulgast ja polaarsusest. Õhuandmeteks kasutas ta Tahkuse õhuseirejaamas mõõdetud aeroioonide liikuvusspektrite pikaajalisi keskmisi.

Primaarsete elektrilaengute mõju eoste õhust väljapesemisele hindasin kaudselt, langevate veepiiskade haardekoefi tsendi abil. Toetusin Tripathi ja Harrisoni (2002) uurimusele. Selles vaadeldud aerosooliosakeste suurus, tihedus ja elektrilaeng ühtisid eoste omadega. Numberarvutustega olid leitud aerosooliosakeste trajektoorid langevat tilka ümbritsevas õhus. Nende põhjal olid arvutatud haardekoefi tsendid. Mudeleostena kasutasin Phellinus tremulae ja P. nigricans kandeoseid, mis on kerajad, diameetriga vastavalt 4.4 (3.9-4.7) µm ja 6.1 (5.6-6.5) µm (Sell 2008), laenguga vastavalt 117 (46-222) e ja 204 (107-496) e (I) ning mille tiheduseks on hinnanguliselt 1.0-1.2 g cm-3.

Ülempiirid eoste ja nende laengute territoriaalse emissiooni kiirustele leidsin juhtudeks, mille puhul seeneliikide jaoks oli täidetud kaks

54 tingimust. 1) Metsa pindalaühikul üheaegselt esinevate viljakehade arvu kohta leidus andmeid kirjanduses. 2) Eoste primaarsed laengud olid doktoriuurimuses määratud (II, Table 4). Näidiseks valisin olukorrad, kus viljakehade arv oli suurim. Haavikus on haavataeliku (Phellinus tremulae) viljakehi kõige rohkem 75-aastases metsas (Tamm 2000, Krigul 1969), hümeniaalset pinda on seal hinnanguliselt 10 m2 ha-1. Tavavahelikul (Paxillus involutus (Batsch) Fr.) ja harilikul kukeseenel (Cantharellus cibarius Fr.) oli viljakehade esinemise tippaeg, uuringu (Lõugas 1980) aastal, septembris. Tavavaheliku hümeniaalset pinda oli siis kanarbiku männi- kasemetsas hinnanguliselt 0.8 m2 ha-1, kukeseenel sambliku männi- kasemetsas 5.0 m2 ha-1. Maksimaalsed hümeniaalse pinna suurused neil liikidel neis metsades on suuremad, sest uuringu aasta oli seenevaene. Eoslavakandjate kogupindala arvutamisel kasutatud viljakehade mõõtmed võtsin kirjandusest või eksperdilt (Ülo Tamm, P. tremulae). Ülempiiri eoste hümeniaalsele emissioonikiirusele tuletasin, kasutades kaht eeldust: 1) Kõikidel liikidel on eoslava eosetoodangu võimel ühesugune ülempiir. Torukestega eoslavakandjal on selle ülempiiri puhul eoste hümeniaalsed emissioonikiirused vahemikus 914-1100 eost cm-2 s-1 (Fomes fomentarius ja Phellinus alni sporulatsioonimaksimumide keskmised Parmasto 1980 järgi). 2) Torukestega eoslavakandjal olev eoslava pindala suhtub lehekestega eoslavakandjal olevasse eoslava pindalasse nagu 3:1, sileda eoslavakandja puhul on suhe 45:1 (Fischer & Money 2010). Arvutused andsid eoste hümeniaalsele emissioonikiirusele ülempiiriks 305-367 eost cm-2 s-1 lehekestega eoslavakandjal ja 20-24 eost cm-2 s-1 siledal eoslavakandjal. Ümbruskonnas kasvavate üht liiki viljakehade sporuleerimise sünkroonsust ja korrapära selgitasin liikidel Fomes fomentarius ja Ganoderma applanatum, mõõtes korduvalt mitmel viljakehal paralleelselt eoste hümeniaalset emissioonikiirust.

Peamised tulemused 1. Eoslavaseente kandeosed on viljakehadelt vabanedes laetud. Ühes eosepopulatsioonis võivad esineda ainult positiivselt või ainult negatiivselt laetud eosed või esineb neid mõlemaid.

Eosekogumi laengutüüp sõltub füsioloogilistest teguritest. Elujõuetult ärakuivamise või kokkuvarisemise eelses seisundis olevalt viljakehalt vabanevates eostekogumites on laengutüüp muutlik või puuduvad laengud üldse. Elujõuliselt viljakehalt vabanevates eoskogumites on laengutüüp ühesugune.

55 Eoskogumi laengutüüp on liigile iseloomulik tunnus, mis ei sõltu välisteguritest. Laengutüüp on ühesugune kõikides eoskogumites, mis vabanevad ühe liigi elujõulistelt viljakehadelt. Laengutüüp ei sõltu aastast, sesoonsest ajast, ööpäevasest ajast, õhu niiskusest, õhu temperatuurist, geograafi lisest asukohast ega substraadist.

Näib, et kõigis taksonoomilistes rühmades alates liigist kuni monofüleetilise sugukonnani on kandeoste primaarsed laengud sama tüüpi.

Eoslavaseentel varieerub eose keskmine erilaengu suurus vahemikus (0.7- 7) × 10-4 C kg-1. Eose erilaengu suurus ei sõltu viljakeha sporuleerimise intensiivsusest. Eose keskmise erilaengu ja eose keskmise ruumala vahel puudub seos.

Haava-tuletaeliku (Phellinus populicola) uuring näitas, et eose keskmise erilaengu suuruse viljakehasisene varieeruvus on sama suur kui liigisisene varieeruvus. Ühe liigi erinevatelt viljakehadelt pärit eosekogumite, aga ka ühelt viljakehalt pärit eosekogumite seas leidub eose keskmise erilaengu suuruse poolest nii üksteisega sarnaseid kui ka üksteisest erinevaid kogumeid. Eose keskmise suuruse looduslik varieeruvus võib olla põhjuseks, miks ühes liigis erinevad eosekogumid üksteisest keskmise erilaengu suuruse poolest.

Eoslavaseentel varieerub keskmine eose laengu suurus vahemikus 20-1000 e. Eose laengu suurus ei sõltu viljakeha sporuleerimise intensiivsusest.

2. Viljakehalt vabanenud eose elektrilaeng hakkab õhus olles kahanema. 23. minutil pärast vabanemist on eosele jäänud laengu suurus 2,7 korda esialgsest laengust väiksem, 46. minutil 7,3 korda väiksem. Laengu kahanemise kiirus ei sõltu eose mõõtmetest ega laengu suurusest.

3. Väikeste kandeostega (läbimõõt 4-5 µm või vähem) eoslavaseentel soodustavad eostel olevad primaarsed laengud (suurus 50 e ja enam) eoste õhust väljapesemist. Positiivne mõju ilmneb vaid väikeste piiskadega (läbimõõt 80 µm ja vähem), s.o. udude, uduvihmade või pilvepiiskadega väljapesemisel. Piirkondades, kus udud ja uduvihmad on sagedased, samuti pilve-metsadega mäestikes soodustavad eoste primaarsed laengud väikeseeoselistel eoslavaseentel eoste õhust väljasadenemist.

56 4. Vana haavikuga kaetud alalt õhku erituvate haavataeliku (Phellinus tremulae) eoste hulk ei ületa määra 108 eost ha-1 s-1. Eosed on positiivsete laengutega. Nende eostega koos õhku erituv positiivne laenguhulk ei ületa määra 1010 e ha-1 s-1.

Sambliku männi-kasemetsaga kaetud alalt seenevaesel aastal õhku erituvate hariliku kukeseene (Cantharellus cibarius) eoste hulk ei ületa määra 107 eost ha-1 s-1. Eosed on negatiivsete laengutega. Nende eostega koos õhku erituv negatiivne laenguhulk ei ületa määra -1010 e ha-1 s-1.

Kanarbiku männi-kasemetsaga kaetud alalt seenevaesel aastal õhku erituvate tavavaheliku (Paxillus involutus) eoste hulk ei ületa määra 106 eost ha-1 s-1. Kolmandik eostest on negatiivselt laetud, kaks kolmandikku eostest on positiivselt laetud. Nende eostega koos õhku erituvad positiivsed ja negatiivsed laenguhulgad ei ületa järgmisi määrasid: vastavalt, 108 e ha-1 s-1 ja -108 e ha-1 s-1.

57 10. ACKNOWLEDGEMENTS

I am grateful to Erast Parmasto for arousing my interest in the primary electric charges of basidiospores. I thank Mart Murdvee and Olev Kerem for their aid in designing and building the measuring device, Matti-Elmar Fischer for his help in solving the problems of instrumental errors and Tõnu Möls for his help with statistical analyses. Many thanks to Kuulo Kalamees and Mall Vaasma for the identifi cation of mushrooms. Also, I thank Ülo Tamm and Thomas B. Jones for sharing their expert knowledge on the Phellinus tremulae and on the triboelectric effect, respectively.

I am also grateful to the offi cial reviewer Kadri Põldmaa for her constructive criticism. Gediminas Mainelis, Philipp Gannibal, Leho Tedersoo, and the other reviewers of the original articles are acknowledged for their valuable comments to the papers.

I would like to express sincere thanks to all my employers and colleagues in the Estonian University of Life Sciences for the positive and favoring attitude in encouraging my personal lifelong learning.

I thank my family and friends for care and support.

58 I

PUBLICATIONS Saar, Maret (2013). Investigation of the electrostatic charge of basidiospores of the Phellinus igniarius group. Central European Journal of Biology, 8 (5), 410 - 422. 
 


!
 
!

!
 
    

!"#$%&'()*%+,"&#(-+*%#&'(+.(!"#$#%&

()*$+%,-"%,.)&./&%0$&$1$2%#.+%"%,2&20"#-$&./& 3"+,4,.+5.#$+&./&%0$&60$11,)7+&,-),"#,7+&-#.75

!"#"$%&'()%*+&,"

!"#$%&'""#!

$2!35)$05"1%"15!088"045'565$"1%"&3'"6(563!("!0#"07'310)$05!(""'$0"$48 4510'!0" 0'7$34'58"1%"'%$""'$0"$48"BCDCE"!3568"4510'! !"&"+-".(/0(12,3(4/546()&&"7*".(/4(1$82$%3(4/59

)>#*%$&*=
"#$%"&'()*+,-")./"0)1.+,2/%"'3"&*+0)*-"%(%4,*+4"4$)*1%5"4)**+%/"6-"6)5+/+'5&'*%5"+.",$%")+*6'*.%"5,),%"7%*%"+.8%5,+1),%/"+."(+8+.1"32.1)(" 3*2+,+.1"6'/+%5"2./%*".),2*)("3'*%5,"4'./+,+'.5"25+.1")"&'*,)6(%"%9&%*+0%.,)("/%8+4%"/%5+1.%/"6-",$%")2,$'*:"#$%"'&%*),+.1"&*+.4+&(%" 
 
 



 
 
 
  
 
 
 

 


 
  

 
5&'*%5"7%*%"/%,%*0+.%/")44'*/+.1",'",$%+*"/%&'5+,+'."5+,%5"'."%(%4,*'/%5";8%*,+4)("0%,)("&(),%5<:"=(,'1%,$%*">>"5)0&(%5"'3"5&'*%5"7%*%" %9)0+.%/"3'*"&'()*+,-?"@A"'3",$%5%"5)0&(%5"";7+,$"@ABC@AD"5&'*%5"&%*"5)0&(%<")(5'"7%*%"25%/",'"4)(42(),%",$%""0%)."5&'*%"4$)*1%E,'E0)55" F2',+%.,")./",$%"0%)."5&'*%"4$)*1%:"#$%"/%,%4,+'."(+0+,5"'3"5&'*%"4$)*1%E,'E0)55"F2',+%.,"8)*+%/"+.",$%"*).1%"3*'0";B:GHI:><
@AEJ",'" ;@:>DHA:>><
@AEB"K"L1E@:"M)5+/+'5&'*%5";5261('6'5%?"50'',$?"/+)0%,%*"'3"BCD"N0<"'3",$%"4('5%(-"*%(),%/";5+6(+.1<"5&%4+%5"  


 ?" 
  ")./" 
  ";O-0%.'4$)%,)(%5?"M)5+/+'0-4',)<"4)**+%/"&'5+,+8%"%(%4,*+4)("4$)*1%5",$),"$)8%"0%)."8)(2%5" 3*'0"BP",'">AJ"%(%0%.,)*-"4$)*1%5:"#$%"+.,*)5&%4+%5"8)*+),+'."'3",$%"5&'*%"4$)*1%"4'2(/"/%&%./"'.",$%".),2*)("8)*+),+'."+.""5&'*%"5+Q%:copy :"3;<%.#=

 

   
 

   
 

 
 

     

 
 
R"S%*5+,)"T&:"Q"':':"

!"#$%&'()*+&,(% 8K:L<=" M.,:" *5-('" -#(" &/+0.1(')" .5" -#(" 3*'*4.$%-" (2(4(%-+" .5" -#(" &/+0#*'7(" 4(0#*%/+4:" /-" /+" 02(*'" -#*-" !"#$%&'(&")(*'+",*+"%((&(&"-."&/+0.1('"*22"3*'*4.$%-" +3.'("'(2(*+("/+"4*/%-*/%(&"6)"+$'5*0("-(%+/.%"5.'0(+=" (2(4(%-+" .5" -#(" 6*22/+-.+3.'/0" &/+0#*'7(" 4(0#*%/+4" N'(+(%-"O%.,2(&7(".%"-#("&/+0#*'7("/+"/%0.432(-(="P.'" .5" 6*+/&/.+3.'(+" 89:;<=" !2'(*&)" *-" -#(" 1(')" 6(7/%%/%7" (@*432(:"/-"/+"%.-"02(*'"#.,"-#(".+4.2)-(+"*'("&(2/1('(&" .5" /%1(+-/7*-/.%+" .5" -#/+" 4(0#*%/+4:" -#(" 3'(+(%0(" .5" C>
C74
B?>A4
BDA5024
8=
0
BD5Q284=C
2>=24=CA0C8>=
C>
02C
(2(0-'.+-*-/0"0#*'7(+".%"6*+/&/.+3.'(+"$3.%"2*$%0#/%7" *+"%$02(/"5.'"-#("0.%&(%+*-/.%".5",*-('"8Q<="M(,"/&(*+"5.'" 5'.4"+-('/74*-*",*+"5.$%&"8>Author's<="?#("%(@-"+-$&)".%"3'/4*')" 0.432(-/%7"-#("O%.,2(&7(".5"-#("6*22/+-.+3.'("&/+0#*'7(" (2(0-'.+-*-/0" 0#*'7(+" .%" 6*22/+-.+3.'/0" 6*+/&/.+3.'(+" <4270=8B<
8=
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

!""$)!'(")!3$5-4!!3$)6-$$ /01

61 !8&'""#

E/%"-.-*2"9;CTY"+3.'(+"3('"+3(0/(+G".'"*"+/%72("6/7"+*432(" *'(" 7'.,/%7" .%" ,(-" +*%&+" 89A<=" ?#(" -(43('*-$'(" E9"#'"0.22(0-/.%".5"+3.'(+G"3('"+3(0/(+:".'"$+/%7"-#("+3.'(+" *%&" #$4/&/-)" .5" */'" ,('(" 4(*+$'(&" 6)" -#("!++4*%%" '(2(*+(&"5'.4"*"3/(0(".5"#)4(%.3#.'(="?#(+("+-$&/(+" 3+)0#'.4(-('"*-"-#("#(/7#-".5"9"4"*6.1("7'.$%&"*%&" *'("+/%72("+3.'*&/0"*--(43-+"-."$%&('+-*%&""(2(0-'.+-*-/0" *-"*"&/+-*%0(".5";"4"5'.4"*"5'$/-/%7"6.&)"E.'",/-#/%"*" 3#(%.4(%*"/%"-#("#/7#('"5$%7/:"*%&",('("%.-"5.22.,(&" &/+-*%0(".5"AY"4:"/5"+(1('*2"5'$/-/%7"6.&/(+",('("+-$&/(&" 6)"+)+-(4*-/0"+-$&/(+".%"3'.62(4+"0.%0('%/%7"-#(".'/7/%" +/4$2-*%(.$+2)G"/44(&/*-(2)"6(5.'("*%&"*5-('"(@3.+$'(" .5"0#*'7(+=" .5"-#("+*432/%7"0#*46('E+G="?#(+("+3.'("0.22(0-/.%+" ?#("*/4+".5"-#("3'(+(%-"+-$&)"*'("6.-#"-#("3.2*'/-)" ,('(" *" 3*'-)" -." *%" /%1(+-/7*-/.%" 0*''/(&" .$-" /%" -#(" *%&" -#(" 4*7%/-$&(" .5" 3'/4*')" (2(0-'.+-*-/0" 0#*'7(" .5" 9UQY+"6)"-#("*$-#.':"*-"-#*-"-/4("*"7'*&$*-("+-$&(%-" 6*+/&/.+3.'(+=" X" '(3.'-" -#(" '(+$2-+" .5" *%" /%1(+-/7*-/.%:" /%"-#("X%+-/-$-(".5"f..2.7)"*%&"V.-*%)".5"-#("!0*&(4)" ,#/0#" ,*+" 6*+(&" .%" 6/7" +3.'(" +*432(+" 0.22(0-(&" /%" .5" I0/(%0(+" .5" e+-.%/*=" !2-.7(-#('" AL" +3(0/(+" .5" %*-$'*2"5.'(+-"0.%&/-/.%+="X"5.0$+"*-"-#("5.$'"02.+(2)"'(2*-(&" #)4(%.4)0(-.$+" !7*'/0.4)0(-(+" ,('(" 0.%+/&('(&:" E+/62/%7G"+3(0/(+"'%))(186(!)1((EV.%&*'-+(1G"N*'4*+-.:" -#("'(+$2-+",('("2(5-"$%3$62/+#(&"$%-/2"-#/+=" /(1(&5(#!16(EP'=G"N="Z*'+-=:"/(3238)(#2)!"M/(4(2[:"*%&" V*+/&/.+3.'(+" .5" -#(" +3(0/(+" +-$&/(&" *'(" /( 75%08)!%" EV.%&*'-+(1G" V.%&*'-+(1" \" N=M=" V.'/+.1" (22/3+./&*2=" g(*%" +3.'(" +/h(+" ,('(" '(0.'&(&" *+" E7'%( '%))(186( (&1(!5(86" 7'.$3:" ])4(%.0#*(-*2(+:" 5.22.,+_" /( !)1((  G
V<
;4=6C7
G
F83C7
0=3" V*+/&/.4)0.-*G=" -#(" D$.-/(%-" .5" 2(%7-#" *%&" ,/&-#" EiG" 5'.4" 9=9" -." 9=;:" /( 1(&5(#!16
T
V<
0=3
'    E-#(" 7'(*-(+-" *%&" *24.+-" 72.6*2" +3.'(+G:" -"#./01',21%&34#5'(+1)*'16 /( 3238)(#2)!
T 
V<
0=3
'  P  
0=3 /( 75%08)!%
T  
V<
0=3
'  P 

C74
-"!#70('16#3%)#60('*43&,(% +4*22(+-"*%&"4.+-"3'.2.%7(&"+3.'(+G"89;:9K<=" I/%0(" V*+/&/.+3.'(+" .5" '%))(186" +3(0/(+" *'(" 3'.&$0(&" 6)" -#(" &(%+/-)" .5" -#(" +3.'(+" '(4*/%(&" *46/7$.$+" 8L<:" 3('(%%/*2" +(++/2(:" $3" -." ;Y" 04" ,/&(" 5'$/-/%7" 6.&/(+" -#("+3.'(+",('("0.%+/&('(&"*+"3'.2*-("(22/3+./&+".5"copy 7'.,/%7".%"-'((+:"*%&"*'("(4/--(&"5'.4"+3'/%7"-."*$-$4%" 0.%+-*%-"+#*3("*%&"+/h(:"#*1/%7"-#("3.2*'"*@/+"(D$*2" 89Y:99<=" I3.'(+" *'(" (4/--(&" -#'.$7#" 0/'0$2*'" 3.'(+" -."-#("4(*%"+3.'("2(%7-#"*%&"-#("(D$*-.'/*2"&/*4(-('" 2>E4A8=6
C74
D=34ABDA5024
>5
5AD8C8=6
1>384B
01>DC
QE4
(D$*2"-."-#("4(*%"+3.'(",/&-#:"*%&"*"&(%+/-)".5"9Y>" 3.'(+"3('"4/22/4(-'("89;= 5'$/-/%7" 6.&):" /-" /+" 5'((" 5*22/%7=" ?#/+" 1('-/0*2" 4.1(4(%-" 0.%-/%$(+" -#'.$7#" *" 2*4/%*'" 2*)('" E*" 6*%&" .5" +-/22" */':" -"-#./01',21%&34#61&8*0 $+$*22)";C>"44"-#/0OG"(@/+-/%7"/44(&/*-(2)"6(%(*-#"-#(" X%"*22"3'(1/.$+"+-$&/(+"8>CL<:"+3.'(+",('("*22.,(&"-."5*22" 5'$/-/%7" 6.&)^" -#('(*5-('" -#(" 4.1(4(%-" .5" -#(" +3.'(" /+" 8=
C74
7><>64=4>DB
7>A8I>=C0;
4;42CA82
Q4;3
=
C74B4
8=RD4=243
1H
08A
R>FB
.9><="V*22/+-/0"6*+/&/.+3.'(+"4.1(" 0.%&/-/.%+:"-#("+3.'("0#*'7("/+"'(2*-(&"-."-#("#.'/h.%-*2" /%" -#(" */'" +/%72):" -#()" &." %.-" -(%&" -." 02$43" -.7(-#('" 1(2.0/-)" .5" -#(" +3.'(:" ,#/2(" -#(" 1('-/0*2" 1(2.0/-)" /+" E3('+.%*2".6+('1*-/.%+G=" '(2*-(&"-."-#("+/h("*%&"4*++="V(-,((%"-#("5.$'"3'(1/.$+" X%"-#("3'(+(%-"+-$&):"-#("+3(0/(+",('("'(3'(+(%-(&" +-$&/(+:"-#(""4(-#.&"$+(&"-.".6-*/%"/%5.'4*-/.%"*6.$-" $%(D$*22)_"/(1(&5(#!16(6)".%("+3.'("+*432(:"/(!)1((6)" +3.'(" -'*B(0-.'/(+" &/55('(&=" ?#(" 4.1/%7" +3.'(+" ,('(" 5.$'"+3.'("+*432(+"E-,."5'$/-/%7"6.&/(+GAuthor's5"/( 3238)(#2)!5" (/-#('" .6+('1(&" 6)" #.'/h.%-*2" 4/0'.+0.3(" -." +((" -#(" 9T" +3.'(" +*432(+" E5.$'" 5'$/-/%7" 6.&/(+G:" *%&" /( &/'(0-/.%" .5" &(02/%*-/.%" .5" -'*B(0-.'/(+" 5'.4" -#(" 1('-/0*2" 75%08)!5"9Y"+3.'("+*432(+"EL"5'$/-/%7"6.&/(+G( E?*62(" 8><:".'",('("3#.-.7'*3#(&"$+/%7"*"4/0'.+0.3("*%&"&*'OC 9G=""'%))(186(!)1((*%&"/( 75%08)!%",('("+-$&/(&"/%"-,." Q4;3
BCA>1>B2>?82
8;;D<8=0C8>=
C>
Q=3
C74
<06=8CD34
2.0*2/-/(+".1('"-,.")(*'+:"/(1(&5(#!16(*%&"/(3238)(#2)!" .5" &(02/%*-/.%" 6)" -#(" 3#.-.7'*3#/0" -'*B(0-.')" 8L<=" ?#(" ,('(" +-$&/(&" /%" .%(" 2.0*2/-)" /%" .%(" )(*'=" X%" 9UQ;:" +3.'("+(&/4(%-".%"-#("6.--.4",*+"$+(&"-."4(*+$'("-#(" +3.'("+*432(+",('("-*O(%"/%"5.'(+-+"/%"-#("`..'(4**" &/+-*%0(".5"&/+32*0(4(%-".5"+3.'("5'.4"-#("1('-/0*2"5*22" a*%&+0*3("N'.-(0-/.%"!'(*"EHb7(1*"S.=:"KTcA>dM"*%&" *%&"-#("'*-/.".5"-#*-"&/+-*%0("-."-#("#(/7#-".5"(2(0-'.&(+" ;LcAKdeG:"*%&"/%"9UQ>"+3.'("+*432(+",('("0.22(0-(&" E*+"-#("1('-/0*2"0.43.%(%-".5"+3.'("-'*B(0-.')G"/%&/0*-(&" /%" -#(" H['1+(2B*" e@3('/4(%-*2" P.'(+-" R/1/+/.%" E?*'-$" -#("&(02/%*-/.%".5"-'*B(0-.')"8K<="?#("(2(0-'.&(+"*%&"-#(" S.=:"KTc9LdM"*%&";Qc9TdeG="?#(+("2.0*2/-/(+"'(3'(+(%-" 6.--.4" 6(-,((%" -#(4" ,('(" 0#(0O(&" 5.'" -#(" 3'(+(%0(" 38554A4=C
;0=3B20?4
A468>=B
C74
QABC
,>>A4<00
;>20;8CH
.'" *6+(%0(" .5" +3.'(" +(&/4(%-+^" -#(" 3.2*'/-)" .5" -#(" 148=6
0
3AD<;8=
Q4;3
F74A4
2D;C8E0C43
3AD<;8=B
0A4
&(+-/%*-/.%" (2(0-'.&(" /%&/0*-(&" -#(" 3.2*'/-)" .5" +3.'(" B4?0A0C43
1H
5>A4BC43
34?A4BB8>=B
>A
;0:4Q;;43
0#*'7("8A<= &(3'(++/.%+" .'" -/22" 32*%(+:" *%&" -#(" +(0.%&" H['1+(2B*" ?#(" (D$/34(%-" $+(&" 5.'" -#(" 4(*+$'(4(%-" .5" -#(" 2.0*2/-)" '(3'(+(%-/%7" *" 2.,2*%&:" ,#('(" 6.77)" 5.'(+-+" +3.'(" 0#*'7(C-.C4*++" D$.-/(%-" 0.%+/+-(&" .5" -#(" +(25C

/00

62 ()*$+%,-"%,.)&./&%0$&$1$2%#.+%"%,2&20"#-$& ./&3"+,4,.+5.#$+&./&%0$&60$11,)7+&,-),"#,7+&-#.75

%XaI`ITG'BUDe IX 4ASVRE' #A`E 5ISE TU* 0U* 4aBY`XA`E 5ESVEXA`aXE9't" &aSIDI`e9'=

!"#$%&'

!" ' (%&)*#'&+$&$9'RIbITG'`XaTQ B*C*D BBCB aTSEAYaXED aTSEAYaXED

!# h h *D DCBD h h

!$ h h *C*D BC h h

BD 6'' (%&)*#YV*9'RIbITG'`XaTQ *BD BDCBDD BCBB BDDCB

!"#&',-'+$&*

BD 6' ./0)%$#YV*9'RIbITG'`XaTQ *BD BBDCBBD BC CD

!"#121)%'+2%$

 '' !21)%)*#0-/3)%$9'Y`aSV *D CBD BCB CD

 h h BD*D DDC BCB C

D h h *D DDCBD BCB DC

B h h *C*D C BCB C

 h h D*D BDDCB BCB C

 ''' !21)%)*#0-/3)%$9'RIbITG'`XaTQ *D CBDB DCB C

 h h *D DDCBD BCB CD

 h h *C*D C BCB DC  h h *D CBDcopyDC C  h h *D DCBDBD BC C

 '6 h *D DDCBD BCB C

 h h *C*D C BCB DC

BDD h h *D CBD DC C

BDB h h *D DCBDBD BC DC

BD 6 !21)%)*#0-/3)%$9'Y`aSV *C*D C BCB DC

BD h h *D CBD DC BC

BD h h *D DCBDBD BC C

BD h h *D CBDB DCB DC

Author's!"#0-/3)%$/ %%& ' !21)%)*#0-/3)%$9'RIbITG'`XaTQ BB*D CBB aTSEAYaXED aTSEAYaXED

%%% h h h BCB h h

%%' '' h h BBCBB h h

BB ''' h B*D CBDB BBCB C

BB h h B*DCB*D BDCBDD BCB C

BB '6 h B*D BCD BCB C

BB h h B*DCB*D BDCBDD BCB C

BB 6 h B*D BCD BCB C

BB 6' h B*D BCD BCB C

BB h h B*DCB*D BDCBDD BCB C

?$>,"(5@((4VUXE'YASVREY'EdASITED'IT'`HE'Y`aDe'UT'`HE'VXISAXe'EREC`XIC'CHAXGEY'UF'BAYIDIUYVUXEY'IT'!4/%%'&)*#',&'$-')*'GXUaV'YUaXCEY9'DA`E'ATD' `ISE'UF'YASVRITGY9'AIX'CUTDI`IUTY'A`'`HE'BEGITTITG'ATD'`HE'ETD'UF'YASVREY*'5HE'YASVREY'SAXQED'IT'BURD'FUT`'STU*'C9'BBDCBBT'cEXE' CURREC`ED'IT'6UUXE'IT'B9'`HE'U`HEX'YASVREY'cEXE'CURREC`ED'IT'(gXbYERPA'IT'B*''''

/02

63 !8&'""#

&(+/7%(&" *%&" C0.%+-'$0-(&" &(1/0(:" *%&" .5" *%" .3-/0*2" -." *&B$+-" -#(" 6.--.4" 32*-(" /%-." -#(" #.'/h.%-*2" 3.+/-/.%" <82A>B2>?4
YWX F8C7
0
3>D1;4
8;;D<8=0C8>=
BHBC4<
E*22.,/%7" &(1/*-/.%+" .5" +4*22('" -#*%" .%(" &(7'((G=" ?#(" >5
CA0=B<8CC43
;867C
0=3
A4R42C43
;867C
F7827
4=01;43
+-*%&",*+"(*'-#(&="!"6*--(')".5"0.%+-*%-"1.2-*7("+.$'0(" -#("&/'(0-"0.$%-/%7".5"-#("+3.'(+"*%&"-#("&(-('4/%*-/.%" ,/-#"%.4/%*2">>Y"1.2-+",*+"$+(&"-."7/1("-#("(2(0-'.&(+" .5" -#(" +(--2/%7" +/-(+" .5" -#(" +3.'(+=" ?#(" .3('*-/%7" (2(0-'/0" 0#*'7(+" .5" (D$*2" 4*7%/-$&(:" 6$-" .5" .33.+/-(" ?A8=28?;4
>5
C74
34E824
50;;8=6
B?>A4B
8=

BC8;;
08A
Q;;43
3.2*'/-)=" F8C7
0
7><>64=4>DB
7>A8I>=C0;
4;42CA82
Q4;3
F0B
C74
+*4("*+"-#*-"$+(&"/%"-#("3'(1/.$+"+-$&/(+="?#("+3.'(" -"9#:;1';,1<#(=#0'(+1)*'16 +(&/4(%-" .%" *%" (2(0-'.&(" ,*+" $+(&" -." 4(*+$'(" -#(" g(*+$'(4(%-" .5" -#(" +3.'(" 0#*'7(C-.C4*++" D$.-/(%-" 1('-/0*2"0.43.%(%-".5"+3.'("-'*B(0-.')"*%&"-#("&/+-*%0(" /%1.21(&" 4*%)" '.$-/%(" (@3('/4(%-*2" 3'.0(&$'(+" 6(-,((%"(2(0-'.&(+:"5.'"-#("#.'/h.%-*2"0.43.%(%-="?#(" 0.%%(0-(&" ,/-#" -#(" +3.'(" +*432/%7:" -#(" +(&/4(%-" &(1/0("0.%+/+-(&".5"-#'(("3*'-+_"*"0#*46(':"*"+-*%&:"*%&" 0#(0O/%7:"*%&"-#("0.43$-*-/.%="!/4+".5"-#("3'.0(&$'(+" *"6*--(')="" 5>A
B438<4=C
2742:8=6
0=3
2>=
F4A4
C>
Q=3
?#(" 0#*46('" *00.44.&*-(&" *%" */'" +3*0(" E#(/7#-" .$-" -#(" 3.2*'/-)" &/+-'/6$-/.%" .5" (2(0-'.+-*-/0" 0#*'7(+"
<<
;4=6C7

<<
F83C7
P 
<<
F8C7
0=
E#('(*5-('" '(5(''(&" -." *+" " j0#*'7(+kG" .5" +3.'(+" /%" -#(" 4;42CA>BC0C82
Q4;3
BCA4=6C7
P
:,
#70('1#63204,%?# *%&" *" #.'/h.%-*2" 4(-*2" 6.--.4" 32*-(="?#(" 4(-*2" ,*22+:" ?#(" 0#*46('" ,*+" 2.0*-(&" *+" %(*'" *+" 3.++/62(" -." 0(/2/%7"*%&"6.--.4"32*-(",('("(*'-#(&="?#("(2(0-'.&(+" -#(" #)4(%.3#.'(:"copy 6$-" %.-" /%" 0.%-*0-" E-#(" &/+-*%0(" F4A4
QG43
8=
?>B8C8>=
1H
E4AC820;
6A>>E4B
2DC
8=
C74
14CF44=

F0B
01>DC
P
<<
0=3
B?>A4B
F4A4
32*+-/0" ,*22+:" *%&" 5.'0(&" -." -#(" -('4/%*2+" 0.1('/%7" -#(" 0;;>F43
C>
50;;
0=3
B4CC;4
8=
QG43
4;42CA820;
0=3
08A
2.,(+-"3*'-".5"-#("7'..1(+="" 0.%&/-/.%+="?#("+3.'(+"+(--2(&".%"-#("(2(0-'.&(+"*%&" ?#(" 4(-*2" +-*%&" #(2&" -#(" 6.--.4" 32*-(" E3'.33/%7" 72*++"+2/&(+="V(5.'("-#("+-*'-".5"*%"(@3.+$'("-."+3.'(+:" -#("0#*46('"$3"*%&"02.+/%7"-#("0#*46('"5'.4"6(2.,G=" -#("0#*46('"%((&(&"-."'(*0#"-#("-(43('*-$'(".5"-#(" 
10;;0=3B>2:4C
7403
F0B
DB43
0B
0
Q=4
A46D;0C>A
BDAA>D=38=6
08A
01>DC
P
<8=DC4B
5C4A
C74
4=3

(&1$27132#6$35 172('13#( 7%&&)6+2('13#(

% 0

/ Author's ! , . -

! 1'#6)6+27132$4#2 $12#%3$4 ! 0%&&8%698(1*:#$2 4#%92172($%69 $120%$$#35

"#$%& '&%($)* +&%(( A+B2%"(5@((#EbICE'FUX'SEAYaXESET`'UF'`HE'YVUXE'CHAXGEC`UCSAYY'WaU`IET`'IT'TA`aXAR'FUXEY`'CUTDI`IUTY'AT'GETEXAR'bIEc'UF'`HE'CHASBEX'RUCA`ED'UT' `HE'BU``US'VRA`E'BT'CXUYYCYEC`IUT'`HXUaGH'`HE'SIDDRE'UF'CHASBEX9'`HE'EREC`XICAR'YCHESE9'ATD'YVUXE'`XAPEC`UXIEY'UF'TUTCCHAXGED'YVUXE' SBT9'YVUXE'cI`H'`HE'CHAXGE'`UU'YSARR'`U'DE`EC`'ST9'ATD'YVUXE'cI`H'`HE'CHAXGE'BIGGEX'`HAT'`HE'DE`EC`IUT'RISI`'ST*

/03

64 ()*$+%,-"%,.)&./&%0$&$1$2%#.+%"%,2&20"#-$& ./&3"+,4,.+5.#$+&./&%0$&60$11,)7+&,-),"#,7+&-#.75

-"A#5(43',&B#),6&',C*&,(%# ?#(" 3.2*'/-)" &/+-'/6$-/.%" .5" -#(" +3.'(" 0#*'7(+" /%" *" B0;;>FB
85
B?>A4B
F4A4
5>D=3
.%2)".%"-#("%(7*-/1("(2(0-'.&(".':"/%"*&&/-/.%:"*2+.".%"   -#("72*++"+2/&(:"-#(%"$%/3.2*'C3.+/-/1("&/+-'/6$-/.%",*+"

 3'.3.+(&^" /5" +3.'(" ,('(" .%2)" 5.$%&" .%" -#(" 3.+/-/1(" (2(0-'.&(".'"*2+.".%"-#("72*++"+2/&(:"-#(%"/-"/+"*"0*+("   

.5"$%/3.2*'C%(7*-/1("&/+-'/6$-/.%^"/5".%"6.-#"(2(0-'.&(+"

   .'"*2+.".%"-#("72*++"+2/&(:"-#(%",("'(5('"-."*"6/3.2*'"    &/+-'/6$-/.%="X5"+3.'(+",('("5.$%&".%2)".%"-#("72*++"      +2/&(:" -#(%" -#(" +*432(" 0.%+/+-(&" (%-/'(2)" .5" %.%C 0#*'7(&"+3.'(+="

  ?#(" 2/4/-" .5" &(-(0-/.%" .5" +3.'(" 0#*'7(+" &(3(%&(&"      .%" -#(" &/+-*%0(" 6(-,((%" -#(" (2(0-'.&(+" *%&" .%" -#("    +/h(".5"-#("+3.'(+="X-",*+"-#("2.,(+-:",#(%"-#("+#.'-(+-" &/+-*%0("6(-,((%"(2(0-'.&(+",*+"$+(&"-."0.22(0-"+4*22(+-"   B?>A4B
8=
C74
5>DA
B?4284B
D=34A
2>=B834A0C8>=
P 
4" /( 75%08)!% G" *%&" -#(" #/7#(+-:" ,#(%" -#(" ,/&(+-" &/+-*%0(" 6(-,((%" (2(0-'.&(+" ,*+" $+(&" 5.'" 7'(*-(+-" +3.'(+"   
  P 
4
/( 1(&5(#!16 G=" ?#('(5.'(:" *0-$*22):" *" %.%C h
            0#*'7(&" 3.'-/.%" .5" +3.'(" +*432(+" 0.$2&" .1(',#(24" %.-" A+B2%"(4@((6EX`ICAR' DIY`XIBa`IUT' UF' YVUXE' YEDISET`' DETYI`e' UT' .%2)"-#.+("+3.'(+"-#*-"&/&"%.-"0*'')"(2(0-'/0"0#*'7(+:"6$-" EREC`XUDEY'YCHESA`IC'XEVXEYET`A`IUT'UF'`HE'SEAT'YVUXE' *2+."-#.+("+3.'(+"-#*-"0*''/(&"3.+/-/1(".'"%(7*-/1("0#*'7(+"  
 
  
 


  


8=BD5Q284=C
5>A
C74
0CCA02C8>=
>5
B?>A4B
C>
E4AC820;
?;0C4B EREC`XUDE* copy -"D##E136*'121%&#(=#&F1#60('1#+F3'?18&(82366# .5"-#("(@3.+$'(:"-#("2*+-"+3.'(+",('("7/1(%"-#("-/4(" G*(&,1%&# -."+(--2("E>"4/%$-(+G="R$'/%7"%.%C(@3.+$'(+:"-#("+2.-" g(*+$'/%7" -#(" 0#*'7(C-.C4*++" D$.-/(%-" .5" *" ,*+" 0.1('(&" 6)" *" 3*3('" 2/&=" V(5.'(" *%&" *5-('" -#(" 4/0'.+0.3/0" 3*'-/02(" 6)" 4(*%+" .5" -#(" &(02/%*-/.%" (@3.+$'(:"-#("1*2$(".5"-#("1.2-*7(".%"-#("-('4/%*2+" .5"/-+"-'*B(0-.')"5'.4"-#("1('-/0*2",#(%"5*22/%7"$%&('" ,*+"0.%-'.22(&= 6A0E8CH
0=3
0
7><>64=>DB
7>A8I>=C0;
4;42CA82
Q4;3
8B
*" ,(22CO%.,%" 4(-#.&" 89LC9T/
QABC
DB43
1H
>??4A
-"@#70('1#32(*%&6#,%#3#632041 *%&" a*6)" /%" 9UA9" 89U<=" X%" 4)" 0*+(:" -#(" &(02/%*-/.%" ?#("(2(0-'.&(+",('("+0*%%(&"$%&('"*"2/7#-"4/0'.+0.3(" 5'.4"-#("1('-/0*2",*+"(D$*2"-."-#("'*-/.".5"El"G_"E'G:" T  
DB8=6

<82A>B2>?82
Q4;3B
?4A
4;42CA>34
4E4=;H
,#('(""/+"-#("&/+-*%0("6(-,((%"-#("(2(0-'.&(+"*%&" +/-$*-(&".1('"-#("+0*%%/%7"*'(*:"5.'4/%7"9K"1('-/0*2"*%&" '"/+"-#("&/+-*%0(".5"-#("+3.'("+(--2/%7"+/-("5'.4"-#(" >A"#.'/h.%-*2"+('/(+G="?#("+3.'(+",('("0.$%-(&"/%"(1(')" $33('" (&7(" .5" -#(" (2(0-'.&(=" ?#(" +3.'(+" +-$&/(&" Q4;3
*74
0<>D=C
>5
B?>A4B
>=
C74
B20==8=6
0A40
Author's",*+" ,('("+4..-#:"+$6+3#('/0*2"*%&"/%"-#("+/h("6)",#/0#" 0.43$-(&"*+"5.22.,+_ -#("&'*7"5.'0("(@('-(&"6)"-#("*/'"-."-#("4.-/.%".5"-#(" ! +3.'("/+"3'(&/0-(&"6)"-#("I-.O(+d"a*,"89T<="?#('(5.'(" 2 -&.//%/0 "" " = × ∑ 1# E9G: -#(" D$.-/(%-" .5" -#(" +3.'(" 0#*'7(" E4G( *%&" -#(" +3.'(" !× 2 $%&'()%*+, #=! 4*++"E0G"0.$2&"6("5.$%&"6)"-#("(D$*-/.%_"

,#('("+0*%%/%7"/+"-#("*'(*"+0*%%(&".%"-#("(2(0-'.&(" "  ! E;G: ; .5">A@99"44 :"<82A>Q4;3
C74
0A40
>5
C74
<82A>B2>?82
Q4;3
; .5"Y=YUL"44 :"7"-#("/%-(7('"(D$*2"-."-#("#(/7#-".5"-#(" ,#('(" 40( /+" -#(" +3.'(" 0#*'7(C-.C4*++" D$.-/(%-" (2(0-'.&("/%"-#("$%/-+".5"4/22/4(-'(:"'"-#("/%-(7('"(D$*2" E4;0G:" &" -#(" *00(2('*-/.%" .5" 7'*1/-):" " -#(" &/+-*%0(" -." -#(" &/+-*%0(" E/%" -#(" $%/-+" .5" 4/22/4(-'(G" 5'.4" -#(" 6(-,((%"-#("(2(0-'.&(+:" "/+"-#("&/55('(%0(".5"3.-(%-/*2" (2(0-'.&(d+"$33('"(&7("-."-#("0(%-'(".5"-#("4/0'.+0.3/0" .%" -#(" (2(0-'.&(+:" *%&" '" /+" -#(" &/+-*%0(" .5" -#(" +3.'("

Q4;3
0=3
@#"-#("4(*%"%$46('".5"+3.'(+"3('"4/0'.+0.3/0" 5'.4"-#("$33('"(&7(".5"-#("(2(0-'.&("8L:9LC9T<="V*0O(&" Q4;3
;>20C43
0C
C74
38BC0=24
'="?#("*4.$%-".5"-#("+3.'(+" .%"-."(D$*-/.%"E;G:"X"&('/1(&"*%"(D$*-/.%"-."0*20$2*-("-#("

.%" -#(" 72*++" +2/&(" E-#(" 4/&&2(" -'*%+1('+*2" 3*'-" .5" -#(" 4(*%"1*2$(".5"-#("+3.'("0#*'7(C-.C4*++"D$.-/(%-"E "! G +2/&("6(/%7"99"44",/&(G",*+"5.$%&"/%"*"+/4/2*'",*)=" .5"-#("0.22(0-/.%".5"+3.'(+"&(3.+/-(&".%"*%"(2(0-'.&(_

/0/

65 !8&'""#

! 3.-(%-/*2"6(-,((%"-#("(2(0-'.&(+:"∆;"-#("'(2*-/1("(''.'" $" #  >5
C74
4;42CA82
Q4;3
BCA4=6C7
20DB43
1H
C74
=>=?0A0;;4;
&' "" " """  E>G: 3.+/-/.%" .5" -#(" (2(0-'.&(+:" ∆ "! o "! %$&%" -#(" '(2*-/1(" (# ! ! #% (''.'" .5" -#(" +3.'(" 0#*'7(C-.C4*++" D$.-/(%-" 0*$+(&" $  " 1H
C74
4364
45542C
>5
C74
4;42CA82
Q4;3
U"
C74
F83C7
>5
"" -#(" +2.-:" ?∆@∆A;" -#(" '(2*-/1(" (''.'" .5" -#(" #.'/h.%-*2"

,#('("&:""*%&" "*'("*+"*6.1(:"7"/+"-#("/%-(7('"E,#.2(" 0.43.%(%-".5"-#("-'*B(0-.')".5"-#("+3.'(:"∆ "! o "! $%#) %$46('G"(D$*2"-."-#("#(/7#-"E44G".5"-#("(2(0-'.&(:"'"-#(" -#("'(2*-/1("(''.'".5"-#("+3.'("0#*'7(C-.C4*++"D$.-/(%-" /%-(7('"(D$*2"-."-#("&/+-*%0("E44G"5'.4"-#("(2(0-'.&(d+" 0*$+(&" 6)" -#(" &(02/%*-/.%" .5" -#(" (2(0-'.&(+" 5'.4"

$33('" (&7(:" "! " -#(" 4(*%" %$46('" .5" +3.'(+" 3('" -#(" -#(" 3.+/-/.%" .5" 1('-/0*2" +-*%&/%7" ,/-#" $33('" (&7(+" <82A>B2>?82
Q4;3
0C
C74
38BC0=24
'="?#("4(*%"1*2$(".5" 6(/%7" #.'/h.%-*2=" X%" -#(" /%+-'$4(%-*2" (''.':" -#(" 4/%.'" -#(" +3.'(" 0#*'7(C-.C4*++" D$.-/(%-" 5.'" 6.-#" 3*'-+" .5" *" 2>=CA81DC8>=B
20<4
5A><
C74
C7A44
QABC
4AA>A
2>=4=CB" +3.'("+*432(:"-#("+3.'(+",/-#"%(7*-/1("0#*'7(+"*%&"-#(" ∆;
>p:"∆ ; ≈9p:"*%&"∆;
Kp:",#('(*+" +3.'(+",/-#"3.+/-/1("0#*'7(+",*+"0*20$2*-(&= C74
<09>A
2>=CA81DC8>=
20<4
5A><
C74
Q5C7
2>=4=C" (∆@A;
A;p=" ?#(" 0.%-'/6$-/.%" .5" -#(" (&7(C -"H#.6&,23&,(%#(=#&F1#2136*'121%
''(' 0.43.%(%-" ,*+" "≈9Yp" *%&" .5" -#(" &(02/%*-/.%C ?#(" 4(*+$'(4(%-" (''.'" .5" -#(" 4(*%" +3.'(" 0#*'7(C 0.43.%(%-:"
9;p="X%"(D$*-/.%"EKG:"-#("3*'-"0.1('(&"6)" -.C4*++" D$.-/(%-" ,*+" (1*2$*-(&" *+" *" +$4" .5" -#(" -#("+D$*'("'..-"+/7%"'(3'(+(%-(&"-#("/%+-'$4(%-*2"'(2*-/1(" /%+-'$4(%-*2"(''.'"*%&"-#("'*%&.4"(''.'=" (''.'=" ?#(" /%+-'$4(%-*2" '(2*-/1(" (''.'" ,*+" 4*@/4$4:"

*74
8=74A4=C;H
D=?A4382C01;4
RD2CD0C8>=
8=
C74
∆! "! o "! ≈AKp:" /%" -#(" (@3('/4(%-+:" ,#('(" -#(" &/+-'/6$-/.%".5"-#("&(%+/-)".5"+3.'("+(&/4(%-*-/.%".%"*%" &/+-*%0(" 6(-,((%" -#(" (2(0-'.&(+" ,*+" 4/%/4$4:"

(2(0-'.&(" ,.$2&" 0*$+(" " '*%&.4" (''.'=" m*%&.4" (''.'" 
<<
0=3
<8=8=B

5.'" ∆;:" (∆@A;:" ∆ "! o "! %$&%" *%& # copy ! ! ∆ "  $ $ "! o ! $%#)" ,('(" 3'.1/&(&" 6)" g*--/Ce24*'" P/+0#('" " " #  #  Ea*6.'*-.')".5"!/'"X.%/h*-/.%"*%&"e2(0-'/0*2"!('.+.2+".5" &'  " "" "  ""  " "  EAG: -#("n%/1('+/-)".5"?*'-$:"/%"-#("9UQY+G= (# # $ !! %&% ! ! $ $  "  " -"I#J34+*43&,(%#(=#&F1#+F3'?1#23?%,&*)1 ""  ""   ?#("4(*%"+3.'("0#*'7("E-#("4(*%"1*2$(".5"-#("0#*'7(" σ ,#('(" "! " /+" -#(" '*%&.4" (+-/4*-/.%" ,#/2(" -#(" .%"-#("+3.'(G"!",*+"0*20$2*-(&"*00.'&/%7"-."-#("(D$*-/.% .-#('"+)46.2+"*'("*+"4(%-/.%(&"3'(1/.$+2)="?#/+"(''.'" "

&(-('4/%(+" 5.'" "!
C74
;8<8CB
>5
C74

2>=Q34=24
"" !

0"
"! "" ELG: /%-('1*2="eD$*-/.%"EAG",*+"3'.1/&(&"6)"?b%$"gJ2+"EX%+-/-$-("

.5"g*-#(4*-/0*2"I-*-/+-/0+".5"-#("n%/1('+/-)".5"?*'-$G= ,#('("0"/+"-#("4*++".5"-#("+3.'("*%&""! "/+"-#("4(*%" ?#(" /%+-'$4(%-*2" (''.'" 0.%+/+-(&" .5" 4*%)" +3.'(" 0#*'7(C-.C4*++" D$.-/(%-=" ?." (@3'(++" 0#*'7(" 2>=4=CB
A4BD;C8=6
5A><
C74
85
1*2$(+"/%"(2(4(%-*')"0#*'7(+"E(G:"-#("'(+$2-+"'(0(/1(&" -#(" *++$43-/.%+" 4*&(" 5.'"Author's -#(" (D$*-/.%" E;G" *%&" 5'.4" /%" IX" $%/-+" E0.$2.46+G" ,('(" &/1/&(&" 6)" -#(" 0.%+-*%-" -#(" /43('5(0-" (@*0-%(++" .5" -#(" 4(*+$'(4(%-+" .5" -#(" 9=LY;
9YC9U"S"(C9="?#("2/4/-+"5.'" !",('("0*20$2*-(&"$+/%7" 5*0-.'+" .5" -#/+" (D$*-/.%=" ?#(" 0.%-'/6$-/.%+" .5" +.4(" C74
;>F4A
0=3
C74
D??4A
;8<8CB
>5
C74

2>=Q34=24

(''.'" 0.43.%(%-+" ,('(" %(72/7/62(" E&:" '(2*@*-/.%:" 'G" .'" /%-('1*2".5" "! =" 6*2*%0/%7"(*0#".-#('"E+3.'("5.'4"*%&"+/h(G".'"/43.++/62(" C>
4E0;D0C4
2>=E42C8E4
08AR>FB
1DC
B8G
2>=4=CB
-"!K##L16&,%?#&F1#),==1'1%+1#C1&<11%#&F1#213%6# ,('("-*O(%"/%-."0.%+/&('*-/.%="!+"-#("0.43.%(%-+",('(" (=#6320416 /%&(3(%&(%-" 5'.4" .%(" *%.-#(':" -#(" /%+-'$4(%-*2" (''.'" ?."*%+,('"-#("D$(+-/.%",#(-#('"+3.'("+*432(+"&/55('(&" ,*+"0*20$2*-(&"*00.'&/%7"-."-#("(D$*-/.% 6)" 4(*%" +3.'(" 0#*'7(C-.C4*++" D$.-/(%-" E.'" 6)" 4(*%" 0#*'7(G:" *" 7" -(+-" 5.'" +*432(+" .5" $%(D$*2" +/h(+" *%&"                """"""EKG:" !!!!! 1*'/*%0(+" ,*+" $+(&=" R(7'((+" .5" 5'((&.4" ,*+" (D$*2"

-."19@1;P 
F74A4
19"*%&"1;",('("-#("-.-*2"%$46('+".5" ,#('(" ∆" ,*+" -#(" *6+.2$-(" (''.'" .5" -#(" &/+-*%0(" +3.'(+".6+('1(&"/%"-#("+*432(+="!6.$-"9YY"-."9Y:YYY" 6(-,((%" -#(" (2(0-'.&(+" *%&" ∆;" -#(" '(2*-/1(" (''.'" .5" +3.'(+" ,('(" .6+('1(&" E0.$%-(&" /%" -#(" 4/0'.+0.3/0" -#("&/+-*%0("6(-,((%" -#("(2(0-'.&(+:"∆ "-#("*6+.2$-(" Q4;3B
?4A
B0A4
C74
346A44B
>5
(''.'" *%&" ∆ ; " -#(" '(2*-/1(" (''.'" .5" -#(" &/55('(%0(" .5" 5A443><
F0B
4@D0;
C>
C74
8=Q=8CH
C
C74
 
;4E4;
>5

/04

66 ()*$+%,-"%,.)&./&%0$&$1$2%#.+%"%,2&20"#-$& ./&3"+,4,.+5.#$+&./&%0$&60$11,)7+&,-),"#,7+&-#.75

B86=8Q20=24
5>A
C74
CF>
C08;43
C4BC
C74
2A8C820;
E0;D4
>5
7" *" 3.+/-/1(" 0#*'7(=" X%" /(3238)(#2)!:" (/-#('" (*0#" +3.'(" /+"9=UL="X5"7
 
C74=
0
B86=8Q20=C
38554A4=24
14CF44=
0*''/(&" *" 3.+/-/1(" 0#*'7(" E>" +*432(+G:" .'" *" '(2*-/1(2)" -#("4(*%+",*+"0.%02$&(&="" +4*22"3*'-".5"%.%C0#*'7(&"+3.'(+"0./%0/&(&",/-#"3.+/-/1(" +3.'(+" E9K" +*432(+G=" X%" /( !)1(" *%&" /( 1(&5(#!16:" -#(" 4*B.'/-)".5"+3.'(+",('("0#*'7(&:"(*0#".5"-#(4"0*'')/%7" 9"#M16*4&6 *"3.+/-/1("0#*'7(=" 9"!#N2(*%&#(=#60('16#,%#6320416 9"9#70('1#+F3'?18&(82366#G*(&,1%&# !2-.7(-#('" >>" +3.'(" +*432(+" ,('(" 0.22(0-(&" /%" -#(" X%" -(%" +*432(+:" -#(" 4(*%" +3.'(" 0#*'7(C-.C4*++"

5.$'" +/62/%7" +3(0/(+" .5" -#(" '%))(186( (&1(!5(86( 7'.$3=" D$.-/(%-" E "! 
F8C7
C74

2>=Q34=24
8=C4AE0;
!4.%7" -#(4" ;>" ,('(" +4*22:" 0.%+/+-(&" .5" 9Y;P >" ,*+" '(0.'&(&" EP/7$'(" >G=" ?#(" 1*'/*-/.%" '*%7(+" .5" A L CA C9 +3.'(+:"*%&"9Y"+*432(+",('("6/7:"0.%+/+-(&".5"9Y P " "!
F4A4
0B
5>;;>FB
  P  
9Y " S" O7 " 5.'" +3.'(+"E?*62(";G="?#("6/7"+*432(+",('("0.22(0-(&"/%"*22" /( !)1(
  P  T CA" S" O7C9" 5.'" /( 1(&5(#!16: +3(0/(+"E.%("+*432("/%"/(!)1((*%&"/(1(&5(#!16:"-,."/%"   P T CA" S" O7C9" 5.'" /( 3238)(#2)!" *%& /(7%508)!%" *%&" +/@" /%" /(3238)(#2)!G=" ?#(" +*432(+" .5"   P 
9YCA"S"O7C9"5.'(/(75%08)!%="?#(+("1*'/*-/.%" /( 75%08)!%" *%&" /( 3238)(#2)!" +3.'(+" ,('(" 4*&(" /%" '*%7(+" .1('2*33(&" (*0#" .-#('="?#(" 1*'/*-/.%" '*%7(" .5" &/55('(%-"+(*+.%*2"E1('%*2"*%&"*$-$4%*2G"*%&"0/'0*&/*%" /(3238)(#2)!"0.1('(&"-#("'*%7(+".5"-#(".-#('+="?#("4(*%"

E%.0-$'%*2:"4.'%/%7:"&/$'%*2"*%&"(1(%/%7G"3('/.&+"*%&"/%" +3.'(" 0#*'7(C-.C4*++" D$.-/(%-" "! " ,*+" *+" 5.22.,+_" /% &/55('(%-" */'" 0.%&/-/.%+=" S.%&/-/.%+" /%02$&(&" -#.+(" -#*-" /(!)1((  Z CA"S"O7C9:"/%"/(1(&5(#!16";=QTq9YCA"S"O7C9: ,('(" '(2*-/1(2)" 0.%+-*%-" */'" -(43('*-$'(" *%&" '(2*-/1(" /( 3238)(#2)!" >=LTq9YCA" S" O7C9" *%&" /%" /( 75%08)!% 7D<838CH
270=68=6
N L
0=3
N 
A4B?42C8E4;H
C>
A=>;q9YCA"S"O7C9"EP/7$'(">:"?*62(">G="""" -#.+(" -#*-" ,('(" 1*'/*62(" /%" -(43('*-$'(" *%&" #$4/&/-)" 
B86=8Q20=C
38554A4=24
&N  
14CF44=
B0

EAcS:" 9Kp" .'" TcS:" 9pG" E?*62(" 9G=" ?#(" +*432(+" .5 /%" "! " .00$''(&" /%" 9o>" .5" +*432(" 3*/'+" *%&" " *" %.%C /( 3238)(#2)!" +3.'(+" *2+." '(3'(+(%-(&" &/55('(%-" B86=8Q20=C
38554A4=24
>22DAA43
8=

>5
?08AB
*01;4
copy AG=" +$6+-'*-(+"E2/1/%7"*%&"&(*&"-'((+G=" >C7
C74
B86=8Q20=C
0=3
=>=B86=8Q20=C
38554A4=24B
.00$''(&" /%" /%-('+3(0/(+" 3*/'+:" /%-'*+3(0/(+" 3*/'+" *%&" 9"-#5(43',&B#),6&',C*&,(% 8=CA0B?428<4=
?08AB
>5
B?>A4
B0
B86=8Q20=C;H
+=8?>;0A?>B8C8E4
38BCA81DC8>=
>5
270A64B
F0B
834=C8Q43
8=
&/55('(&"/%-('+3(0/(+"3*/'+".5"/(3238)(#2)!(C(/(1(&5(#!16:" *22"+*432(+"E?*62(";G="X%"/(75%08)!%:"(*0#"+3.'("0*''/(&" /( 3238)(#2)!( C( /( !)1(:" /( 3238)(#2)!( C( /( 75%08)!%:

1CCaXXETCE'UF

4ASVRE'ATD'RISI`Y'UF'DE`EC`IUT SUaT`'UF'YVUXEY'IT'#5TEG6#5TUT9'5VUY 5&/, 5&2& 512*

!"#$%&)*

%&$'9$@'9'9''98@ 0U 7EY 7EY D9'BD9'BD'9YASVRE'TU*'%&$@

!"#&',-'+$&*

Author's   %&#'9$@ 0U 7EY 7EY D9'BD 9'BD '9TU*'%&#@

!"#121)%'+2%$

D9'BD9'BD'9TU*'('@'D9'BD9'BD'9TU*'(%9'((@' (%'9 @'(('9!@'9'("9'%&'9'BD'9"@)(''9$@'' 0U 7EY 7EY D9'BD9'BD'9TU*'("9'%&'@

!!9'9'BDD9'BDB'9!@'9'9'BD9'BD'9"@ 0U 7EY'SbEXe'FEcT 7EY D9'bEXe'FEc9'BD'9TU*'!!@

D'9 @''9!@''9"@' 0U 0U 7EY

!"#0-/3)%$/ BB9'%%*9'BB9'BB'9"@'BB9'BB9'%%!'9#@'BBD9'BBB9'BB' 0U 0U 7EY D9'D9'BD'9%%*@'D9'D9'BD'9%%!@ 98@

?$>,"(4@((4VUXE'YASVREY'EdASITED'IT'`HE'Y`aDe'UT'`HE'VXISAXe'EREC`XIC'CHAXGEY'UF'BAYIDIUYVUXEY'IT'!4/%%'&)*#',&'$-')*'GXUaV'RISI`Y'UF'DE`EC`IUT'

UF'`HE'YVUXE'CHAXGEC`UCSAYY'WaU`IET`'`HE'UCCaXXETCE'UF'VUYI`IbERe'CHAXGED9'TEGA`IbERe'CHAXGED'ATD'TUTCCHAXGED'VUX`IUTY'S0VUY9'0TEG'

ATD'0TUTT*'$dAC`'ASUaT`Y'UF'YVUXEY'cEXE'DE`EXSITED'IT'`ET'YASVREY'SYASVRE'TaSBEX'IY'SAXQED'IT'BURD'FUT`T9'UXDEXY'UF'`HE'SAGTI`aDE'UF' `HEYE'ASUaT`Y'AXE'GIbET'IT'`HE'XIGH`'CURaST*'5HE'U`HEX'YASVREY'CUTYIY`ED'UF'BD'i'BD'YVUXEY*')AXQY'FUX'RISI`Y'UF'DE`EC`IUT'S
BDC'"'QGCBT 'i'B*DqD*9'!'i'B*qD*D'UX'B*qD*B9'"'i'B*qD*B9'#'i'D*DqD*B9'$'i'D*qD*'UX'D*DqD*D9'8'i'FXUS'B*' `U'D**'

/05

67 !8&'""#       

         
   

  
  



    
! 


    

  


   
   copy


  

        

A+B2%"(9@(()EAT'YVUXE'CHAXGEC`UCSAYY'WaU`IET`'ScHI`E'BAXYT'IT'`HE'YVUXE'YASVREY'aYED'FUX'`HE'Y`aDe'UF'BAYIDIUYVUXEY'IT'!4/%%'&)*#',&'$-')*'GXUaV' ATD'`HE'RISI`Y'UF'DE`EC`IUT'SBRACQ'BAXYT'AVVRIED'IT'YASVREY*

:8 739'
89:;#<#=, 79'/ #A`AYE`    

XI`HSE`IC' 4`ATDAXD' B XI`HSE`IC' 4`ATDAXD' SATD'I`Y'YIfET 3ATGEA bAXIA`IUT 9= 3ATGEA AbEXAGE DEbIA`IUTB AbEXAGE DEbIA`IUTB ',&'$-')*CGXUaV' B*BDp*D * B*B  pD B  BD'YASVREY'TU*'9'B9'9'9' 9'BD9'BD9'BD9'BB9'ATD'BB !"#121)%'+2%$ Author'sB*Bp*D * B*  p B  'YASVREY'TU*'9'B9'9'9' 'ATD'BD '''FXaI`ITG'BUDe'UF'!"#121)%'+2%$# B*Bp*D * B*  p B 

'YASVREY'TU*'9'B'ATD'

!"#0-/3)%$/# *p* * D*  BpBD BB 

'YASVREY'TU*'BB'ATD'BB

?$>,"(9@(()EAT'YVUXE'CHAXGEC`UCSAYY'WaU`IET`'S73T'ATD'SEAT'YVUXE'CHAXGE'S7T'IT'`HE'YE`Y'UF'`HE'YVUXE'YASVREY'XEVXEYET`ITG'`HE'',&'$-')*CGXUaV9' `HE'YVECIEY'UF'!"#121)%'+2%$'ATD'!"#0-/3)%$/6'ATD'A'FXaI`ITG'BUDe'UF'!"#121)%'+2%$*'

' $
& 
/+&
)'%
+!
$'.)
$"%"+
'
'&2&
"&+)-$
'
+!
*%$$*+
-$,
"&
+!
*+
+'
+!
,(()
$"%"+
'
'&2&
"&+)-$
'
+!
" *+
-$,
"&
+!
*+





# 
+&)
-"+"'&
+-
&
' 2"&+
'
-)"+"'&
-
.)
$,$+
0
+!
3,&"*4
%+!'
'&*")"&
+!
*"1
'
+!
+*+
)*'&,#04/#>2%%2?'&,#>2-3)%$*@# ###


&


.!)
/
"*
+!
-) 
'
+!
+*+
&
+!
*"1
'
+!
+*+
&

/06

68 ()*$+%,-"%,.)&./&%0$&$1$2%#.+%"%,2&20"#-$& ./&3"+,4,.+5.#$+&./&%0$&60$11,)7+&,-),"#,7+&-#.75 6' B*B B* D*B D*B D* B*B B* B* B* !"#0-/3)%$/ ''' B*B D* D*DB D* +,'+ ',*% ',+$ +,&! ',(" D*B D*D D* B*D D*B D* ',"+ B* B*D B* D*B B* D* D*B +,!( +,+% ',"' +,#* !"#&',-'+$&* !"#$%&' 6 B*B B*B D* B* B*D B* ',%" ',+$ ',%" 'GXUaV'bARaEY'UF'`'ACCUXDITG'`U'`HE'ITDEVETDET`'`cU'YASVRE' !4/%%'&)*#',&'$-')* '6 D* D* D* B* B* B* D* ',** ',!& copy ''' D*D B*D D* ',($ ',%& ',"% ',** +,%" +,&+ !"#121)%'+2%$ D* B* D* B* D* ',&' '' D* B*D D*D B* D* D* ',%" ',+$ +,&+ +,%" +,+# +,*$ Author's D*B B* D* +,+& ',*# ',!' ',!& +,*$ +,+#    B  BB BB BD BD BD 4ASVRE 'SBBBBT B'ST 'SBDBBT 'SBT 'SBT BD'ST BD'SBT BD'SBT BB'SBT BB'SBBT h h '' 6 ''' ''' 6' '6 BUDe %XaI`ITG' #IFFEXETCE'BE`cEET'`HE'YVUXE'YASVREY'IT'SEAT'CHAXGEC`UCSAYY'WaU`IET`'SABUbET'ATD'SEAT'YVUXE'CHAXGE'SBERUcT'IT'`HE' `'`EY`'FUX'`HE'YASVREY'UF'aTEWaAR'YIfE'ATD'aTEWaAR'bAXIATCE* ,%)*
"&
)#+*

%',&+
'
*(')*
"&-'$-
"&+'
+!
$,$ +"'&
'
+!
%& 3 3 3 3 3 3 !"#$%&' 4VECIEY !"#0-/3)%/$ !"#&',-'+$&+/ !"#121)%'+2%$ ?$>,"(C@(( '
&
'$

+ 

*" &"2&+
" )&
 

/07

69 !8&'""#

/(75%08)!%(C(/(!)1("*%&".5"/(75%08)!%(C(/(1(&5(#!16" P 
4
5>A
/(1(&5(#!16
P 
4
5>A
/(3238)(#2)!"*%& >22DAA43
*74
>22DAA4=24
>5
?08AB
>5
=>=B86=8Q20=C;H
 P 
4
5>A( /(75%08)!%( E(" /+" -#(" (2(4(%-*')" 0#*'7(" &/55('(&" +*432(+" /%" (1(')" -,.C0.46/%*-/.%" 5'.4" 5.$'" (D$*2" -." -#(" 0#*'7(" .5" *%" (2(0-'.%" /%" 3.+/-/1(" +/7%:" +3(0/(+" +#.,(&" -#*-" -#(+(" +3(0/(+" &/&" %.-" &/55('" 9=LY;q9YC9U" SG=" ?#(" *6+.2$-(" 1*2$(+" .5" 4(*%" +3.'("

5'.4" (*0#" .-#('" /%" "! =" P$'-#('4.'(:" -#(" '(+$2-+" /% 0#*'7("!",('("*+"5.22.,+_"/%"/(!)1("9YT"(:"/%"/(1(&5(#!16" /(3238)(#2)!
B7>F43
C70C
=>=B86=8Q20=C
38554A4=24B
8=
;YA" (:" /( 3238)(#2)!" 9K;" (" *%&" /( 75%08)!%" 99Q" ("

"! ".00$''(&"6(-,((%"-#("+*432(+".'/7/%*-/%7"5'.4"6.-#" EP/7$'("A:"?*62(">G= *"0('-*/%"5'$/-/%7"6.&)"*%&"&/55('(%-"5'$/-/%7"6.&/(+="?#(" 
B86=8Q20=C
38554A4=24

&N  
14CF44=
B0

B0<
4027
>C74A
>22DAA43
"!
>22DAA43
8=

>5
B0=B86=8Q20=C
6.-#"/%"-#("+3(0/(+"*%&"/%"*"5'$/-/%7"6.&)="?#("'*%7(+".5" &/55('(%0(" .00$''(&" /%" ;o>" .5" 3*/'+" E?*62(" AG=" V.-#" -#(" /%-'*+3(0/4(%" E,/-#/%" *" 5'$/-/%7" 6.&)G" 1*'/*-/.%" *%&" .5" B86=8Q20=C
0=3
=>=B86=8Q20=C
38554A4=24B
>22DAA43
8=

-#("/%-'*+3(0/(+"E,/-#/%"*"+3(0/(+G"1*'/*-/.%".5" "! ",('(" /%-('+3(0/(+"3*/'+:"/%-'*+3(0/(+"3*/'+"*%&"/%-'*+3(0/4(%"

*0-$*22)"-#("+*4("E?*62(">G=" 3*/'+".5"+3.'("+*432(+="!+"/%"-#("0*+(".5" "! :"-#("'(+$2-+" +#.,(&:"-#*-"-#("&/55('(%0(+"6(-,((%"*22"-#(+("+3(0/(+"/%" 9">#JF3'?1#23?%,&*)1 !
F4A4
=>=B86=8Q20=C
=
/( 3238)(#2)! :" -#(" +*432(+" ?#(" *6+.2$-(" 1*2$(+" .5" 4(*%" +3.'(" 0#*'7(" E !G" ,('(" 38554A8=6
B86=8Q20=C;H
5A><
4027
>C74A
>22DAA43
1>C7
8=
C74
5.$%&" /%" -#(" -(%" +*432(+" EP/7$'(" AG=" e1(')" 1*2$(" +3(0/(+" *%&" /%" *" 5'$/-/%7" 6.&):" *%&" -#(" '*%7(+" .5" F0B
2>=B834A43
F8C7
C74

2>=Q34=24
8=C4AE0;
/%-'*+3(0/4(%"1*'/*-/.%"*%&".5"-#("/%-'*+3(0/(+"1*'/*-/.%" ?#(" 1*'/*-/.%" '*%7(" .5" !
F0B
P 
4
5>A
/( !)1(: .5" !",('("*0-$*22)"-#("+*4("E?*62("AG="""   


    copy          

          



  

         

  

  Author's 

   

      

     





   
   

A+B2%"(C@(()EAT'YVUXE'CHAXGEY'ScHI`E'BAXYT'IT'YVUXE'YASVREY'aYED'FUX'`HE'Y`aDe'UF'BAYIDIUYVUXEY'IT'!4/%%'&)*#',&'$-')*'GXUaV'ATD'`HE'RISI`Y'UF' DE`EC`IUT'SBRACQ'BAXYT'AVVRIED'IT'YASVREY*'4VUXEY'AXE''AYYaSED'`U'HAbE'`HE'SEAT'YVUXE'YIfE'ATD'`HE'DETYI`e'UF'cA`EX*

/08

70 ()*$+%,-"%,.)&./&%0$&$1$2%#.+%"%,2&20"#-$& ./&3"+,4,.+5.#$+&./&%0$&60$11,)7+&,-),"#,7+&-#.75

>"#O,6+*66,(% "(632586" #*+" *" 4$0#" 2.,('" 1*2$(" EY=YUq9YCA" S" O7C9G="

*74
A4BD;CB
>5
C78B
BCD3H
P
0

J "! r" /+" /%" -#(" '*%7(" .5" >"!#5(43',&B#),6&',C*&,(%#   P T CA" S" O7C9" /%" '%))(186( !)1(5( /(1(&5(#!165( X%" /( 75%08)!%5( /( 3238)(#2)!( *%&( /( !)1(:" -#(" 3.2*'/-)" /( 3238)(#2)!( *%&" /( 75%08)!%
P
2>8=28343
F8C7
C74
&/+-'/6$-/.%".5"+3.'("0#*'7(+"*33(*'(&"-."6("/%&(3(%&(%-" 4*B.'/-)".5"-#("(*'2/('"'(+$2-+"E0.%-'*+-(&"-."-#("'(+$2-+"/%" .5" +3*-/*2" .'" -(43.'*2" 5*0-.'+:" */'" 0.%&/-/.%+:" *%&" X7%5621()(!(3%53)%@!1#%"*%&"7&!5(#86""(632586G="

+$6+-'*-(+="?#/+"0.''.6.'*-(&"-#("'(+$2-+".5"W'(7.')"8A<( ?#(" &/55('(%0(" 6(-,((%" -#(" (@-'(4(+" .5" r "! r" ,*+" *%&"I,/%6*%O"%7(!)/"8K<",#."+#.,(&"-#("+*4("3.2*'/-)" ;=>TC5.2&"/%"/(3238)(#2)!"E%.="TT:"%.="U;G"*%&(9=>;C5.2&" &/+-'/6$-/.%" /%" *22" +*432(+" .5" %538)!( )!#5A0!16( E-#(" /%(/(75%08)!%(E%.="99A:"%.="99TG="?#(+("&/55('(%0(+",('(" $%/3.2*'C%(7*-/1("&/+-'/6$-/.%"/%"+(1('*2"+3.'("+*432(+" (D$*2"-."*";=AYC5.2&"&/55('(%0("/%"7/(#!03%675(6
QABC
0=3
5'.4".%("5'$/-/%7"6.&)G"*%&".5"!12$%50!(!33)!1!780" -#/'&"+*432(+G"8L<"*%&"-."*"9=UQC5.2&"&/55('(%0("6(-,((%"

E-#(" $%/3.2*'C3.+/-/1(" &/+-'/6$-/.%" /%" *6.$-" &.h(%" -#(" (+-/4*-(+" .5" r "! r" 5.'" %538)!( )!#5A0!16" 7/1(%" /%" +*432(+"0.4/%7"5'.4"+(1('*2"5'$/-/%7"6.&/(+"*-"+(1('*2" 8K<" *%&" 8L<=" X-" ,*+" +$33.+(&" 8L<" -#*-" -#(" 4*/%" 5*0-.'" 7(.7'*3#/0*2"2.0*-/.%+"&$'/%7"-,.")(*'+G="" '(+3.%+/62("5.'"*"2*0O".5"+/4/2*'/-)"6(-,((%"-#("1*2$(+"

?#("'(+$2-+"/%"/(!)1(:(/(1(&5(#!165(/(3238)(#2)!"*%&" .5"r "! r"5.'"/()!#5A0!16"5.$%&"6)"&/55('(%-"'(+(*'0#('+" /(75%08)!%"+#.,(&".%2)".%("-)3(".5"3.2*'/-)"E3.+/-/1(" 4*)"6("-#("&/55('(%0(".5"-(0#%/D$(+".5"(@3('/4(%-+="!" $%/3.2*'/-)G" 5.'" *" 7'.$3" .5" 3#)2.7(%(-/0*22)" 02.+(2)" =0CDA0;
?74=><4=>=
>5
0
B?4284B
P
C74
E0A80C8>=
>5
B8I4B
'(2*-(&"+3(0/(+="?#/+"+$33.'-(&"-#("'(+$2-+"/%"E235(186( >5
B?>A4B
P
2>D;3
14
0<>=6
C74
502C>AB
A4B?>=B81;4
5>A
'(!6#%16
A

"0=64

E235(1%))86('(!6#%16"EP'=G" C74
8=CA0B?428Q2
E0A80C8>=
>5
C74
<40=
B?>A4
270A64BC> m(&#(*&" %7( !)=G" *%&" E235(186( 0(#!#%86" EV$22=G" P'= 4*++"D$.-/(%-"E+(("-#("+(0-/.%".5"0#*'7("4*7%/-$&(G="" 
E235(1%))86( 0(#!#%86" EV$22=G" `/27*2)+" %7( !)=G" 8A<=" X%" -#.+("+-$&/(+"/-",*+"&(4.%+-'*-(&"-#*-"-#("+3(0/(+".5"-#/+" >"9#JF3'?1#23?%,&*)1# '(2*-/1(2)"+4*22"4.%.3#)2(-/0"7(%$+"E235(1%))865(+#.,(&" ?#("+3(0/(+".5"-#("(&1(!5(86C7'.$3"E/(!)1(:(/(1(&5(#!165( -#("+*4("-)3(".5"-#("3.2*'/-)"&/+-'/6$-/.%"E-#("6/3.2*'/-)" /(3238)(#2)!copy"*%&"/(75%08)!%G"#*&"4(*%"+3.'("0#*'7(+" ,/-#"-#("3'(1*/2/%7"%(7*-/1("3*'-G="?#("$%/5.'4/-)"/%"-#(" 8=
C74
A0=64
>5
P 
4
*78B
06A443
F8C7
C74
<09>A8CH
-)3(" .5" -#(" 3.2*'/-)" &/+-'/6$-/.%+" +#.$2&" /%&/0*-(" -#*-" .5"-#("(*'2/('"3$62/+#(&"'(+$2-+"8K:L<"+#.,/%7"*"'*%7(".5" -#(" +*4(" 0#(4/0*2" +$6+-*%0(+" *'(" /%1.21(&" /%" -#(" P 
4
5>A
C74
4867C
B?4284B
0=3
2>=CA0BC43
C>
C74
3'.0(++".5"0#*'7("5.'4*-/.%"/%"3#)2.7(%(-/0*22)"02.+(2)" '(+$2-+" /%" -,." +3(0/(+:" Q" (" 5.'" 7&!5(#86( "(632586" *%&" '(2*-(&"+3(0/(+="?#('("*'("2/O(2)"-."6("-,."(%&.7(%.$+" L:Q9Y"("5.'"7%5621()(!(3%53)%@!16/" 3'.0(++(+",#/0#"0*%"#.+-"-#(+("+$6+-*%0(+="P/'+-2):"-#(" X%" -#(" 0*20$2*-/.%+" .5" 0#*'7(+:" -#'((" *++$43-/.%+" 5.'4*-/.%".5"-#("V$22('d+"&'.3="R/55('(%-"3#)+/0.0#(4/0*2" ,('("4*&("-#*-"*22"0.%0('%(&"-#("4*++".5"5*22/%7"+3.'(+_" 3'.0(++(+"*'("/%1.21(&"/%"0.%&(%+*-/.%".5",*-('"$3.%" 9=" !" +3.'(" 4.1(+" *2.%(:" ,/-#.$-" -#(" V$22('d+" &'.3=" -#("+3.'("+$'5*0(="I(0.%&:"-#("'$3-$'(".5"-#("0.%-*0-/%7" ;="?#("&(%+/-)".5"*"+3.'("/+"(D$*2"-."-#("&(%+/-)".5",*-('="" ,*22+" .5" -#(" +3.'(" *%&" +-('/74*=" ?#(" +3.'(" 7*/%+" *%" >="I3.'(+"#*1("-#("+*4("+/h(="!"&/+0$++/.%".5"(*0#".5"-#(+(" (2(0-'/0"0#*'7("*-"-#("4.4(%-".5"(B(0-/.%"5'.4"+-('/74*=" *++$43-/.%+"*%&"-#("0.''(+3.%&/%7"/432/0*-/.%+"5.22.,+= *78B
4;42CA8Q20C8>=
2>D;3
>22DA
3D4
C>
C74
4;42CA82
?." 0*20$2*-(" -#(" 4*@/4$4" 3.++/62(" (''.'" .5" r !r: 3.2*'/h*-/.%" /%" +$6+-*%0(+" +$6B(0-/%7" -." 4(0#*%/0*2" -#(" ,#.2(" *4.$%-" .5" 2/D$/&" 4$+-" 6(" 0.%+/&('(&=" !%" BCA08=
CA81>4;42CA8Q20C8>=
%A
3D4
C>
C74
B4?0A0C8>=
>5
Author's(+-/4*-("5.'" /(3%53)%@!16:"*++$4/%7"-#("&'.3"-."#*1(" &/55('(%-"2*)('+"0.%+/+-/%7"-#("(2(0-'/0*2"&.$62("2*)('"/%" -#("&/*4(-('".5"9Y"44:"/+"7/1(%"/%"8L<_"-#("/7%.'/%7".5"-#(" -#("+3*0("6(-,((%"-,."+(3-*="!%"(%&.7(%(.$+".'/7/%" 2/D$/&".5"-#("V$22('d+"&'.3".%"-#("5*22/%7"+3.'(+"4*O(+" 5.'" 3'/4*')" (2(0-'.+-*-/0" 0#*'7(+" .5" 6*+/&/.+3.'(+" #*+" -#("0#*'7("Y=L9C5.2&"+4*22('=" 6((%"+$77(+-(&"(*'2/('"8K:;Y<"6$-"%."(1/&(%0("-."+$33.'-" e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r !r",('("%.-".1('(+-/4*-(&:"6$-"-#()"0.$2&"#*1("6((%" I/%7(':"!5!60(86(25%!$%6"EV.2-.%G"P'=:"#'(B23'A))80( $%&('(+-/4*-(&"+2/7#-2)"EY=T>C5.2&"/5"-#("+3.'("&(%+/-)"/+" #20081%"P'=:"%538)!()!#5A0!16:"*%&"8())86()87%86"Ea=G" (D$*2"-."9=;9q9Y>"O7"4C>G= CA C9 m.$++(2"#*1("r "! J
8=
C74
A0=64
>5
 P T "S"O7 ^ ?#("%*-$'*2"1*'/*-/.%".5"-#("+3.'("+/h("E2(%7-#"*%&" ,#('(*+"7%5621()(!(3%53)%@!1#%(E?'(4(22.4)0(-(+G"#*+" ,/&-#G" /%" *" +/%72(" +*432(" E*G" *%&" -#(" 1*'/*-/.%" .5" -#(" *" 4$0#" #/7#('" 1*2$(" E9;=Tq9YCA" S" O7C9G:" *%&" 7&!5(#86( 4(*%" +3.'(" +/h(" /%" +*432(+" E6:" 0G" ,/22" 6(" &/+0$++(&"

/21

71 !8&'""#

6(2.,=" ?#(" &/+0$++/.%" /+" +$33.'-(&" 6)" -#(" 7(%('*2" *74
38B2DBB8>=
01>DC
C74
8=RD4=24
>5
C74
&(+0'/3-/.%" 7/1(%" 6)" N*'4*+-." *%&" N*'4*+-." 8;;<" .%" *++$43-/.%+" 4*&(" 5.'" -#(" 4*++" .5" -#(" 5*22/%7" +3.'(" -#("1*'/*-/.%".5"6*+/&/.+3.'(+"/%"-#("])4(%.4)0(-(+= +#.,+"-#*-"-#("4(*%"+3.'("0#*'7(+",('("0.%+('1*-/1(2)" E*G" ?#(" 5'(D$(%0)" &/+-'/6$-/.%" .5" -#(" +3.'(" +/h(" .5" *%&" %*''.,2)" (+-/4*-(&" /%" -#(" 3'(+(%-" +-$&)=" X-" *2+." *"+*432("/+"*33'.@/4*-(2)"%.'4*22)"&/+-'/6$-(&:"*"+4*22" +#.,+" -#*-" -#(" %*-$'*2" 1*'/*-/.%" .5" -#(" +3.'(" +/h(" /+" 3.+/-/1(" +O(,%(++" .00$'+" 8;;<=" R$(" -." -#(" +O(,:" -#(" .61/.$+2)"*%"/43.'-*%-"5*0-.'".5"/%-'*+3(0/(+"1*'/*-/.%+" 1*2$(+".5"r !r",('("+2/7#-2)"$%&('(+-/4*-(&"/%"-#("3'(+(%-" .5"+3.'("0#*'7(+=" +-$&)=" F.$2&"-#("0#*'7(+"*55(0-"-#("'(2(*+(:"-'*%+3.'-:".'" E6G" ?#(" +3.'(" 4(*%" +/h(" /+" %.-" 0.%+-*%-" /%" -#(" 34?>B8C8>=
>5
C74
B?>A4B
70A64B
3>
=>C
8=RD4=24
C74
+*432(+" .'/7/%*-(&" 5'.4" .%(" 5'$/-/%7" 6.&)" /%" &/55('(%-" '(2(*+(".5"+3.'(+"5'.4"-#("5'$/-/%7"6.&)"8L<="?#("e*'-#d+" )(*'+:" .%" &/55('(%-" &*)+" .'" *-" &/55('(%-" -/4(+=" X%" 508AF40C74A
4;42CA82
Q4;3
8B
38A42C43
3>F=F0A3B
0=3
8CB
?>;H?>A4B
C74
B?>A4
B8I4
70B
0
RD2CD0C8=6
=0CDA4
*1('*7("1*2$("%(*'"-#("7'.$%&"/+"*6.$-"9>Y"`"4S ="?#$+" (/%/:" 1*'/*-/.%" ,/-#.$-" 02(*'" 0.44.%" -(%&(%0/(+=" 8;;<=" -#("+3.'(+",/-#"*"3.+/-/1("0#*'7("*'("&'/5-/%7"&.,%,*'&+" M(72(0-/%7"-."0.%+/&('"-#("1*'/*-/.%".5"-#("4(*%"+3.'(" *%&"1/0("1('+*"5.'"%(7*-/1("0#*'7(="].,(1(':"-#("&'/5-" B8I4
2>D;3
14
A4B?>=B81;4
5>A
C74
B86=8Q20=C
38554A4=24B
+3((&" /+" 2.," 5.'" -)3/0*2" +3.'(+=" P.'" (@*432(:" *" +3.'(" 6(-,((%"-#("4(*%"+3.'("0#*'7(+"/%"-#("XX"5'$/-/%7"6.&)" F8C7
0
380<4C4A
>5

V<
0=3
F8C7
0
270A64
>5

4
.5"/(3238)(#2)!="S.%+/&('/%7"UU=Qp".5"-#("1*'/*-/.%".5" &'/5-+" *-" -#(" +3((&" .5" Y=Y;" 44" +S =" I$0#" *" +3((&" /+" -#("4(*%"+3.'("+/h(:"(/(%/
C74
A0=64
>5
.<40=
P

T
;4BB
C70=
>A38=0AH
RD2CD0C8>=B
>5
F8=3
E4;>28CH
0=3
8B
+-*%&*'&" &(1/*-/.%G:" E4(*%" s" >" q" +-*%&*'&" &(1/*-/.%G<" %."&(-('4/%/%7"5*0-.'"5.'"-#("+(&/4(%-*-/.%".5"+3.'(+=" E?*62("KG:"-#("7"-(+-"+#.,(&:"-#*-"%(72(0-/%7"-#("1*'/*-/.%" S#*'7(+" .5" 4*7%/-$&(" t9YA
4
?4A
B?>A4
>5

V<
.5" -#(" 4(*%" +3.'(" +/h(" 0.$2&" 6(" '(+3.%+/62(" 5.'" -#(" 380<4C4A
0A4
8=BD5Q284=C
C>
8=RD4=24
34?>B8C8>=
C>
B86=8Q20=C
38554A4=24B
>5
C74
<40=
B?>A4
270A64B 32*%-" +$'5*0(+" 8;>/
-74C74A
C74
270A64B
8=RD4=24
E0G"?#("1*'/*-/.%".5"-#("+3.'("4(*%"+/h("&(3(%&+".%" -#("0.%&(%+*-/.%".5",*-('".%"-#("+$'5*0(".5"+3.'(+".'" C74
B?4284B
*74
<40=
E0A80C8>=
2>45Q284=C
8B
 
C74
2>06D;0C8>=
copy >5
B?>A4B
F8C7
Q=4
3A>?B
8=
5>6
0=3
5.'" -#(" 4(*%" +3.'(" 2(%7-#" *%&" K=AC9>=Y" 5.'" -#(" 4(*%" 3A8II;4
C7DB
8=RD4=28=6
C74
F0B7>DC
>5
B?>A4B
5A><
C74
+3.'(" ,/&-#=" 8;;<=" !4.%7" -#(" 5.$'" +3(0/(+" +-$&/(& *-4.+3#('(G:"*'("D$(+-/.%+")(-"-."6("+-$&/(&="""" E(!)1(:"/(1(&5(#!16:"/(3238)(#2)!:"*%&"/(75%08)!%G:"-#(" 4(*%" +3.'(" 1.2$4(" /+" 2(*+-" 1*'/*62(" /%" /( 3238)(#2)!" E&/55('(%0(" 6(-,((%" -#(" (@-'(4(+" .5" -#(" +3.'(" 4(*%" @"#J(%+4*6,(%6#

1.2$4(" ! 4*@"_! 4/%" /+" 9=KC5.2&G" *%&" 4.+-" 1*'/*62(" /% /(!)1((E;=;C5.2&G="?."0.%+/&('"UU=Qp".5"-#("1*'/*-/.%".5" X%" -#(" 7'.$3" .5" -#(" +3(0/(+" +-$&/(&" E+/62/%7" +3(0/(+" -#(" 4(*%" +3.'(" +/h(" /%" *%" (+-/4*-/.%" .5" 0#*'7(+:" -#(" '%))(186( !)1(5( /( 1(&5(#!165( /( 3238)(#2)!5" *%& ;>F4A
;8<8C
>5
2>=Q34=24
8=C4AE0;
>5
J !r"5.$%&"/%"-#("0*+(" /(75%08)!%G:"*22"+*432(+".5"6*+/&/.+3.'(+"*'("(2(0-'/0*22)" .5" -#(" 4(*%" +3.'(" +/h(" E+#.,%" .%" P/7$'(" AG" 4$+-" 6(" 0#*'7(&="!22"(2(0-'.+-*-/0"0#*'7(+".%"+3.'(+"*'("3.+/-/1(= <034
P 5>;3
;>F4A
0=3
C74
D??4A
;8<8C
;3
78674A
*01;4
KG="X%"*22"+3(0/(+",/-#" C74
A0=64
>5
  P T CA" S" O7C9=" X%" /(3238)(#2)!:" 6*+/&/.+3.'(+" .5" 2.," 1*'/*6/2/-)" E/%02=" -#(" +3(0/(+" C74
E0A8018;8CH
F0B
7867
E0A80C8>=
2>45Q284=C

F8C78=
+-$&/(&G:"-#(+("%$46('+"0.$2&"6("$+(&"5.'"*"3'(2/4/%*')"Author's-#("+3(0/(+"*%&"K>p:",/-#/%"*"5'$/-/%7"6.&)G:"-#("'*%7(" (+-/4*-(".5"-#("1*'/*-/.%"'*%7(".5"r ! r".00$''/%7"&$("-." .5" /%-'*+3(0/(+" 1*'/*-/.%" 0./%0/&(&" ,/-#" -#(" '*%7(" .5" -#("%*-$'*2"1*'/*-/.%".5"+3.'("+/h(+= /%-'*+3(0/4(%" 1*'/*-/.%/" ?#(" +3(0/(+" +-$&/(&" &/&" %.-"

4VECIEY A'q''r' Bq''r' CSIT@C CSAdC )EAT'YVUXE'CHAXGE' 3ATGE'UF'`HE'SEAT' 4`DEb'SsST 4`DEb'SsST SET' YVUXE'CHAXGE'SET'

!"#$%&' *'q'D* *BB'q'D* D* B* BD i

!"#&',-'+$&* *'q'D* *'q'D* D* B* D i

!"#121)%'+2%$ *'q'D* *'q'D* D*BB B*B B i

!"#0-/3)%$/ *'q'D*B *D'q'D*D D* B*D BB i

?$>,"(0@((3ATGE'UF'SEAT'YVUXE'CHAXGEY9'CUTYIDEXITG'`HE'TA`aXAR'bAXIA`IUT'UF'SEAT'YVUXE'YIfE9'IT'FUaX'YVECIEY'UF'2HERRITaY'IGTIAXIaY'GXUaV*'3ATGE'  


 
 

 

 






 

 

'')#*

5
%&
*(')
$& +!
+-
5
*+&)
-"+"'&

5
%&
*(')
."+!

5
%&
*(')
-'$,%
3'&#D#%2?/-#%'3'0#2>#04/#3/$&#*12-/# -'$,%
3$E
5
!" !)
$"%"+
'
+!
%&
*(')
-'$,%
$,*
'



&
+-
)
*
"&
 F"

/20

72 ()*$+%,-"%,.)&./&%0$&$1$2%#.+%"%,2&20"#-$& ./&3"+,4,.+5.#$+&./&%0$&60$11,)7+&,-),"#,7+&-#.75

38554A
B86=8Q20=C;H
5A><
4027
>C74A
A460A38=6
C74
<40=
N+P%(<41)?121%&6# 0#*'7(C-.C4*++"D$.-/(%-+=" ?#(" 4(*%" +3.'(" 0#*'7(+" 1*')" /%" -#(" '*%7(" .5" e'*+-" N*'4*+-." /+" 7'*-(5$22)" *0O%.,2(&7(&" 5.'" P 
4

=
/(3238)(#2)!:" 1*'/*6/2/-)" /%" !" ,*+" #/7#:" *'.$+/%7"4)"/%-('(+-"/%"-#("3'/4*')"(2(0-'/0"0#*'7(+".5" ,#('(*+" -#(" '*%7(+" .5" /%-'*+3(0/4(%" 1*'/*-/.%" *%&" .5" 10B838>B?>A4B

C70=:
#0AC
#DA3E44
0=3
%;4E
!4A4<
-#("/%-'*+3(0/(+"1*'/*-/.%"0./%0/&(&/"?#("+3(0/(+"+-$&/(&" 5.'"-#(/'"*++/+-*%0("/%"&(+/7%/%7"*%&"6$/2&/%7"-#("&(1/0(:" 383
=>C
38554A
B86=8Q20=C;H
5A><
4027
>C74A
A460A38=6
C74
g*--/Ce24*'" P/+0#('" 5.'" #/+" #(23" /%" /%+-'$4(%-*2" (''.'+" 4(*%" +3.'(" 0#*'7(+=" " V.-#" -#(" &/55('(%0(+" *%&" -#(" 3'.62(4+:" *%&" ?b%$" gJ2+" 5.'" #/+" #(23" ,/-#" +-*-/+-/0*2" +/4/2*'/-/(+" /%" !" 6(-,((%" +3.'(" +*432(+" .00$''(&" /%" *%*2)+(+"*%&"H**%"I*24"5.'"0*20$2*-/.%".5"&'/5-"+3((&=" /%-('+3(0/(+:"/%-'*+3(0/(+"*%&"/%-'*+3(0/4(%"3*/'+="?#(" g*%)" -#*%O+" -." W(&/4/%*+" g*/%(2/+" 5.'" 0.44(%-+" .%" /%-'*+3(0/(+"1*'/*-/.%".5"-#("4(*%"+3.'("0#*'7("0.$2&" -#("4*%$+0'/3-="?#*%O+"*'("&$("-."n'1("g*'-/%+.%"*%&" &(3(%&".%"-#("%*-$'*2"1*'/*-/.%".5"-#("4(*%"+3.'("+/h(=" g*22"?*44"5.'"2/%7$/+-/0"#(23="

!"D"%"8&"#

89<" g.%()" M=N=:" g.'(" 7d+" -#*%" -#(" I3*0(" I#$--2(_" 

 P 
8=
(DBB80=
F8C7
BD<<0AH
8=
10;;8BC>B?>A4
38B270A64
#H2>;>680

 
 P e%72/+#G" KKT 899<" ?*44" u=:" !+3(%" /%" e+-.%/*:" e(+-/" a..&$+5.-.:" 8;<" N'/%72("!=:"N*-(O"I=M=:"P/+#('"g=:"I-.2h("H=:"g.%()" ?*'-$:";YYY:"E/%"e+-.%/*%:",/-#"+$44*')"/%"e%72/+#G"""""""" M=N=:" ?#(" 0*3-$'(&" 2*$%0#" .5" *" 6*22/+-.+3.'(:" 89;<" M/(4(2[" ?=:" N.2)3.'(+" /%" P/%%2*%&" *%&" e+-.%/*:" #H2>;>680


P e(+-/"a..&$+5.-.:"?*'-$:";YYT:"E/%"e+-.%/*%G 8><" V$22('" !=]=m=:" m(+(*'0#(+" .%" P$%7/:" `.2=" 9:" . /
44A8=6
(
>=6

(0<1>

#>=4H
$&
8AR>F
copy a.%74*%+:"a.%&.%:"9UYU 3*--('%+"*'.$%&"4$+#'..4+"*%&"-#(/'"'(2*-/.%+#/3" 8A<" W'(7.')" N=]=:"e2(0-'.+-*-/0" 0#*'7(+" .%" +3.'(+" .5" -."+3.'("&/+3('+*2:"g)0.2.7/*:";YY9:"U>:"Q>;CQ>L 5$%7/:"M*-$'(:"9UKQ:"9>Y:">>Y" 89A<" !'.2&" X=:" e+-.%/*%" 2*%&+0*3(+:" ?*'-$" u2/O..2/" 8K<" I,/%6*%O" N=:" ?*77*'-" H=:" ]$-0#/%+.%" I=!=:" ?#(" Z/'B*+-$+:"?*'-$:";YYK:"E/%"e+-.%/*%:",/-#"+$44*')" 4(*+$'(4(%-" .5" (2(0-'.+-*-/0" 0#*'7(+" .%" +3.'(+" /%"e%72/+#G .5"g('$2/$+"2*0')4*%+"EF$25=G"P'=:"!%%"V.-Ca.%&.%:" 89K<" I(22" X=:" ?*@.%.4)".5" -#(" +3(0/(+" /%" -#(" N#(22/%$+"  

P  86=80A8DB
6A>D?
#H2>C0G>=


P  8L<" F(6+-('" H=:" N'.0-.%" g=S=P=:" R*1()" m=!=:" 89L<" P$0#+"M=!=:"?#("4(0#*%/0+".5"*('.+.2:"!0*&(4)".5" g(*+$'(4(%-" .5" -#(" (2(0-'/0*2" 0#*'7(" .%" +.4(" I0/(%0(+".5"-#("nIIm:"g.+0.,:"9UKK:"E/%"m$++/*%G 6*+/&/.+3.'(+"*%&"*%"*++(++4(%-".5"-,."3.++/62(" 89Q<" W'((%" ]=:" a*%(" F=:" N*'-/0$2*-(" 02.$&+_" &$+-+:" 4(0#*%/+4+" .5" 6*22/+-.+3.'(" 3'.3$2+/.%:" ?" V'/-" +4.O(+" *%&" 4/+-+=" Z#/4/)*" N'(++:" a(%/%7'*&:" #H2>;
)>2

 
 P 9UQ;:"E/%"m$++/*%G" 8Q<" g.%()"M=N=:"P/+0#('"g=F=P=:"V/.4(0#*%/0+".5"+3.'(" 89T<" I(/%5(2&"H=]=:"N*%&/+"I=M=:"!-4.+3#('/0"0#(4/+-')" '(2(*+("/%"3#)-.3*-#.7(%+:"X%_"e++('"Z="Ee&=G:"?#("Author's*%&"3#)+/0+_"5'.4"*/'"3.22$-/.%"-."02/4*-("0#*%7(:" g)0.-*:"`.2="K:"!"0.43'(#(%+/1("-'(*-/+(".%"5$%7/" ;%&"(&=:"H.#%"F/2()"\"I.%+:";YYL *+" (@3('/4(%-*2" +)+-(4+" 5.'" 6*+/0" *%&" *332/(&" 89U<" ].33('" `=R=:" a*6)" ?=]=:" ?#(" (2(0-'.%" 0#*'7(:" N" '(+(*'0#:" R(+/%7" ]=V=" Ee&=G:" N2*%-" '(2*-/.%+#/3+:" (>H
)>2
">=3>=

 

P  ;%&"(&=:"I3'/%7('C`('2*7:"V('2/%"](/&(26('7:";YYU . /
)0E8;4
%
)?>A4
38B270A64
8=
10B838>A
&
=3A404
#%
&KB27;
+

D=8Q43
C74>AH
)284=24


  S.%-'/6$-/.%".5"5$%7/"-."3'/4*')"6/.7(%/0"*('.+.2+" 8;9<" W'(7.')"N=]=:"g/0'.6/.2.7)".5"-#("*-4.+3#('(:";%&" /%"-#("*-4.+3#('(_",(-"*%&"&')"&/+0#*'7(&"+3.'(+:" (&=:" H.#%" F/2()" \" I.%+:" M(," v.'O" *%&" ?.'.%-.:" 0*'6.#)&'*-(+:"*%&"/%.'7*%/0"/.%+:"!-4.+="S#(4=" 9UQ> N#)+=:";YYQ:"Q:"AKLUCAKTT"" 8;;<" N*'4*+-."e=:"N*'4*+-."X=:"`*'/*-/.%".5"6*+/&/.+3.'(+" ./
)4B0AC82

0;;0Q>A
*$
;>10;
5D=60;
B?>A4
8=
C74
H<4=>
C748A
(4/++/.%:"'(1/(,"*%&"+)%-#(+/+".5"2/-('*-$'("&*-*:" -*@.%.4):"V/62/.-#="g)0.2=:"9UTQ:"99K:"9C9LT V/.7(.+0/(%0(+:";Y99:"T:"99T9C99U;" 8;><" g0S*'-%()"]=!=:"V*/%V'/&7("!=:"a(77"V=H=:"e2(0-'/0" 89Y<" N*'4*+-."e=:"R(1(2.34(%-"*%&"+3.'("&/+0#*'7(" 0#*'7(" *%&" -#(" &(3.+/-/.%" .5" +3.'(+" .5" 6*'2()" .5" -#(" 5'$/-C6.&/(+" .5" N.2)3.'*0(*(:" e(+-/" MI`" 4/2&(,"e')+/3#("7'*4/%/+:"!-4.+="e%1/'.%=:"9UT;:" ?(*&$+-(" !O*&((4/*" ?./4(-/+(&:" V/.2..7/*:" 9L:"99>>C99A>""""""""

/22

73

II

75 Saar, Maret & Salm, Jaan (2014). Emission rate of charged spores in basidiomycetous fungi and the relaxation time of their electric charges. Aerobiologia, 30 (1), 71 - 89. personal copy

Aerobiologia (2014) 30:71–89 DOI 10.1007/s10453-013-9310-6

ORIGINAL PAPER

Emission rate of charged spores in basidiomycetous fungi and the relaxation time of their electric charges

Maret Saar • Jaan Salm

Received: 18 December 2012 / Accepted: 20 June 2013 / Published online: 4 July 2013 Ó Springer Science+Business Media Dordrecht 2013

Abstract Basidiospores are one of the main com- 8.6 9 107 charged spores ha-1 s-1. Calculations ponents of coarse fraction of atmospheric aerosol. showed that a spore charge diminished sevenfold Majority of them, the ballistospores of 20,000 species within 47 min. Ecologists, health and agricultural of Basidiomycotina, carry electrostatic charges when scientists could be interested in this information. It getting airborne. To study the polarity and magnitude could be useful by investigating the role of microor- of primary charges and the hymenial emission rate of ganisms in meteorological phenomena and in atmo- charged spores, 128 spore samples of 31 species of spheric processes in general. Agaricomycetes were collected in natural conditions. A portable device was placed under the fruiting body Keywords Agaricomycetes Ballistospore Á Á and the freely falling charged spores were extracted Charge-to-mass quotient Electric charge from the air by a horizontal homogeneous electrostatic Emission rate Charge relaxationÁ time Á Á field. The charge polarity distribution was the same in all intraspecies spore samples; it was unipolar- positive, unipolar-negative, or bipolar, depending on the species. The mean spore charge magnitude was 21–981 e, and it was not related to the emission rate of 1 Introduction charged spores. The hymenial emission rate was fluctuating, and the maximum value was 715 charged Contribution of fungal spores to the biogenic aerosol spores cm-2 s-1. To estimate the territorial emission particles in the atmosphere has been studied for a long rate of charged spores, area of the hymenial surface time (Gregory 1973); at present, it is carried out at the per hectare of forest was calculated for three species level of continent, forest, and fruitbody (Galante et al. and the maximum values were 11 m2 ha-1 and 2011; Winiwarter et al. 2009; Zhang et al. 2010). There is a swath of research on spore concentrations in the ambient air often put in the context of human M. Saar (&) health and indoor air quality (Delfino et al. 1997; Fairs Department of Botany, Institute of Agricultural and Environmental Sciences, Estonian University of Life et al. 2010; Neas et al. 1996). This shows, for example, Sciences, 51014 Tartu, Estonia that fungal spores, especially basidiospores, are e-mail: [email protected] among the important triggers of asthma exacerbations needing emergency visits (Dales et al. 2000). But the J. Salm Institute of Physics, University of Tartu, 50090 Tartu, role of fungal spores and other microorganisms in the Estonia meteorological phenomena and atmospheric processes

123

77 Author's personal copy

72 Aerobiologia (2014) 30:71–89 in general is an underexplored component in retain a substantial portion of this primary charge researches in atmospheric and climate sciences (Mor- during spore transport by air flows, and the height of ris et al. 2011). It is important to go beyond the the atmospheric layer in which these spores could be description of abundance of microorganisms in the present. Information about these factors is absent or atmosphere toward an understanding of their dynam- scanty in the literature. The emission rate of charged ics in terms of both biological and physicochemical spores and the height of atmospheric layer have properties and of their relevant transport processes at received no attention until now. The retaining or different scales (Morris et al. 2011). relaxation time is assessed for pollen grains (Bowker Atmospheric aerosol particles modify the chemical and Crenshaw 2003) but not for spores. The spore composition of atmospheric gas-phase and particulate charges measured in the whole and healthy fruitbodies matter due to the collision–coalescence processes and growing in their natural habitats are shown for only the absorption of molecules from surfaces. These five species (Swinbank et al. 1964; Saar 2013). In the effects of atmospheric aerosol particles are dependent present article, we explored all these factors, except on their concentration, size, chemical composition, the height of atmospheric layer. and electric charge (Seinfeld and Pandis 2006; To study the relaxation time, we developed a Hirsikko et al. 2010). Charged airborne molecules, theoretical model and made an estimation using data molecule clusters, and the finest aerosol particles on air ions in Estonia. To explore the charges and the (\7 nm in diameter) are typically singly charged emission rate of charged spores, we integrated avail- (Hirsikko et al. 2010). The largest particles, being in able information from several investigations carried the steady state of bipolar charging with atmospheric out earlier in natural forest conditions in Estonia on the ions in the atmosphere where the equal concentrations following subjects: the magnitude and polarity of of positive and negative ions occur, carry several spore charges in hymenomycetous Agaricomycetes, elementary charges on average. Particles with the relative amount of trees affected by a polypore and diameter of 2 lm carry *3 e, particles with 5 lm fruitbody number per tree, seasonal dynamics of carry *5 e, and particles with 10 lm carry *7e fruitbody production in mushrooms and spore pro- [according to the Boltzmann equilibrium equation duction in polypores. Based on these data, we tested (Roos 1996)]. These charges are distributed symmet- the following hypotheses: (1) all ballistosporic fungi rically in sign. Ballistic basidiospores [2–16 lm in of Agaricomycetes exhibit primary electric charges on diameter (Ingold 2001)] (further in the text as basidiospores; (2) these charges depend on the fungal ‘‘spores’’), at the moment of release from a fungal species; (3) fruitbodies emit charged spores (and fruitbody into the atmosphere, carry on average tens to charges) at variable rates; (4) emission rate of charged thousand elementary charges; in a spore sample, the spores from a unit area of forest depends on the type of polarity distribution of charges is unipolar-positive or forest. unipolar-negative, or bipolar (Saar 2013). Anemoph- ilous pollen grains (10–100 lm in diameter) carry thousands elementary charges on average at a moment 2 Materials and methods of release from an inflorescence; in a grain sample, the polarity distribution of charges is bipolar (Bowker and 2.1 Collection of spores Crenshaw 2007). Essential differences between old atmospheric Spores were collected in chambers that were placed aerosol particles and freshly emitted basidiospores adjacent to the hymenial surface of fruitbodies grow- and pollen grains occur in the polarity distribution of ing in natural forest conditions of Ja¨rvselja (58°160N, charges and the magnitude of charges. Whether these 27°180E) in 1973, in Tammistu (58°270N, 26°550E) in biological particles could be capable of modifying the 1974, and in Koke (58°180N, 26°550E) in 1978 as electrical characteristics of the lowest layer of the described in Saar (2013). Spore collecting lasted from atmosphere is potentially interesting topic of study. To 15 to 1,500 min and was performed during different investigate the contribution of spores, we have to annual (vernal, estival, autumnal) and circadian (noc- know the emission rate of charged spores in space unit, turnal, morning, diurnal, evening) periods. Altogether charges on spores, the time during which the spores 31 macrofungi species inhabiting standing and

123

78 personal copy

Aerobiologia (2014) 30:71–89 73 downed wood, litter, woody debris, decomposed volume, and between the magnitude of the spore organic matter, and soil were included in the present charge and the spore volume were present or not. study. These species represented the Agaricales, Boletales, Cantharellales, Corticiales, Hymenochae- 2.3 Hymenial emission rate of charged spores tales, Polyporales, Russulales, and Thelephorales and charges orders (Agaricomycetes, Basidiomycota), covering 19 families and 26 genera. Three types of hymeno- The hymenial emission rate of charged spores shows a phore (configuration of the spore-producing surface) number of charged spores released per second per cm2 were represented: the poroid hymenophore by Fomes of hymenial surface area, i.e., per unit area of outer fomentarius (L.) J. Kickx f., Fomitopsis pinicola (Sw.) surface (neglecting the internal hymenial structure). It P. Karst., F. rosea (Alb. & Schwein.) P. Karst., was calculated using the numbers of positively Ganoderma applanatum (Pers.) Pat., Junghuhnia charged spores and negatively charged spores released nitida (Pers.) Ryvarden, Phellinus alni (Bondartsev) from the area of 0.44 cm2 of the hymenial surface Parmasto, P. nigricans (Fr.) P. Karst., P. populicola during the collection of spore sample. The hymenial Niemela¨, P. tremulae (Bondartsev) Bondartsev & P.N. emission rate of charges was computed multiplying Borisov, Piptoporus betulinus (Bull.) P. Karst., Pol- the hymenial emission rate of charged spores by the yporus badius (Pers.) Schwein., and Suillus luteus (L.) number of elementary charges on the mean charged Roussel, the lamellate hymenophore by Amanita spore. muscaria (L.) Lam., Cantharellus cibarius Fr., Clito- cybe sp., Cortinarius sp., Lactarius torminosus 2.4 Variability and correlation in charges (Schaeff.) Gray, L. turpis (Weinm.) Fr., Lepista nuda and hymenial emission rates (Bull.) Cooke, Paxillus involutus (Batsch) Fr., Pholi- ota sp., Pleurotus ostreatus (Jacq.) P. Kumm., Russula The variation coefficients (v) of both the charges and vinosa Lindblad, Stropharia hornemannii (Fr.) S. the emission rate were found. By that, as the classical Lundell & Nannf., Tricholoma equestre (L.) P. estimator v = 100(s/a) gives underestimated values in Kumm., and T. imbricatum (Fr.) P. Kumm., and the the case of the small set size (amount of samples in a flat hymenophore by Coniophora puteana (Schu- species, n), the unbiased estimator using the small set mach.) P. Karst., Dendrothele griseocana (Bres.) correction proposed by Haldane (1955) was calculated Bourdot & Galzin, Stereum rugosum Pers., Thelepho- of the form ra terrestris Ehrh., and Vesiculomyces citrinus (Pers.) v 100 s=a 1 1=4n ; 1 E. Hagstr. ¼ ðÞðþ Þ ð Þ where s is the standard deviation and a is the arithmetic average of the emission rates or charges 2.2 Charge-to-mass quotient and charge of spores in a set. The Pearson’s correlation coefficient (r) was computed to see whether correlation between the For each sample, the mean charge-to-mass quotients charge magnitude and the hymenial emission rate was for negatively and positively charged spores were present or not. determined and the mean positive charge of spore and the mean negative charge of spore were calculated as 2.5 Synchrony and difference in hymenial described in Saar (2013). Length and width of spores emission rates were taken equal to the mean values given by Niemela¨ (2008) or were computed by us as means of the To get preliminary data about the variation ranges, minimum and maximum values given by Kalamees synchrony, and differences in the hymenial emission (1971b, 1972) and others (Corner 1968; Eriksson and rates of charged spores, the repeated spore collections Ryvarden 1975; Eriksson et al. 1984; Gilbertson and was carried out in a group of fruitbodies in Fomes Hemmes 1997; Nakasone 2006; Sell 2008). The fomentarius and Ganoderma applanatum; in Fomit- Pearson’s correlation coefficients (r) were computed opsis pinicola, the spore collections were carried out to see whether correlations between the magnitude of in different groups of fruitbodies at different times the spore charge-to-mass quotient and the spore (Tables 1, 2). The Friedman rank test, Spearman’s

123

79 Author's personal copy

74 Aerobiologia (2014) 30:71–89 rank-order correlation, and Student’s test were used to 2.8 Territorial emission rate of charged spores analyze the data. In t tests, due to limited set size and charges (n B 6), we considered p = 0.10 as significance level. Due to a wide variation in the values of emission The territorial emission rate of charged spores shows rate, we used the logarithmic transformation for the number of charged spores released per second per emission rate. No transformation was used for hectare of forest. It was calculated multiplying the charge-to-mass quotient and charges. hymenial emission rate of charged spores by the area of hymenial surface per hectare of forest. The 2.6 Fruitbody number on forested territory territorial emission rate of charges was computed by multiplying the territorial emission rate of charged For three fungal species involved in the present study spores and the number of charges on the mean charged on spore charges, empirical data on the seasonal spore. dynamics of fruitbody numbers per hectare were available in the literature—for Cantharellus cibarius 2.9 Charge relaxation time and Paxillus involutus in pine and pine-birch forests (Lo˜ugas 1980) and for Phellinus tremulae in aspen As described above, the freshly emitted basidiospores stands (Tamm 2000). We computed the fruitbody and pollen grains can carry high electric charges, numbers per hectare for C. cibarius and P. involutus in which considerably exceed the steady-state charges of the peak period of the fruitbody production (Septem- atmospheric aerosol particles. However, the atmo- ber) and for P. tremulae, in stands 40 years old and spheric air is ceaselessly ionized and steadily contains older. negative and positive air ions. The highly charged basidiospores and pollen grains attract oppositely 2.7 Area of hymenial surface on forested territory charged air ions, and thus, their charge diminishes or relaxes during time. Let us estimate the rate of such The area of hymenial surface per hectare of forest was charge relaxation. computed multiplying the number of fruitbodies The diffusion charging processes of aerosol parti- growing on this unit area by the hymenial surface cles are described by Salm and Tamm (2011). If the area of the mean fruitbody. The fruitbody’s hymenial initial charge of an aerosol particle considerably area was considered to have shape of an annulus with exceeds the steady-state charge, then we may take the outer diameter of 11.5 cm in Paxillus involutus into account only the oppositely charged air ions. Let and 6.5 cm in Cantharellus cibarius and with the inner us consider a spherical aerosol particle with a high diameter of 1.5 cm in both [the mean width of pileus charge of ie, where e is the positive elementary charge and stipe according to Kalamees (1971b)]. In Phelli- and i is the number of elementary charges on a particle. nus tremulae, the fruitbody’s hymenial area was In the case of negative charges, i is negative. considered to have shape of an ellipse with axes of 4 The strength of the electric field on the surface of and 5 cm (U¨ lo Tamm, personal communication). the charged particle is

Table 1 Study setup in Fomes fomentarius and Ganoderma applanatum (for the repeated spore collections, carried out in a group of fruitbodies) and in Fomitopsis pinicola (for the spore collections, carried out in different groups of fruitbodies at different times) Species A series of spore collections Number of fruitbodies studied simultaneously Number of Number of spore Duration of Time interval collections samples per collection involved per series fruitbody period

Fomes fomentarius 6 6 15–36 min 69 h 4 Ganoderma 4 4 45–60 min 50 h 3 applanatum Fomitopsis pinicola 4 1 50–168 min 179 h 6 (in three collections) and 4 (in one collection)

123

80 Author's personal copy

Aerobiologia (2014) 30:71–89 75

Table 2 Study subjects and results in Fomes fomentarius and (the spore collections, carried out in different groups of Ganoderma applanatum (the repeated spore collections, car- fruitbodies at different times) ried out in a group of fruitbodies) and in Fomitopsis pinicola Species Study subject Results

Fomes fomentarius Variation range of the fruitbody The minimum range stayed within 1 order of HER magnitude (in fruitbody III, 116–715 spores cm-2 s-1), the maximum range extended over 3 orders of magnitude (IV, 2–587 spores cm-2 s-1) F. fomentarius Variation range of the group HER Extended over 2 orders of magnitude [the group of fruitbodies I–IV, 19 spores cm-2 s-1 (95 % confidence interval of 4–100 spores cm-2 s-1) in period b to 517 (416–644) spores cm-2 s-1 in period d] F. fomentarius Synchrony of dynamics of HER Absent in five pairs of fruitbodies (in I&II, I&III, between fruitbodies I&IV, II&IV, and III&IV the Spearman’s rank- order correlation coefficient rs = 0.2–0.7, number of time periods n = 6, p = 0.1) and present in one pair (II&III, rs = 0.943, n = 6, p = 0.02) F. fomentarius Difference between fruitbodies in Nonsignificant [the Friedman rank test statistic chi- the mean fruitbody HER square = 5.4 for four fruitbodies (df = 3), p = 0.14] F. fomentarius Difference between periods in the Significant [the Friedman rank test statistic chi- group HER square = 13.6 for six time periods (df = 5), p = 0.02] Ganoderma applanatum Variation range of the fruitbody Extended over 2 orders of magnitude in one HER fruitbody (II, 9–57 spores cm-2 s-1) and over 3 orders in the rest of fruitbodies (I, 8–343; III, 8–296) G. applanatum Variation range of the group HER Extended over 2 orders of magnitude [the group of fruitbodies I–III, 10 spores cm-2 s-1 (95 % confidence interval of 7–15 spores cm-2 s-1) in period a to 113 (42–299) spores cm-2 s-1 in period d] G. applanatum Synchrony of dynamics of HER Absent in all pairs of fruitbodies (in I&II, I&III, between fruitbodies II&III the Spearman’s rank-order correlation coefficient rs = 0.0–0.8, number of time periods n = 4, p = 0.1) G. applanatum Difference between fruitbodies in Nonsignificant [the Friedman rank test statistic chi- the mean fruitbody HER square = 1.5 for three fruitbodies (df = 2), p = 0.47] G. applanatum Difference between periods in the Nonsignificant [the Friedman rank test statistic chi- group HER square = 5.4 for four periods (df = 3), p = 0.14] Fomitopsis pinicola Variation range of the group HER Extended over 2 orders of magnitude [9 spores cm-2 s-1 (95 % confidence interval of 4–21 spores cm-2 s-1) in the group of four fruitbodies in period h to 23 (8–67) spores cm-2 s-1 in period e in the group of six fruitbodies] Fomitopsis pinicola Difference between periods in the Nonsignificant [t =-0.3… ? 1.4; for four periods group HER (df = 10 for periods where the groups of 4 and 6 fruitbodies were studied, and df = 8 for periods where the groups of 6 fruitbodies where studied), p = 0.1]

Marks. HER—hymenial emission rate of charged spores; I, II, …—fruitbodies (see Figs. 1, 2); a, b, …–spore collection periods (see Figs. 1, 2); the mean fruitbody HER—average hymenial emission rate of charged spores over all periods studied per fruitbody; the group HER—average hymenial emission rate of charged spores over all fruitbodies studied simultaneously per period

123

81 Author's personal copy

76 Aerobiologia (2014) 30:71–89

ie originating from a certain species, displayed the same E 2 2 ¼ 4pe0rp ð Þ polarity distribution (Figs. 1, 2, 3). The absolute value of charges was in the range of orders of magnitude of where e0 is the electric constant and rp is the radius of 10–1,000 e (Table 4). the particle. The hymenial emission rate of charged spores was The electric current density of oppositely charged in the range of orders of magnitude of 0.1–100 spores air ions through the surface of the charged particle is cm-2 s-1 (Figs. 1, 2, 3; Table 5). In all bipolar species r ie (except Thelephora terrestris and Cantharellus ciba- J E À 3 À rÀ 2 ; rius), both components of the charged spores had ¼ ¼ 4pe0rp ð Þ emission rates in this range. In T. terrestris and C. where rÀ is the polar conductivity of air due to the cibarius, the positive spores had emission rate in the oppositely charged air ions. order of magnitude of 0.001 spores cm-2 s-1. The The electric current strength through the entire maximum found was 343 spores cm-2 s-1 for positive surface of charged particle spores and 715 spores cm-2 s-1 for negative spores. 2 Variability of charge magnitudes was several times IÀ 4pr JÀ ierÀ=e0: 4 ¼ p ¼ ð Þ lower than that of the hymenial emission rates: v of If we consider the charge number i as a continuous charges 18 % and v of emission 75 % in Fomes variable, then the current strength may be expressed fomentarius; 15 and 198 % in Fomitopsis pinicola; 26 through the derivative of i with respect to time t. The and 118 % in Ganoderma applanatum; 23 and 123 % obtained current strength is equal to the current in Lactarius turpis. Correlation between the charge strength expressed in Eq. (4), and thus, we result in a magnitude and the hymenial emission rate was absent simple differential equation and its solution (r = 0.01; set of 26 spore samples in F. fomentarius t and r = 0.03; 28 samples in F. pinicola). Correlation i i0 exp ; 5 between the magnitude of spore charge-to-mass ¼ s ð Þ  quotient and the spore volume was absent (r = where i0 is the initial charge number of a particle, and s -0.07; set of 49 pairs listed in Table 4, df = 47, is the charge relaxation time, p = 0.63). Correlation between the magnitude of s e0=rÀ: 6 spore charge and the spore volume was significant ¼ ð Þ (r = 0.50; set of 49 pairs listed in Table 4, df = 47, The initial charge number diminishes about 2.7 p = 0.0002, 95 % confidence level 0.26–0.69). times during a time interval of s and 7.3 times during 2s. The rate of charge relaxation does not depend on the size of particles. Equation (6) is derived also in the 3.2 Synchrony and difference in hymenial monograph (Chalmers 1967) with somewhat different emission rates context. If the initial charge is not considerably higher than steady-state charges, then the theory needs The hymenial emission rate of charged spores was refinements (Salm and Tamm 2011). characterized by constant change—this was shown by the series of repeated samples in F. fomentarius and G. applanatum (Figs. 1, 2). The fruitbodies, although 3 Results studied simultaneously, showed uneven extents of the variation ranges of the hymenial emission rate of 3.1 Spore charge and hymenial emission rate charged spores (Table 2). Dynamics of the hymenial of charged spores emission rate were asynchronous in fruitbodies (Table 2). Differences between fruitbodies in the In total, 128 spore samples from 81 fruitbodies of 31 mean fruitbody hymenial emission rate were nonsig- species were studied (Table 3). All these samples nificant (Table 2). Differences between periods in the exhibited a majority of charged spores. The polarity group hymenial emission rate were nonsignificant in distribution of charges in a sample was unipolar- G. applanatum and F. pinicola, but significant in F. negative or unipolar-positive, or bipolar. All samples, fomentarius (Table 2).

123

82 Author's personal copy

Aerobiologia (2014) 30:71–89 77

Table 3 Study on the hymenial emission rate of charged spores in 31 species of Agaricomycetes: number of fruitbodies, time periods, spore samples, repeated collections and simultaneous collections per species Speciesa Number of Number of repeated Number of simultaneous samplings per fruitbody samplings per time period fruitbodies time spore periods samples

Fomes fomentarius 4 8 26 8, 6, 6, and 6 4(6) (the same four fruitbodies in six periods) Fomitopsis pinicola 26 8 28 2 6, 6, 6, 4, and 2(2) F. rosea 4 1 4 non 4 Ganoderma applanatum 3 5 13 5, 4, and 4 3(4) Lactarius turpis 8 3 10 2 and 2 6, 2, and 2(2) Paxillus involutus 4 3 7 2, 2, and 2 3(2) Phellinus populicola 4363 4 Clitocybe sp. 2 1 2 non 2 Coniophora puteana 1 2 2 2 non Cortinarius sp. 2 2 3 2 2 Phellinus tremulae 2 2 2 non non Pholiota sp. 1 2 2 2 non Stropharia hornemannii 2232 2 Tricholoma imbricatum 1 2 2 2 non Vesiculomyces citrinus 1 2 2 2 non a The other 16 species were represented by one spore sample (Table 4)

Fig. 1 Hymenial emission rate of charged spores in the spore ‘‘Phellinus populicola’’—Fruitbodies: white I, light gray II, samples where the polarity distribution of spore charges was dark gray III, black IV. Times: all samplings were performed in unipolar-positive. Marks to indicate time and duration of spore 1973; a sampling date 9 July, start time 9:24, duration 7 h 6 min; collection and fruitbodies examined on the chart ‘‘Ganoderma b 25 June, 22:33, 9 h; c 30 September, 16:00, 2 h 55 min. applanatum’’—Fruitbodies: white I, gray II, black III. Times: all ‘‘Phellinus alni etc.’’—Fruitbodies: black I, white II. Time samplings were performed in 1973; a sampling date 5 July, start periods: all samplings were performed in 1973; a 31 May, 18:34, time 19:08, duration 60 min; b 5 July, 23:10, 60 min; c 6 July, 3 h 46 min; b 31 May, 23:10, 11 h 40 min; c 5 Oct, 17:10, 1 h 21:45, 45 min; d 7 July, 10:00, 60 min; e 7 July, 19:50, 60 min. 50 min; d 8 Oct, 16:30, 2 h 30 min

3.3 Area of hymenial surface on forested territory. grows older (Table 6). The number of fruitbodies reaches Territorial emission rate of charged spores its maximum in 75-year-old stands, corresponding to the and charges hymenial area of about 10 m2 ha-1 in predominantly aspen stands. In such stand and older by the hymenial The fungal fruitbodies of Phellinus tremulae occur on emission rate and spore charges measured by us, aspen (Populus tremulae)stemsover30yearsold.The emission of about 3 9 105 positively charged spores percentage of trees with fruitbodies increases as the stand s-1 ha-1 and 3 9 107 es-1 ha-1 occurred (Table 7).

123

83 Author's personal copy

78 Aerobiologia (2014) 30:71–89

Fig. 2 Hymenial emission rate of charged spores in the spore 9:34–9:44, 55 min; h 11 June, 18:10, 2 h. ‘‘Fomitopsis rosea’’— samples where the polarity distribution of spore charges was Fruitbodies: white I, light gray II, dark gray III, black IV. Times: unipolar-negative. Marks to indicate time and duration of spore all samplings were performed simultaneously in 1973 on 9 July, collection and fruitbodies examined on the chart ‘‘Fomes start time 9:24, duration 7 h 6 min (a). ‘‘Lactarius turpis’’— fomentarius’’—Fruitbodies: white I, light gray II, dark gray Fruitbodies: black square I, dark gray square II, light gray III, black IV. Times: all samplings were performed in 1973; square III, white square IV, black ring V, dark gray ring VI, a sampling date 1 June, start time 20:27, duration 36 min; b 1 light gray ring VII, white ring VIII. Times: all samplings were June, 21:36, 20 min; c 2 June, 17:13, 29 min; d 2 June, 18:10, performed in 1973; a sampling date 15 September, 9:00, 10 h 20 min; e 2 June, 18:50, 15 min; f 2 June, 19:35, 25 min; g 4 15 min; b 21 September, 18:00, 2 h; c 22 September, 17:45, 2 h. June, 17:11, 30 min; h 4 June, 18:14, 45 min. ‘‘Fomitopsis ‘‘Amanita muscaria, etc.’’—Fruitbodies: black I, white II. pinicola’’—Fruitbodies: black XVII, white XXI, X–26 different Times: samplings were performed in 1973 (a–e) and 1978 (f, g); fruitbodies, each with a single sample. Times: samplings were a 19 September 1973, 16:20, 3 h 30 min; b 30 September 1973, performed in 1973 (a) and 1974 (b–h); a sampling date 21 May, 16:20–16:30, 2 h 30 min–2 h 40 min; c 5 October 1973, 17:00, start time 22:33, duration 50 min; b 2 June, 17:15, 65 min; c 3 2 h 10 min; d 6 October 1973, 16:55–17:15, 1 h 30 min–2 h; e 7 June, start in the interval of 9:11–9:53, duration in the interval of October 1973, 17:10–17:40, 1 h 10 min–1 h 40 min; f 15 55–65 min; d 4 June, 8:42–9:16, 60 min; e 7 June, 9:27–9:49, September 1978, 8:55, 22 h 5 min; g 16 September 1978, 8:15, 50–65 min; f 8 June, 17:57–18:50, 2–2 h 48 min; g 9 June, 23 h 5 min

Production of fruitbodies of Paxillus involutus and ha-1, and 2 9 105 negatively charged spores s-1 ha-1 Cantharellus cibarius strongly varied in pine and pine- and -6 9 107 es-1 ha-1 occurred. birch forests (Table 8). The P. involutus fruitbodies In pine and pine-birch forests in a year of a quite were most numerous in the paludified Calluna pine- poor yield of mushrooms, the group of edible fungi birch forest. There the peak time of fruitbody production corresponded to the hymenial area of 11–12 m2 ha-1 corresponded to the hymenial area of about 2 m2 ha-1. at the peak time of the fruitbody production (Table 8, By the hymenial emission rate and spore charges the nonbog forests). In mature pine forests, in years of measured by us, emission of about 5 9 104 positively poor yield, the ratio of the number of inedible charged spores s-1 ha-1 and 6 9 106 es-1 ha-1, and fruitbodies/the number of edible fruitbodies varies 2 9 104 negatively charged spores s-1 ha-1 and from 0.8 to 2.2 (Kalamees and Silver 1988). Basing on -3 9 106 es-1 ha-1 occurred (Table 9). Contrary to these data and assuming equality of the groups of P. involutus, which was among dominant species in all edible and inedible fungi in a mean hymenial area of forests studied, C. cibarius belonged to the dominants fruitbody, we got 20–42 m2 ha-1 for an estimation only in the lichen pine-birch forest. There the peak time of the area of the hymenial surface of mushrooms in of its fruitbody production corresponded to the hymenial mature pine and pine-birch forests in a year of a poor area of about 5 m2 ha-1. By the hymenial emission rate yield. In the eutrophic Hepatica boreo-nemoral spruce and spore charges measured by us, emission of about forest, at the peak time of fruitbody production of 500 positively charged spores s-1 ha-1 and 60,000 e s-1 mushrooms, edible and inedible fungi altogether

123

84 Author's personal copy

Aerobiologia (2014) 30:71–89 79

Fig. 3 Hymenial emission rate of charged spores in the spore 17:50, 1 h 30 min; c 5 October 1973, 16:30, 2 h 55 min; d 6 samples where the polarity distribution of spore charges was October 1973, 16:40, 2 h 25 min; e 7 October 1973, 16:35, 2 h bipolar. Marks to indicate time and duration of spore collection 35 min; f 8 October 1973, 18:00, 1 h 30 min; g 19 September and fruitbodies examined on the chart ‘‘Paxillus involutus’’— 1978, 10:00, 23 h; h 20 September 1978, 10:30, 7 h 45 min. Fruitbodies: black I, dark gray II, light gray III, white IV. Times: ‘‘Cantharellus cibarius, etc.’’—Fruitbodies: one fruitbody per all samplings were performed in 1973; a sampling date 23 species. Times: samples were performed in 1973 (a–d) and 1978 September, 16:00, 3 h; b 9 October, 18:00, 14 h; c 10 October, (e–g); a 17 Sept, 16:55, 2 h; b 5 October, 16:10, 3 h 15 min; c 8 16:10, 3 h. ‘‘Pholiota sp., etc.’’—Fruitbodies: the same marks October, 18:00, 1 h 30 min; d 9 October, 16:40, 3 h; e 16 mean the same fruitbody. Times: samplings were performed in September, 8:15, 23 h 5 min; f 17 September, 9:40, 24 h 1973 (a–f) and 1978 (g, h); a sampling date 30 September 1973, 20 min; g 18 September, 11:30, 22 h sampling start 15:55, sampling duration 3 h; b 4 October 1973, corresponded to the hymenial area of about 1.1– 4 Discussion 1.6 m2 ha-1 (our estimation according to 21 dominant species in Vooremaa in September 1970; Kalamees 4.1 Presence of charges and Kollom 1971). Studying the primary electric charges of ballistic basidiospore, we did not found spore samples consist- 3.4 Charge relaxation time ing of electrically neutral spores or having a majority of neutral spores. Such spore samples have not been Let us estimate the charge relaxation time in real reported in earlier studies (Buller 1909; Gregory 1957; atmospheric conditions near the ground level. Equa- Swinbank et al. 1964; Webster et al. 1988). Altogether tion (6) contains the electric constant e0 8:854 more than 150 spore samples and 100 fruitbodies 12 -1 ¼ Â 10À Fm and the polar conductivity of air rÀ: The covering 43 species, 33 genera, 23–25 families, and 9 polar conductivity of air can be estimated on the basis orders (Auriculariales plus those eight listed in of the regular long-term air ion measurements carried ‘‘Materials and methods’’) of Agaricomycetes and out at the Tahkuse Observatory, Estonia. Tahkuse one species of Tremellomycetes (Basidiomycota) Observatory with coordinates 58°310N, 24°560E is (Webster et al. 1988) have exhibited the presence of located in a sparsely populated rural region. The air the electric charges on basidiospores. The charged was sucked into the mobility spectrometers through an spores were released by mature and healthy macro- opening in the south gable of the building at a height of scopic fruitbodies (Gregory 1957; Swinbank et al. about 5 m from the ground. A thoroughly analyzed 1964; present study), small pieces of the macroscopic measurement period lasted from September 1, 1993 to fruitbodies (Buller 1909; Webster et al. 1988), and a October 27, 1994 (Ho˜rrak et al. 2000). The average microscopic basidiomycetous fungus—a basidiomy- polar conductivity rÀ for this period equals to 6.18 fS cete yeast (Webster et al. 1988). These results show, m-1. Taking into account this value of polar conduc- firstly, that the charged spores have been observed at tivity, the charge relaxation time can be calculated by different times within 80 years, at different places means of Eq. (6) as s = 1,430 s. Thus, the initial apart from each other up to thousands of kilometers, charge number diminishes about 2.7 times during a in different environmental conditions from forests time of 1,430 s. to laboratories, and in the majority of orders of

123

85 Author's personal copy

80 Aerobiologia (2014) 30:71–89 16 17–59 19–75 8 55–116 8 26–92 Charge (e) Mean 95 % confint Stdev Range ) 3 m l ( Shape Volume m) l ) Sizes ( 1 - C kg 4 - 2.03 1.66–2.39 0.26 0.84–3.27 5.75 3.5 I E 37 47 38–55 2.78 1.41–4.153.68 2.69–4.684.32 non 3.15–5.481.65 1.25 1.56–1.74 non 0.841.65 1.15–6.60 0.06 1.56–1.742.03 2.23–6.26 5.58 1.66–2.39 0.75–2.55 6.55 4.99 0.06 4.76 5.75 0.26 5.86 4.08 0.75–2.55 VIII 3.5 0.84–3.27 VIII 8.5 VIII E 8.5 I E E 5.6 5.6 66 118 I E 43 I 152 204 E 37 120–184 117 104–305 E 86–149 140 52 non 38 140 36–40 23 48–273 non 144 61–170 136–151 176 145–208 6 23 65–222 73–285 7.32 3.82–10.82 non non 4.8 1.6 IV C 10 44 23–65 non non 2.12 1.10–3.14 non non 5.94 5.11 VIII E 81 107 56–159 non non 0.74 0.38–1.10 non non 9 7 I E 231 107 55–158 non non 1.88 0.86–2.901.68 1.32–2.041.53 non 1.08–1.97 0.48 non 0.61 0.48–3.48 0.52–3.87 9 7 9 5.5 4.75 5.5 I I I E E E 143 83 143 149 118–181 136 97 96–176 44–150 43 non 54 43–310 non 46–344 2.66 2.30–3.03 0.59 0.87–4.87 8 5.75 I E 138 230 199–262 51 75–421 0.93 0.56–1.300.89 0.52–1.255.78 non 5.38–6.186.09 non 5.77–6.425.13 non 1.04 4.68–5.58 non 0.89 2.65–8.94 0.46 3.14–11.00 11 17.8 7.6 3.34–7.08 11 5.4 9 5.8 3.9 9 IV 2.4 III IV III E IV E E E 272 C 467 61 467 981 26 271 913–1,049 230 162–379 258 218–243 176 151–365 84 77–91 non 450–1,517 33 non non 119–416 non 2.18 1.87–2.482.63 1.56–3.701.53 0.56 0.89–2.17 non 0.92–4.62 non non 8.2 non 5.7 4.2 IV 4.2 2.6 2.6 O IV IV 118 C C 161 22 138–183 22 41 37 22–52 21 68–341 12–30 non non non non 4.57 3.15–5.991.65 0.98–2.333.24 non 2.63–3.85 non2.32 non 0.44 1.95–2.70 non 1.49–5.25 0.33 9 4.4 0.90–3.85 9 3.5 7.5 5.25 5.25 I I 4.5 I I E E E E 125 28 125 80 355 57 245–466 128 46–68 76–181 115 non 97–134 non non non 17 45–191 3.24 2.63–3.852.32 0.44 1.95–2.70 1.49–5.25 0.33 7.5 0.90–3.85 5.5 15 I 8 E I 119 E 240 503 195–285 729 33 610–848 110–389 105 282–1,208 1.56 0.81–2.301.46 1.13–1.79 non 0.24 non 0.72–2.26 10.5 10 6.5 7.5 II I E E 232 295 212 165–260 286 149–423 34 non 105–328 non Mean 95 % confint Stdev Range Length Width Source Charge-to-mass quotient (10 ? - - - - ? - - ? - ? ------L U ? ? - ? ? - L U - L U L U ? sp. sp. ? sp. sp. Magnitude of spore charge in 31 species of Agaricomycetes: computation on the base of spore charge-to-mass quotient (found out in natural forest conditions in sp. sp. sp. sp. ? Pholiota Pholiota Phellinus populicola Phellinus tremulae Pholiota Pholiota Piptoporus betulinus Phellinus nigricans Lepista nuda Paxillus involutus P. involutus Phellinus alni Lactarius torminosus D. griseocana Fomes fomentarius Fomitopsis pinicola Fomitopsis rosea Ganoderma applanatum Junghuhnia nitida J. nitida Lactarius turpis C. cibarius Clitocybe Cortinarius Clitocybe Cortinarius Dendrothele griseocana Coniophora puteana Cantharellus cibarius Amanita muscaria Table 4 Estonia) and spore sizes (taken fromSpecies literature) and polarity of spore charges

123

86 Author's personal copy

Aerobiologia (2014) 30:71–89 81 in ), VI— 1984 92 33–89 17–46 1 37–117 ), E—ellipsoid, C—cylinder, O— ), V—Eriksson et al. ( Clitocybe, Cortinarius glaucopus 2008 ( in ¨ Charge (e) Mean 95 % confint Stdev Range ) 3 m ), IV—Niemela l ( 2006 Clitocybe cerussata Shape Volume ), III—Nakasone ( 1997 m) l Clitocybe geotrapa, Cortinarius collinitus, Pholiota aurivella ) ) Sizes ( 1 - 2008 C kg ), II—Gilbertson and Hemmes ( 4 - 1972 ), VIII—Sell ( 1975 ), U—a species with the biggest spores ( ) or Kalamees ( 1971b Pholiota 1.83 1.40–2.26 0.31 1.10–2.98 4.5 4.5 VII E 48 55 42–67 0.99 0.88–1.10 0.08 0.56–1.53 4.5 4.5 VII E 48 29 26–33 1.96 1.33–2.591.74 non 0.89–2.591.40 0.71–2.102.24 non non 2.05–2.421.77 non 1.73–1.81 non 0.13 10 non 0.03 1.09–3.46 0.87–2.71 6.5 7.5 6.5 6.5 6.5 VI 4.5 4.5 4.5 4.5 I I E I I 295 E E E E 360 69 69 245–476 69 69 75 non 96 38–111 60 88–104 76 non 30–90 74–78 non 6 non non 47–149 non 1.84 0.90–2.781.64 0.75–2.521.89 non 1.16–2.621.50 non 0.52–2.481.03 non non 0.70–1.362.17 non 0.87 1.19–3.151.39 non 0.29 0.74–2.041.70 0.48–3.76 7.8 non 1.03–2.361.32 0.43–2.08 7.8 12 non 0.84–1.81 3.6 9.5 non 12 non 3.6 non 6.75 non IV 7.5 non 6.75 IV I 9.5 non I I E 9.5 E 3.75 9 E 3.75 9 O 53 E V 53 V 286 3.5 250 286 3.5 C 61 I C 269 30–92 54 I 105 295 93–444 183 25–83 E 105 181–409 124–243 E non 142 155 non non 58 78–206 non 52 91 85–672 58 non non 48–134 77–372 non 61 non 37–85 48 non 30–65 non non non non non 3.12 1.60–4.64 non non 9.5 3.5 I C 91 178 91–265 non non 2.77 1.44–4.10 non non 9.5 3.5 I C 91 158 82–234 non non 11.44 4.68–18.20 non non 10 7.5 VI E 295 2,103 860–3,346 non non Mean 95 % confint Stdev Range Length Width Source Charge-to-mass quotient (10 negative polarity, L—a species with the smallest spores in a genus in Estonia ( in ------? - ? - ), VII—Eriksson and Ryvarden ( ? ? ? ? ? ? positive polarity, ? continued 1968 ? V. citrinum Vesiculomyces citrinum T. terrestris Tricholoma equestre T. equestre Tricholoma imbricatum T. imbricatum P. badius Russula vinosa Stropharia hornemannii S. hornemannii Stereum rugosum S. rugosum Suillus luteus S. luteus Thelephora terrestris Pleurotus ostreatus P. ostreatus Polyporus badius Corner ( Cortinarius, Pholiota adiposa Marks: ovoid, mean—the arithmetic average ofconfidence a interval single of mean, sample stdev—standard orof deviation, of the range—interval the highest from set value, the I—Kalamees of lower ( all border samples of representing the 95 a species, % non—absent confidence interval (one of spore the sample smallest per value species), in 95 a % setup to confint—the the 95 upper % border Species and polarity of spore charges Table 4

123

87 Author's personal copy

82 Aerobiologia (2014) 30:71–89

Table 5 Hymenial emission rate of charged spores: statistics 4.2 Charge-to-mass quotient and charge of spore of sets of several (n [ 2) spore samples in 9 species of Agaricomycetes Our results in spore charge-to-mass quotient and in Species and Hymenial emission rate charges accorded with the measurements made in -2 -1 polarity of (spores cm s ) basidiospores earlier. The ranges of absolute values of spore charges Geometric average Range these two characteristics found by Swinbank et al. (1964) andWebster et al. (1988)—(0.09 7 13) 9 10-4 Ckg-1 Average 95 % -17 confidence and 7 7 6,710 e [originally (0.12 7 107.50) 9 10 interval C]—are wider than the ranges of our results and overlap ours entirely. All types of polarity distributions—the Cortinarius sp. - 1.2 0.0–4.7 0.6–4.3 unipolar-positive, unipolar-negative, and bipolar— Fomes fomentarius - 177 175–178 2.4–715 occur in charges of basidiospores that was seen by Fomitopsis pinicola - 21 20–23 3.8–471 Buller (1909) and Gregory (1957) and by us. Earlier Fomitopsis rosea - 28 26–30 6.7–81 results show the absence of intraspecies variation in the Ganoderma applanatum ? 46 44–48 7.9–343 polarity distribution in four species: Agaricus campestris Lactarius turpis - 1.0 0.0–2.8 0.3–6.5 and Polyporus squamosus, due to identical results of Paxillus involutus- & ? 2.8 0.3–5.2 0.5–10.4 Buller (1909) and Gregory (1957); Ganoderma applan- Phellinus populicola ? 16 13–19 4.3–251 atum (Gregory 1957)andSerpula lacrymans (Swinbank Stropharia 6.3 4.1–8.5 2.8–10.6 et al. 1964). This coincides in our results that showed hornemannii- & ? constancy in the polarity distribution in 15 species (listed Remarks. A set consisted of all samples representing a species, in Table 3). for the number of samples see Table 3. All the samples in a set The main shortcoming of our study concerns had the same polarity distribution of charges, unipolar-positive (?) or unipolar-negative (-), or bipolar (- & ?) Cantharellus cibarinus and Thelephora terrestris. In these species, relative importance of one unipolar part was extremely small (0.3–0.4 % of all charged spores in a spore sample), compared with the other bipolar Agaricomycetes. The results show, secondly, that the species studied by us (5–48 %). In T. terrestris, the charged spores occur in different classes of basidio- magnitude of mean charge of the positive spores was mycetous fungi and that hymenium is not a precondi- extremely big (2,103 e), compared with other species tion for the presence of the charges on basidiospores studied by us (21–981 e). We were backed on single by their release into the air. Considering both circum- spore samples. More samples should be examined. stances, we concluded that the primary electric Earlier and our results of the constancy in the charges occur on basidiospores in all ballistosporic polarity distribution concern 18 species covering 12 fungi of the Basidiomycota. families and five orders of Agaricomycetes. In seven

Table 6 Area of hymenial Stand Tree Relative amount of Fruitbody Fruitbody Area of surface of Phellinus age number trees with fruitbodies, number per number per ha, hymenial tremulae on forested (years) per ha, P K tree, R P 9 K 9 R surface per territory in Estonia: ha (m2) estimation based on the literary data of numbers of 40 1,000 0.1 5 500 0.785 trees in aspen stands and fruitbodies, and on the area 1,200 0.1 5 600 0.942 of hymenial surface of a 55 750 0.6 7.6 3,420 5.37 2 mean fruitbody (15.7 cm ) 900 0.6 7.6 4,104 6.44 75 530 0.9 11.5 5,486 8.61 Data for K were taken from 700 0.9 11.5 7,245 11.37 (Tamm 2000), for P from [90 470 0.92 11.9 5,146 8.08 (Krigul 1969), and for R 600 0.92 11.9 6,569 10.31 (Tamm 2000)

123

88 personal copy

Aerobiologia (2014) 30:71–89 83

Table 7 Territorial emission rate of charged spores and rate of spores and charges of spores (mean 117 e, 95 % charges in Phellinus tremulae in aspen stands in Estonia: confidence interval 86–149 e, range 61–170 e) estimation based on the empirical data of hymenial emission Age of stand (years) Territorial emission rate of Spores Charges (106 e ha-1 s-1) (103 spores ha-1 s-1) Mean 95 % conf int Range

Hymenial emission rate of 3.04 charged spores cm-2 s-1a 40 24 2.79 2.05–3.56 1.46–4.06 55 163 19.1 14.0–24.3 10.0–27.8 75 262 30.6 22.5–39.0 16.0–44.5 [90 246 28.7 21.1–36.6 15.0–41.8 40 29 3.35 2.46–4.27 1.75–4.87 55 196 22.9 16.8–29.2 11.9–33.3 75 346 40.4 29.7–51.5 21.1–58.8 [90 313 36.7 27.0–46.7 19.1–53.3 Hymenial emission rate of 752 charged spores cm-2 s-1a 40 5,903 691 507–881 361–1,004 55 40,383 4,725 3,463–6,011 2,474–6,877 75 64,748 7,575 5,566–9,647 3,958–11,008 [90 60,762 7,109 5,220–9,054 3,711–10,340 40 7,084 829 609–1,056 433–1,205 55 48,429 5,666 4,156–7,223 2,944–8,237 75 85,503 10,004 7,347–12,740 5,220–14,545 [90 77,532 9,071 6,679–11,552 4,723–13,185 a The value of 3.04 was found by us, 752 was taken from (Parmasto 1981) considering all released spores as charged ones species, the spore sample originated from different produce smaller drops (Stolze-Rybczynski et al. fruitbodies and from 2–3 geographical sites and years. 2009). Correlation between area of the hilum (the Based on this phylogenetically diverse set of species place of spore insertion on the sterigma) and spore size and the spatiotemporally diverse sets of spore samples has not been reported, but it is logical to suppose a per species, we conclude that, in basidiomycetous positive correlation between them. These results fungi, the polarity distribution of the primary electric support the hypotheses of endogenous origin of the charges of ballistosporic basidiospores is species- primary electrostatic charges of ballistosporic basi- specific. diospores reported in (Saar 2013). Factors responsible for the magnitude of spore charge, as well as the charge formation process itself 4.3 Magnitude of the hymenial emission rate are not known (Webster et al. 1988). Our correlation of charged spores analyses showed that the charge magnitude does not depend on the sporulation intensity of fruitbody. The As the hymenial emission rate of charged spores has big charges tend to be on larger spores, and the smaller not been reported earlier, we had to evaluate our charges on smaller spores. The charge magnitude results using data on the spore production (Table 10). could partially (25 % of it) be explained by spore size The data of production include both charged and (volume) or by something, which correlates with it. noncharged spores. Consideration of noncharged Studies of the spore discharge mechanism in the spores could not give much higher rates than the rates Basidiomycota revealed that large spores tend to of charged spores, because the relative amount of produce larger Buller’s drops and small spores noncharged spores is quite low (in our spore samples,

123

89 Author's personal copy

84 Aerobiologia (2014) 30:71–89

Table 8 Area of hymenial surface of Paxillus involutus and the peak period of the production of fruitbodies: an estimation Cantharellus cibarius and of the group of species of dominant based on the empirical data of number of fruitbodies in edible fungi in pine and pine-birch forests in North Estonia in Lahemaa National Park in 1975, in a year of quite poor yield Species or group of species Fruitbody number per ha Area of hymenial surface per ha (m2) Sept I Sept II Sept III Sept I Sept II Sept III

Bog pine forest, Oruveski P. involutus 24 19 25 0.24 0.20 0.25 Dominantsa 1,143 929 1,186 4.7 3.8 4.8 Paludified Calluna pure pine forest, Oruveski P. involutus 75 50 92 0.78 0.51 0.95 Dominantsb 2,199 1,446 2,693 8.9 5.9 10.9 Paludified Calluna pine-birch forest, Ka¨smu P. involutus 197 175 149 2.02 1.79 1.53 Dominantsc 2,247 1,992 1,702 9.6 8.5 7.3 Lichen pine-birch forest, Oruveski C. cibarius 1,025 1,616 732 3.2 5.1 2.3 P. involutus 50 79 36 0.52 0.81 0.37 Dominantsd 2,008 3,164 1,434 7.7 12.1 5.4 Calluna pine-birch forest, Ka¨smu P. involutus 60 83 44 0.62 0.85 0.45 Dominantse 1,743 2,398 1,278 8.3 11.4 6.0 Remarks. Numbers of fruitbodies—according to the data in (Lo˜ugas 1980). Sept I, II, III—at the end of the first, second, and third 10-day period. Area of the hymenial surface of fruitbody was computed using mean sizes of fruitbodies found according to size variation ranges given by Kalamees (1971b, 1972); 31.4 cm2 in C. cibarius and 102.1 cm2 in P. involutus Dominants (ordered according to the number of fruitbodies, the most yielder species first)—a Lactarius rufus (Scop.) Fr., Russula emetica (Schaeff.) Pers., Lactarius helvus (Fr.) Fr., Rozites caperatus (Pers.) P. Karst., and Paxillus involutus (Batsch) Fr.; b Lactarius rufus, Paxillus involutus, Russula decolorans (Fr.) Fr., R. emetica, and R. paludosa Britzelm.; c Lactarius rufus, Paxillus involutus, Leccinum aurantiacum (Bull.) Gray, L. scabrum (Bull.) Gray, Tricholomopsis rutilans (Schaeff.) Singer, and Russula fragilis var. fragilis Fr.; d Cantharellus cibarius Fr., Lactarius rufus (Scop.) Fr., Suillus bovinus (Pers.) Roussel, Paxillus involutus, Sarcodon imbricatus (L.) P. Karst., Leccinum aurantiacum, and Suillus variegatus (Sw.) Kuntze.; e Rozites caperatus, Lactarius rufus, Suillus variegatus, Paxillus involutus, Boletus edulis Bull., and Russula aeruginea Fr it was mostly 0–10 % and rarely 30–40 % of all 4.4 Variation and fluctuation in the hymenial spores). In F. fomentarius, F. pinicola, G. applanatum, emission rate of charged spores and Phellinus populicola, the range of earlier data overlaps our rates partially, their highest rates being 2- Fluctuation of the hymenial emission rate of charged to 3-fold greater than our highest rate. In Phellinus spores and large variation ranges of this rate in a alni, P. nigricans, P. tremulae, and Piptoporus Fomes fomentarius fruitbody—these our results betulinus, the earlier data contrast our rates, being accord with earlier observations of spore production 28- to 1,722-fold greater. These differences might be (Rockett and Kramer 1974; Votintseva 2005). explained by the temporal scale of the studies and by Absence of synchrony between Fomes fomentarius the inter-fruitbodies variation in productivity. The fruitbodies in the hymenial emission rate, which was earlier studies focused on 24-h spore samples col- observed by us, coincides with the earlier observations lected across a sporulation period (Parmasto 1981) or a on spore production (Votintseva 2005). High variabil- year (Kadowaki et al. 2010) or many successive days ity of the hymenial emission rate of charged spores in a or months (Meyer 1936; Buchwald 1938; Rockett and Ganoderma applanatum fruitbody accords with ear- Kramer 1974), while we focused on short time (tens of lier observations on spore production (Haard and minutes) spore collections in a random day or some Kramer 1970; Kadowaki et al. 2010). Asynchronous random days. spore release in G. applanatum fruitbodies, which was

123

90 personal copy

Aerobiologia (2014) 30:71–89 85

Table 9 Territorial emission rate of charged spores and on empirical data of fruitbody numbers, and of charge charges in Paxillus involutus and Cantharellus cibarius in magnitude and hymenial emission rate of spores for negative pine and pine-birch forests in North Estonia: estimation based and positive components Forest, site Time Territorial hymenial rate of negative spores negative charges positive spores positive charges (104 spores ha-1 s-1) (104 e ha-1 s-1) (104 spores ha-1 s-1) (104 e ha-1 s-1) Mean 95 % conf int Mean 95 % conf int

Paxillus involutus Bog pine forest, Oruveski Sept I 0.25 37 29–45 0.56 77 54–99 Sept II 0.21 31 24–37 0.47 64 45–83 Sept III 0.26 38 30–47 0.59 80 56–103 Paludified Calluna pure pine forest, Oruveski Sept I 0.80 120 95–145 1.83 249 176–323 Sept II 0.53 78 62–95 1.20 163 115–211 Sept III 0.98 146 115–177 2.23 304 214–393 Paludified Calluna pine-birch forest, Ka¨smu Sept I 2.08 310 246–377 4.75 646 456–835 Sept II 1.84 275 218–334 4.21 572 404–740 Sept III 1.58 235 186–285 3.60 489 345–633 Lichen pine-birch forest, Oruveski Sept I 0.54 80 63–97 1.22 166 117–215 Sept II 0.83 124 98–151 1.90 259 183–335 Sept III 0.38 57 45–69 0.87 118 83–153 Calluna pine-birch forest, Ka¨smu Sept I 0.64 95 75–116 1.46 166 140–256 Sept II 0.88 130 103–158 2.00 259 192–352 Sept III 0.46 69 55–84 1.06 118 102–186 Cantharellus cibarius Lichen pine-birch forest, Oruveski Sept I 10.62 3,772 2,603–4,951 0.03 3.7 2.2–5.2 Sept II 16.93 6,011 4,148–7,890 0.05 5.9 3.5–8.3 Sept III 7.64 2,711 1,871–3,558 0.02 2.6 1.6–3.7 Remarks. Hymenial emission rate of spores was taken equal to 1.03 negative and 2.35 positive spores per cm2 per second in P. involutus (geometric mean of seven spore samples, Fig. 3), and 3.32 negative and 0.009 positive spores per cm2 per second in C. cibarius (single sample, Fig. 3). For charges, look Table 4 and for territorial hymenial area, Table 8

observed by us, accords with earlier observations shows that—despite asynchronous dynamics of the (Kadowaki et al. 2010). Altogether these data exhibit hymenial emission rate in fruitbodies—the group that fruitbodies of these polypores release the charged hymenial emission rate fluctuates significantly within spores in fluctuating mode, independently from each a 24-h period. The fluctuations onto the highest level other, and at the hymenial emission rate varying very seem to be possible. To find the probability of occur- largely within a day. rence of the situations when all fruitbodies release spore The most interesting result in the group hymenial at near-maximum intensity, the repeated simultaneous emission rate of charged spores concerns the significant measurements of the hymenial emission rate should be difference between time periods in F. fomentarius. This carried out using short-term (\20 min) periods.

123

91 Author's personal copy

86 Aerobiologia (2014) 30:71–89

Table 10 The earlier data of spore production expressed in the units of spores per s per cm2 of hymenial surface Species Spore production Number of Author fruiting bodies Daily mean Hourly mean

Fomes fomentarius 903 – 1 Meyer (1936) F. fomentarius 1,609 – 1 Buchwald (1938) F. fomentarius 36 0.4–105 ? Lebkova (1972) F. fomentarius 486 (914) – 4 Parmasto (1981) Fomitopsis pinicola 1.3 0.4–66 ? Lebkova (1972) F. pinicola 127 (752) – 4 Parmasto (1981) Ganoderma applanatum 141–499 – 30 Kadowaki et al. (2010) Phellinus alni 220 (1,100) – 5 Parmasto (1981) Phellinus nigricans 93 (370) – 3 Parmasto (1981) Phellinus populicola 150 (718) – 4 Parmasto (1981) Phellinus tremulae 185 (752) – 7 Parmasto (1981) Piptoporus betulinus 1,105 1,034–8,611 1 Rockett and Kramer (1974) Parmasto (1981) gives the daily mean at average and at maximum, while Kadowaki et al. (2010) give the daily mean at minimum and at maximum. All these data but those of Lebkova (1972) represent the days of strong or moderate emission (‘‘a 24-h period when spore production was near maximum,’’ Rockett and Kramer 1974; days ‘‘when the fruitbody produced more than 1 9 107 spores per 24 h,’’ Parmasto 1981; ‘‘weak seasonality in spore release,’’ Kadowaki et al. 2010). Lebkova’s data represent four consecutive 24-h periods in the II half of June, in the time when the strongest emission is over

4.5 Upper limit for the maximum hymenial 914:45 = 20 to 1,100:45 = 24 spores cm-2 s-1 in the emission rate of charged spores group of fruitbodies with flat hymenophore. These theoretical upper limits are greater than the To estimate contribution of basidiospores to the phys- maximum values in our experimental data: 20–24 spores icochemical processes of the atmosphere, one must cm-2 s-1 (upper limit) versus 8.4 spores cm-2 s-1 know the upper limit for the total amount of spores (Stereum rugosum) in the group of fruitbodies with flat released into a unit space in a time unit. To estimate this hymenophore, 305–367 spores cm-2 s-1 versus 269 limit for a forest, one must know many factors, including spores cm-2 s-1 (Pleurotus ostreatus) in the group of the maximum hymenial emission rate of spores for fruitbodies with lamellate hymenophore, 914–1,100 every species inhabiting the forest. Values of hymenial spores cm-2 s-1 versus 715 spores cm-2 s-1 (Fomes emission rate are reported for species with poroid fomentarius) in the group of fruitbodies with poroid hymenophores, for polypores with narrow pores (listed hymenophore. Despite that, our experimental results in 4.3). Two of the highest values are 1,100 and 914 showed that the spore production potential of hyme- spores cm-2 s-1. Using these data, it is possible to nium similar to that in Phellinus alni or F. fomentarius estimate the upper limit for the maximum hymenial may occur in the groups of lamellate hymenophore emission rate in fruitbodies with both lamellate and flat (in P. ostreatus, for example) and flat hymenophore hymenophore. The maximum hymenial emission rate (S. rugosum). depends on the potential of the hymenium to produce This estimation was carried out on the assumption spores and on the ratio (area of hymenium):(area of that the spore production potential of the hymenium is fruitbody undersurface). In poroid fruitbodies with similar to that in Fomes fomentarius and Phellinus narrow pores, this ratio is threefold greater than in alni. We overlooked the diversities of composition of lamellate fruitbodies, and 45-fold greater than in flat the hymenium in Agaricomycetes, arrangement of fruitbodies [approximation according to the data of configuration of hymenophore within each hymeno- Fischer and Money (2009)]. So, the upper limit is phore configuration group of fruitbodies, life strate- 914:3 = 305 spores cm-2 s-1 to 1,100:3 = 367 spores gies of fungi, and environmental factors. Due to these cm-2 s-1 in the group of lamellate fruitbodies, and simplifications, our estimate is very rough. But, it

123

92 Author's personal copy

Aerobiologia (2014) 30:71–89 87 might be acceptable in preliminary studies of contri- 4.7 Territorial emission rate of charged spores bution of the primary electric charges of basidiospores to the physicochemical processes of the atmosphere. Our results on the territorial emission rate of charged Phellinus tremulae spores in aspen stand represented a weak sporulation in all fruitbodies. In the case of the 4.6 Number of fruitbodies: number of spore upper limit of the hymenial emission (752 spores sources cm-2 s-1; Parmasto 1981) in old aspen stands 2 -1 (Sforest = 11.4 m ha ), the territorial emission rate The composition of species and productivity (both of charged Phellinus tremulae spores should corre- numbers of fruitbodies and biomass) of mushrooms spond to the value of 8.5 9 107 positively charged strongly vary from year to year within the same forest spores ha-1 s-1. stand (Kalamees and Silver 1988; Krastina 2000). The In Cantharellus cibarius and Paxillus involutus, our study Lo˜ugas (1980) represents, according to an results represented a weak sporulation in a year of empirical estimation of the author, a year of a quite quite poor fruitbody production. The upper limits of poor production, and the study Kalamees and Kollom the territorial emission rate (according to 367 spores (1971) represents a normal year production. There- cm-2 s-1) in such year should be 5 9 104 positively fore, the numbers of fruitbodies per hectare were not and 1.9 9 107 negatively charged spores ha-1 s-1 in 2 -1 6 overestimated in the present study. C. cibarius (Sforest = 5.1 m ha ), and 5 9 10 pos- While we computed territorial emission rate, all the itively and 2 9 106 negatively charged spores fruitbodies were considered as the spore sources. But ha-1 s-1 in P. involutus (2.0 m2 ha-1) at most densely not always all fruitbodies do sporulate. The relative populated areas. amount of nonsporulating fruitbodies varies through In a year of normal fruitbody production in the boreo- the season. In the mushrooms, this amount could be nemoral spruce forest, the group of dominant litter- great (even equal to 100 %) during a few days at the inhabiting species with lamellate fruitbodies (edible and beginning and at the end of season, and it is small inedible fruitbodies together) should emit up to (even equal to 0 %) during the remaining time of the (367 spores cm-2 s-1) 9 (1.6 m2 ha-1) = 5.9 9 106 season. Here, we were supported by the information charged spores ha-1 s-1. In a year of quite poor on the development, growth, and sporulation of fruitbody production in the lichen pine-birch forest, fruitbodies given by Haard and Kramer (1970), the fruitbodies of dominant litter-inhabiting species Kalamees (1971a), and Moore et al. (2008). Conse- (edible and inedible fruitbodies together) should emit up quently, we did not overestimate the number of spore to (367 spores cm-2 s-1) 9 (12.8 m2 ha-1 ? 12.8 sources nor the territorial hymenial area in Cantha- m2 ha-1) = 9.4 9 107 charged spores ha-1 s-1. These rellus cibarius, Paxillus involutus and dominant results show that tenfold differences between forests groups in the peak time of the seasonal dynamics of occur in territorial emission rates of charged spores of fruitbody production, because it was reasonable for mushrooms. this time to consider all fruitbodies as spore sources. In At present, there are many gaps in our knowledge to the wood-inhabiting fungi, bias between the number of consider all basidiomycetes. The number of fruitbod- fruitbodies and the number of spore sources could ies and the area of hymenial surface in dominant occur if the seasonal dynamics of sporulation depends wood-inhabiting species should be studied. Only after on the age of fruitbodies. This dependence is known in that, we could estimate the order of magnitude of the Fomes fomentarius and Fomitopsis pinicola (Votints- total number of charged spores and charges emitted eva 2005). A P. tremulae fruitbody sporulates from from a unit area of forested territory in a time unit. spring to autumn, irregularly performing some-day- long and some-week-long interruptions (Tamm 2000). 4.8 Relaxation time Therefore, it is obvious all fruitbodies in a forest stand do not sporulate simultaneously. Our results of spore The estimated above value of the charge relaxation sources and of the territorial hymenial area in Phel- time 1,430 s means that highly charged spores can linus tremulae should be considered as the theoretical spread over a considerably area depending on the wind maxima. velocity. Formerly, Bowker and Crenshaw (2003)

123

93 Author's personal copy

88 Aerobiologia (2014) 30:71–89 published somewhat similar analysis of charge relax- Dales, R. E., Cakmak, S., Burnett, R. T., Judek, S., Coates, F., & ation time as in present article, but they found an Brook, J. R. (2000). Influence of ambient fungal spores on emergency visits for asthma to a regional children’s hos- estimation of 440 s for the time. The origin of such a pital. American Journal of Respiratory and Critical Care short time came of the overestimated value of air Medicine, 162(6), 2087–2090. conductivity. In addition, they considered total con- Delfino, R. J., Zeiger, R. S., Seltzer, J. M., Street, D. H., Matteucci, ductivity instead of polar conductivity. R. M., Anderson, P. R., et al. (1997). The effect of outdoor fungal spore concentrations on daily asthma severity. Envi- ronmental Health Perspectives, 105(6), 622–635. Eriksson, J., Hjortstam, K., & Ryvarden, L. (1984). The Cor- 5 Conclusion ticiaceae of North Europe (Vol. 7). Oslo: Fungiflora. Eriksson, J., & Ryvarden, L. (1975). The Corticiaceae of North Europe (Vol. 3). Oslo: Fungiflora. Extensive information about airborne spores and about Fairs, A., Wardlaw, A. J., Thompson, J. R., & Pashley, C. H. spore release had been collected in former publica- (2010). Guidelines on Ambient Intramural Airborne Fun- tions in the field of aerobiology. However, the gal Spores. Journal of Investigational Allergology and knowledge on electrostatic charges released by basi- Clinical Immunology, 20(6), 490–498. Fischer, M. W. F., & Money, N. P. (2009). Why mushrooms form diospores into air was insufficient up till now. As the gills: Efficiency of the lamellate morphology. Mycological main results of this study, the data on the polarity and Research,.doi:10.1016/j.mycres.2009.10.006. magnitude of the primary electrostatic charges of Galante, T. E., Horton, T. R., & Swaney, D. P. (2011). 95% of ballistic basidiospores were measured, registered, and basidiospores fall within 1 m of the cap: a field- and modeling-based study. Mycologia, 103(6), 1175–1183. analyzed. Secondly, the magnitude of the hymenial Gilbertson, R. L., & Hemmes, D. E. (1997). Notes on Hawaiian emission rate of charged spores was measured and Coniophoraceae. Mycotaxon, 65, 427–442. analyzed. The upper limit for the emission rate was Gregory, P. H. (1957). Electrostatic charges on spores of fungi. estimated. Third, the equation for the charge relaxa- Nature, London 130, 330. Gregory, P. H. (1973). The microbiology of the atmosphere (2nd tion time of spores was derived, and corresponding ed.). New York: Wiley. relaxation time was estimated. Haard, R. T., & Kramer, C. L. (1970). Periocity of spore discharge in the Hymenomycetes. Mycologia, 62(6), 1145–1169. Acknowledgments This research was supported by the Haldane, J. B. S. (1955). The measurement of variation. Evo- Doctoral School of Earth Sciences and Ecology created under lution, 18, 484. the auspice of European Social Fund and, in part, by the Estonian Hirsikko, A., Nieminen, T., Gagne´, S., Lehtipalo, K., Manninen, Science Foundation through grant 8342. Thanks are due to Kuulo H. E., Ehn, M., Ho˜rrak, U., Kerminen, V.-M., Laakso, L., Kalamees and U¨ lo Tamm for consultations, Leho Tedersoo for McMurry, P. H., Mirme, A., Mirme, S., Peta¨ja¨, T., Tam- critical remarks and Urve Martinson for linguistic help. met, H., Vakkari, V., Vana, M., & Kulmala, M. (2010). Atmospheric ions and nucleation: a review of observations. Atmospheric Chemistry and Physics Discussions, 10(10), 24245–24324. http://www.atmos-chem-phys-discuss.net/ 10/24245/2010. Accessed 9 January 2011. References Ho˜rrak, U., Salm, J., & Tammet, H. (2000). Statistical charac- terization of air ion mobility spectra at Tahkuse Observa- Bowker, G. E., & Crenshaw, H. C. (2003). The influence of fair tory: Classification of air ions. J Geophys Res Atmospheres, weather electricity on the charging of wind-dispersed 105, 9291–9302. pollen. Proceedings of 12th international conference on Ingold, C. (2001). Range in size and form of basidiospores and atmospheric electricity, 9–13 June 2003, Versailles, ascospores. Mycologist, 15(4), 165–166. France, Vol. I, 361–364. Kadowaki, K., Leschen, R. A. B., & Beggs, J. R. (2010). Peri- Bowker, G. E., & Crenshaw, H. C. (2007). Electrostatic forces in odicity of spore release from individual Ganoderma fruit- wind-pollination–part 1: Measurement of the electrostatic ing bodies in a natural forest. Australasian Mycologist, 29, charge on pollen. Atmospheric Environment, 41(8), 17–23. 1587–1595. Kalamees, K. (1971a). Peculiarities in the growth and devel- Buchwald, N. F. (1938). Om Sporenproduktiones storrelse hos opment of fruit-bodies in some fungal species. Estonian Tøndersvampen Polyporus fomentarius (L.) Fr. Friesia, Contribution to the International Biological Programme, 2(1), 42–69. (Cited after Votintseva 2005). III (pp. 47–62). Tartu: Eesti NSV Teaduste Akadeemia. Buller, A. H. R. (1909). Researches on fungi (Vol. 1). London: Kalamees, K. (1971b). The key to Estonian fungi. 1. Tartu: Tartu Longmans. Riiklik U¨ likool. (In Estonian). Chalmers, J. A. (1967). Atmospheric electricity (2nd ed.). Kalamees, K. (1972). The key to Estonian fungi. 2. Tartu: Tartu Oxford: Pergamon Press. Riiklik U¨ likool. (In Estonian.). Corner, E. J. H. (1968). A monograph of Thelephora. Basidio- Kalamees, K., & Kollom, A. (1971). On the biological productivity mycetes. Nova Hedwigia. Beiheft, 27, 1–100. of Agaricales in forest ecosystems. Estonian contributions to

123

94 Author's personal copy

Aerobiologia (2014) 30:71–89 89

the International Biological Programme: progress report III Rockett, T. R., & Kramer, C. L. (1974). Periodicity and total (pp. 29–46). Tartu: Eesti NSV Teaduste Akadeemia. spore production by lignicolous basidiomycetes. Mycolo- Kalamees, K., & Silver, S. (1988). Fungal productivity of pine gia, 66(5), 817–829. heaths in North-West Estonia. Acta Botanica Fennica, 136, Roos, R. A. (1996). The forgotten pollution. Dordrecht: Kluwer 95–98. Academic Publishers. Krastina, I. (2000). Species composition and dynamics of spo- Saar, M. (2013). Investigation of the electrostatic charge of rocarp production of macrofungi in pine forests with basidiospores of the Phellinus igniarius group. Central Vaccinium myrtillus in northern Latvia. Folia Cryptog- European Journal of Biology, 8(5), 410–422. amica Estonica, 37, 39–50. Salm, J., & Tamm, E. (2011). Dependence of the ion-aerosol Krigul, T. (1969). Forest surveyor’s reference book. Tartu: Eesti equivalent attachment coefficient on the ratio of polar Po˜llumajanduse Akadeemia. (In Estonian.). conductivities in a steady state. Aerosol and Air Quality Lebkova, G. N. (1972). Sporulation of polyporous fungi in Research, 11, 211–217. Siberian pine forests in western Sayans. Algae and fungi in Seinfeld, J. H., & Pandis, S. N. (2006). Atmospheric chemistry Siberia and Far East, 2(4), 147–151. (In Russian). and physics: from air pollution to climate change (2nd ed.). Lo˜ugas, T. (1980). About the resources of edible fungi in some Hoboken: Wiley. forest site types of Lahemaa National Park. In Year-book of Sell, I. (2008). Taxonomy of the species in the Phellinus ig- the Estonian Naturalists’ Society, volume 68 (pp. 32–49). niarius group. Mycotaxon, 104, 337–347. Tartu: Eesti Loodusuurijate Selts. (In Estonian, with sum- Stolze-Rybczynski, J. L., Cui, Y., Stevens, M. H. H., Davis, D. mary in English.). J., Fischer, M. W. F., & Money, N. P. (2009). Adaptation of Meyer, H. (1936). Spore formation and discharge in Fomes fo- the spore discharge mechanism in the basidiomycota. PLoS mentarius. Phytopathology, 26, 1155–1156. (Cited after ONE, 2009(4), e4163. doi:10.1371/journal.pone.0004163. Votintseva 2005). Swinbank, P., Taggart, J., & Hutchinson, S. A. (1964). The Moore, D., Gange, A. C., Gange, E. G., & Boddy, L. (2008). measurement of electrostatic charges on spores of Merulius Fruit bodies: their production and development in relation lacrymans (Wulf.) Fr. Annals of Botany, 28, 239–249. to environment. In: L. Boddy, J. C. Frankland, P. van West Tamm, U¨ . (2000). Aspen in Estonia. Tartu: Eesti Loodusfoto. (In (Eds.), Ecology of Saprotrophic Basidiomycetes (pp. Estonian, with summary in English.). 79–103). Amsterdam. eScholarID:130295. Votintseva, A. A. (2005). Ecological characteristics and biotic Morris, C. E., Sands, D. C., Bardin, M., Jaenicke, R., Vogel, B., linkages in mono- and dicaryotic polyporous fungi. 03.00.16– Leyronas, C., et al. (2011). Microbiology and atmospheric Ecology. Dissertation for the Candidate degree in Biology. processes: Research challenges concerning the impact of Scientific supervisor V. A. Muhin, PhD. The Ural State airborne micro-organisms on the atmosphere and climate. University named after A. M. Gorky, Ekaterinburg, Russia. Biogeosciences, 8(1), 17–25. Manuscript. UDK 574:581.524.1:582.284.5. (In Russian.). Nakasone, K. K. (2006). Dendrothele griseocana (Corticiaceae) Webster, J., Procton, M. C. F., Davey, R. A., & Duller, G. A. and related taxa with hyphal pegs. Nowa Hedwigia, Beiheft (1988). Measurement of the electrical charge on some 83(1–2), 99–108. (Stuttgart). basidiospores and an assessment of two possible mecha- Neas, L. M., Dockery, D. W., Burge, H., Koutrakis, P., & nisms of ballistospore propulsion. Transactions of the Speizer, F. E. (1996). Fungus spores, air pollutants, and British Mycological Society, 91(2), 193–203. other determinants of peak expiratory flow rate in children. Winiwarter, W., Bauer, H., Caseiro, A., & Puxbaum, H. (2009). American Journal of Epidemiology, 143(8), 797–807. Quantifying emissions of primary biological aerosol par- Niemela¨, T. (2008). Polypores in Finnland and Estonia. Tartu: ticle mass in Europe. Atmospheric Environment, 43(7), Eesti Loodusfoto. (In Estonian.). 1403–1409. Parmasto, E. (1981). Quantitative characteristics of sporulation Zhang, T., Engling, G., Chan, C., Zhang, Y., Zhang, Z., Lin, M., in wood-destroying polyporous fungi. Vtoraja vseso- Sang, X., Li, Y. D. & Li, Y. (2010). Contribution of fungal juznaja konferencija po biopovrezˇdenij. Tezisy dokladov. spores to particulate matter in a tropical rainforest. Envi- Cˇ ast II (pp. 138–139). Gor’’kij (Russia). (In Russian.). ronmental Research Letters, 5(2), 024010 (9 pp).

123

95

III Saar, Maret & Parmasto, Erast (2014). Primary basidiospore charge and taxonomy of Agaricomycetes. Central European Journal of Biology, 9 (9), 874-887.  &+
,)

#'$
1

1

1
  
 *    /

!"#$%&'()*%+,"&#(-+*%#&'(+.(!"#$#%&

+#.,"#9&/"*.0.-*1-#$&23"#4$&"50&%"6-5-,9& ' 0!)#'%. + *

!"#"$%&'((%)*&+" !"#$%&&""#'(&)#"*%&+"#,"*%-&

$2!35)$05"1%"15!088"045'565$"1%"&3'"6(563!("!0#"07'310)$05!(""'$0"$48 4510'!0" 0'7$34'58"1%"'%$""'$0"$48"BCDCE"!3568"4510'!

!"&"*,"-(40(.$E(4/59/((&&"0)"-(54(1$23$%E(4/5C

(7#)%$&)6
"" ! "%!(*"+*% "$!"!*"%!%""**(! !%!("(%* "!%!"%*"(%*+""3% !("3% + * "%!*"!*"3*%!"""!" 3+* 6" !*" * " $**" (*(!*+" 3%" %(" %" !%3*" +*#(*" 3**%!" !*" 3% +*" %+" %$" !*" !" %" " %" &!%" %#% $%*(
")'%())!
0"
')
.
+')!"
&'"""
&"$
")'%(
 *(
) 
-&'!#$)"
()*&
$"
)%
!$+()! )
) 
&'!#'.
 ' (
) 
 ' (
&'($)
%$
(&%'(
!##!)".
)'
) 
'"(
'%#
(&%'
'!$
""(
&%'(
%

(!!%#(
%

(&!(
%
.#$%#.)%*(
*$ !
,'
%"")
!$
+'!%*(
$)*'"
%$!)!%$(
 
$%$)*' ($)
'.!$
*&
%""&(!$
%'
(!$
)%
(&%'*")
(!!%#(
,'
-"*
'%#
) 
)-%$%#!"
$".(!(
) 
 
)*' ($)
(!!%#(

(&%'
(#&"(
%

(&!(
,'
)-%$%#!"".
$"./
 (
(&!(
'&'($)
! )
%''(
 '!%#.)(
(!!%#.%)
%+'!$

#!"!(
$

$'
 
$".(!(
( %,
) )
) 
(&%'
 ' (
,'
!()'!*)
%'!$
)%
) 
&%"'!).
(!#!"'".
!$
""
(#&"(
!$

(&!(
$
!$

$*(
$
#%()
((
"(%
$'
%
%$
#!".

) 
(#
).&
%
&%"'!).
!()'!*)!%$
%
(&%'
 ' (
%((!".
""
)-
'%#
(&!(
)%
#%$%& .")!
#!"!(
'
 ')'!/
.
(&!0
).&
%
&%"'!).
%
) 
&'!#'.
")'%())!
 ' 
%
(!!%(&%'(
&$!$
%$
) 
)-%$%#!"
'%*&
""
(&%'
 ' (
,'
$ )!+

&%(!)!+
%'
%)
$ )!+
$
&%(!)!+
 ' (
,'
&'($)
! "%!"("+"3*"" *"""#* !"!*"3% ! ("+ (%*"*(% "%+""( !"(!"*9*#*"%*%6"

4"EF5%-#6

    

 

  

 

 
 

  
 


  

 

   

 

1
'(!)
&
/
%%

!"#1642#7"6'21# *"#" ./#)#$+#" -0" +"%/5#)" -$" *"#" 1%<-/(*4" -0" ).-/#)3" &2*" $-*" *"#" -++2//#$+#" -0" &(.-'%/" ,()*/(&2*(-$" -0" ).-/#" !"#$"%"&%''()*(+"&%)(,(-).-/#"'(&#/%*#)"0/-1"%"&%)(,(213" +"%/5#)"($"%''").#+(#)6"G))#$*(%''4")(1('%/"%..%/%*2)"9%)" *"#" ).-/#" 2)2%''4" +%//(#)" %$" #'#+*/-)*%*(+" +"%/5#6" DB43
8=
C74
Z4;3
?;028=6
C74
0??0A0CDB
D=34A
5D=60;
7#5($%',"82''#/"9"-",()+-:#/#,"*"#"."#$-1#$-$3"0-2$," 0/2(*($5"&-,(#)3"&%)(,(-1#)B3"%$,"*"#",#$)(*4"-0",#.-)(*" *"%*"($"%''"+%)#)";*"#"1%<-/(*4"-0").-/#)"/#+#(:#".-)(*(:#" -0" ).-/#)" %,"#/($5" *-" *"#" +"%/5#," .'%*#)" 1%($*%($($5" -/" $#5%*(:#" #'#+*/(+" +"%/5#)" -0" ,(00#/#$*" )*/#$5*")3" C74
4;42CA82
Z4;3
F0B
>1B4AE43
%4BD;CB
8=
$%&'!()*( 9"(')*"%"+#/*%($"$21&#/",-"$-*"&#+-1#"+"%/5#,"%*"%''=" (&+,#*-'!*( C63" 3/,'!4#00)*( 5!&*(#4*
A
%437403
>?@6"A#"-&)#/:#,"*"#"0%''"-0"&%)(,(-).-/#)"&#*9##$"*"#" H('5%'4)" I" J-$+%':-3" 3"( +!(&(#)*
D;;
)8;60;HB
#'#+*/(+%''4"+"%/5#,"%$,":#/*(+%''4".'%+#,".%/%''#'".'%*#)" A-..'#"I"K%+L6"K-"$)-$3".5/0!/-&(5!%50&46#4*!*
$42:
1H
<82A>B2>?4
8B
Z6DA4
86DA4

8=
-?@B")"-9)"2)"*"%*" 
&<

4B;4A
."(*2)&''/*&
)07;
$
D<<
0=3
*"#".-)(*(:#'4"%$,"$#5%*(:#'4"+"%/5#,").-/#)"-++2//#," ./01,/')*( *2)&+/*)*
D3B
A
F4A4
B8<8;0A
C>
C70C
)(12'*%$#-2)'43" !"#"" *"#" &(.-'%/" ,()*/(&2*(-$" -0" +"%/5#)" 0-2$,"&4"82''#/M"&-*"".-)(*(:#'4"%$,"$#5%*(:#'4"+"%/5#," -++2//#,"($"*"#").-/#")%1.'#)6"A#"-&)#/:#,").-/#)"-0" ).-/#)"9#/#"/#'#%)#,"0/-1"%"&%)(,(-1#")(12'*%$#-2)'46" $%&'!()*((&+,#*-'!*"C63"./01,/')*(*2)&+/*)*
D3B
N$" )-1#" ).#+(#)3" *"#" .-)(*(:#'4" +"%/5#," ).-/#)" A
0=3
>C74A
D=<4=C8>=43
B?4284B
#1B4AE0C8>=B
./#:%('#,3"%)"-..-)#,"*-"*"#"$#5%*(:#'4"+"%/5#,").-/#)" 1%,#"&4"D"('(."E/#5-/4"-$")#:#$").#+(#)">F.
2>=ZA<43
8=
>C74AB
&?>A4B
>5
7&4/6#'+&( &,,0&4&-)+
$4AB

!""$)!'(")!3$5-4!!3$)6-$$ 76/ !"#$%&'"%()#%'* +,-".,#* +#%' / 0122123 4563 78

99 !7&&""#(&)7&+"#,"*%-&

D%*6"+%//(#,".-)(*(:#"+"%/5#)"-$'46"N$"*"()").#+(#)3"%")#*" /#L2(/#,"*-"./-<#+*"*"#").-/#"-00"*"#"&%)(,(21")"-9#," >5
>1B4AE0C8>=B
F0B
20AA843
>DC
8=E>;E8=6
0
3>I4=
C70C
C74
BDA5024
4=4A6H
>5
C74
3A>?
2>D;3
14
BD5Z284=C
).-/#" )%1.'#)" 0/-1" )#:#/%'" &%)(,(-1#)" ($" ,(00#/#$*" C>
?4A5>A<
C74
F>A:
8=E>;E43
-V@6"O"#""4.-*"#)()"%&-2*" .'%+#)" ,2/($5" *9-" 4#%/)B6" O"#" /#)2'*)" ($,(+%*#," *"%*" *"#" #'#+*/-)*%*(+" /#.2')(-$" &#*9##$" *"#" ).-/#" %$," *"#" %" 2$(.-'%/P.-)(*(:#" ,()*/(&2*(-$" -0" ).-/#" +"%/5#)" +-2'," &%)(,(21"($:-':#,"*"#"+'%(1"*"%*"*"#"+"%/5#"-$"*"#").-/#" 14
0
2>=B8BC4=C
540CDA4
>5
C78B
B?4284B

D=8?>;0A 0=3
C74
10B838D<
0A4
4@D0;
8=
1>C7
<06=8CD34
0=3
$#5%*(:#" ,()*/(&2*(-$" -0" ).-/#" +"%/5#)" 9%)" 0-2$," &4" .-'%/(*4B"%*"*"#"1-1#$*"-0",()+"%/5#">V@6"N$"$($#").#+(#)" $4C4A
&F8=10=:
#-( &0"" ($" 8#',)0&( 0&('1+&4*
*D;54=
>5
60A82>A4
270A64C><0BB
@D>C84=C

&27APC
-Q@6" O"#)#" ,%*%3" .2&'()"#," &#*9##$" ?RSRP 9%)" 1#%)2/#," %$," *"#" +"%/5#" 1%5$(*2,#" ,#*#/1($#," 
0=3
2>E4A8=6
4867C
B?4284B
>5
7H<4=>DB
>V@6" O"#" 1#%$" :%'2#" :%/(#)" -:#/" %" 9(,#" /%$5#3" 0/-1" 60A82>
5A06<4=C0;
C70C
A46>AH

4
0B
 
a
` "Y"($">V@"0-/"9&'&*+!)*(/'#&6#*" 78
2>=2;D34
8=

C70C
X;8CC;4
8B
:=>F=
>;C>=
A
C>

4
0B

a
` "Y"($">V@"0-/"$%&'!()*( %&-2*" *"#" ."#$-1#$-$=" >@3" !"#"
F4
70E4
8=BD5Z284=C
:!*,/')*"C6B6"Y%'+2'%*(-$)")"-9#,"*"%*"(0"*"#")-2/+#"-0" :=>F;4364
01>DC
C74
?>;0A8CH
0=3
<06=8CD34
01B>;DC4
#$#/54"0-/"*"#",()+"%/5#"()"#'#+*/-)*%*(+3"*"#"1%W(121" :%'2#B"-0"*"#"./(1%/4"+"%/5#)3"*"#"#'#+*/-)*%*(+"+"%/5#)" ,()+"%/5#",()*%$+#"-0"*"#").-/#"()"0%/")"-/*#/"*"%$"*"%*" +%//(#,"-$"*"#"&%''()*(+"&%)(,(-).-/#)"(11#,(%*#'4"%0*#/" 1#%)2/#,"#W.#/(1#$*%''4">V.
'74
F>A:B
>=
C74
270A64
'(&#/%*(-$"0/-1"%"0/2(*($5"&-,46" 1%5$(*2,#" >Q3V@" ,(," $-*" 5(:#" ($0-/1%*(-$" %&-2*" 0%+*-/)" !06=8CD34
>5
C74
?A8<0AH
270A64B
F0B
ZABC
<40BDA43
8=[D4=28=6
C74
<06=8CD34
>5
C74
B?>A4
270A64
8=

8=
>=4
B?4284B
-Q.

=460C8E4
4;42CA>BC0C82
O"#" 1#+"%$()1" -0" +"%/5($5" -0" *"#" &%''()*(+" 270A64
>5

4
0B
- b S b .a `" #6)626" ($" >Q@B" 10B838>B?>A4
8B
D=:=>F=
0B
C74
BD1942C
70B
=>C
144=
9%)" 0-2$," -$" %" T1#%$U" ).-/#" -0" 8#',)0&( 0&('1+&4*" BCD3843
H4C
&><4
7H?>C74B4B
01>DC
C74
>A868=B
70E4
*D;54=

&27APC
'74
270A64
<06=8CD34
F0B
0;B>
144=
<034
'74
270A64
8B
?82:43
D?
5A><
C74
4=E8A>=<4=C
1#%)2/#,">V@"($"*"#".#/(-,"9"#$"*"#")*2,(#)"*"%*"'#%,"*-" %0*#/" *"#" /#'#%)#" >V@6"O"#" ).-/#" &#+-1#)" +"%/5#," ,2#" *"#"+-//#+*"($*#/./#*%*(-$"-0"*"#"&%''()*-).-/(+",()+"%/5#" *-"%"1#+"%$(+%'")#.%/%*(-$"-0"*"#"1%*2/#").-/#"0/-1"*"#" 1#+"%$()1" ($" &%)(,(-14+#*#)" 9#/#" .#/0-/1#,6" O"#$3" )*#/(51%">Q@"-/"&4"0/(+*(-$"%*"*"#"1-1#$*"-0"/#'#%)#">?Q@6" 8=
C74
 B
4E834=24B
>=
C74
;8@D83B
>=
C74
B?>A4
F4A4
O"#"+"%/5#"()"5#$#/%*#,"&4";)*/#%1($5".-*#$*(%'="%)"*"#" ./-:(,#,3" %$," *"#" )2/0%+#" *#$)(-$" "4.-*"#)()" %&-2*" '(L2(," -0" *"#" ,/-." ()" 0-/+#," *"/-25"" %" 1#1&/%$#" > @6" *"#"&%''()*-).-/#",()+"%/5#"1#+"%$()1"9%)"%,:%$+#," 
?>BB81;4
B>DA24
>5
C74
B?>A4
270A64
8B
0
18>;>6820;;H
0=3
C74
4;42CA>BC0C82
A4?D;B8>=
7H?>C74B8B
A4942C43
0-/1#," #'#+*/#*Z.-'%/" 1-'#+2'#)" -/(#$*#," ($" +#''" 9%'')3" >A
D;;4AYB
3A>?
C70C
34E4;>?B
0C
0
E4AH
B<0;;
1DC
%''")#**($5"2."12*2%'"/#.2')(-$3".2''($5"*"#"1%*2/#").-/#" +-1.'#W"-/5%$"-0"*"#").-/#">.
F7827
4GC4A=0;;H
;>>:B
0/-1"*"#")*#/(51%"> @6"O"#)#""4.-*"#)#)""%:#"$-*"&##$" ;8:4
0
<8=DC4
?A>942C8>=
0C
C74
B?>A4
0=3
8B
20;;43
78;0A
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
;B>
C74
4G?0=B8>=
>5
%+L2(/#," &4" .%/*(+'#)" ($" *"#" %*1-)."#/#" >?V@6" O"#)#" 82''#/U)",/-."()",/(:#$"&4"*"#"#W*/2)(-$"-0"%""45/-)+-.(+" ."#$-1#$%".-($*#,"*-"*"#"#$,-5#$-2)"-/(5($"-0").-/#" )2&)*%$+#" 0/-1" *"#" ,)4(-)+( 0&('1+&4*
C74
?>8=C
>5
270A64B
*4
?A4BD<4
C70C
CF>
>A
<>A4
?A>24BB4B
0A4
0-/1%*(-$"-0",/-."($(*(%'B3"/#)2'*($5"($"%"/%.(,"%&)-/.*(-$" /#).-$)(&'#" 0-/" *"#" ).-/#" +"%/5#3" %$," %&'#" *-" #W.'%($" -0"9%*#/":%.-/"0/-1"*"#"%(/">?SP?F.
&>
50A
=>C78=6
F0B
*"#" &(.-'%/" ,()*/(&2*(-$" -0" ./(1%/4" +"%/5#)" ($" ).-/#" :=>F=
01>DC
C74
;8@D83
>=
C74
B?>A4
F0;;
">F
C74
.-.2'%*(-$)6" O"#" 1(+/-1#+"%$(+%'" ./-+#))#)" ($:-':#," 4E834=24
B7>F43
C70C
0
[0C
;8@D83
?>>;
0
Z;<
>5
;8@D83
>A
($" *"#" &%''()*-).-/(+" ,()+"%/5#" 1#+"%$()1" %/#" )(1('%/" 0
[0C
3A>?
20;;43
0=
030G80;
3A>?
34E4;>?B
0C
C74
B?>A4
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
C74
B?>A4
0=3

C78B
[D83
<>E4<4=C
20DB4B
C74
A0?83
)*#/(51%*%"%$,"*"#"&%)(,(-).-/#)3"-/"($"*"#"&%''()*-).-/(+" /#,()*/(&2*(-$"-0"1%))3"+/#%*($5"'($#%/"1-1#$*21"($"*"#" ,()+"%/5#3"-/"&-*"6"N0"*"#"&(-+"#1(+%'"./-+#))#)"5(:($5" 38A42C8>=
0F0H
5A><
C74
BC4A86<0
C74
B?>A4140A8=6
/()#"*-"*"#").-/#"+"%/5#"%/#"*"#")%1#"9(*"($"%").#+(#)3" ./-<#+*(-$" -0" &%)(,(21B3" '#%,($5" *-" )#.%/%*(-$" -0" *"#" *"#$" *"#" .-'%/(*4" ,()*/(&2*(-$" -0" +"%/5#)" ($" %" ).-/#" ).-/#"0/-1"*"#"&%)(,(21">X@6"Y%'+2'%*(-$)"-0"*"#"#$#/54" )%1.'#" )"-2'," $-*" &#" ,#.#$,#$*" -$" &%)(,(-1#)6""

764 !"#$%&'"%()#%'* +,-".,#* +#%' / 0122123 4563 78

100 )#%).
*##'*(')
")!
&
+-'&'%.
'
!)#'%. + *

N0"*"#"./-+#))#)"%/#"*"#")%1#"($"%"5#$2)3"*"#$"*"#".-'%/(*4" .-)(*(:#"+"%/5#)"9#/#"#W*/%+*#,"-$*-"*"#"$#5%*(:#".'%*#" ,()*/(&2*(-$" )"-2'," $-*" &#" ,#.#$,#$*" -$" *"#" ).#+(#)" %$," *"#" ).-/#)" +%//4($5" $#5%*(:#" +"%/5#)3" -$*-" *"#" 9(*"($"(*3"#-(6"O"#"./#)#$*".%.#/"%,,/#))#)"*"#"+"%/5#" ?>B8C8E4
?;0C4
'74
B?>A4B
70E8=6
270A64B
8=BD5Z284=C
.-'%/(*4"")(1('%/(*(#)""9(*"($"&%)(,(-1#)3").#+(#)3"5#$2)3" *-" &#" #W*/%+*#," -$*-" *"#" .'%*#)3" %$," *"#" 2$+"%/5#," 0%1('(#)"%$,"-/,#/)6"O"()"($0-/1%*(-$"+-2',"&#"2)#02'"($" B?>A4B
20;;43
=>=270A643
B?>A4B
54;;
>=C>
C74
[>>A
($:#)*(5%*($5" *"#" &%''()*-).-/(+" ,()+"%/5#" 1#+"%$()1" &#*9##$"*"#".'%*#)6"O"#",#.-)(*"-0").-/#)"%,"#/($5"*-" %$,"0-/"+-$)*/2+*($5""(5"#/P'#:#'"."4'-5#$46" C74
?;0C4B
F0B
>1B4AE43
1H
<82A>B2>?4
G
 
DB8=6
#DA
F>A:
8B
10B43
>=
C74
30C0
2>;;42C43
8=
C74
 B
A4[42C43
;867C
0=3
C74
CA0=B<8CC43
;867C
F0B
DB43
5>A
0=3
 B
0C
C74
=BC8CDC4
>5
,>>;>6H
0=3
>C0=H
>5
C74
1>CC><
6;0BB
B;834B
&?>A4B
5>D=3
>=
1>C7
?;0C4B
C74
20345
&284=24B
>5
BC>=80
,
'74=
C74
($,(+%*#," *"%*" %" ).-/#" )%1.'#" "%," &(.-'%/" ,()*/(&2*(-$" ./(1%/4" +"%/5#" -0" &%)(,(-).-/#)" ($" "41#$-14+#*-2)" >5
270A64B
#22DAA4=24
>5
B?>A4B
>=
>=4
?;0C4
0=3
60A82>;0A
38BCA81DC8>=
>5
270A64B
<>D=C
>5
B?>A4B
9#/#" .2&'()"#," > 3?R@6" O"#)#" .%.#/)" %,,/#))#," *"#" .#/".'%*#"9%)",#*#/1($#,"&4"+-2$*($5"*"#").-/#)"> @3" ).-/#"+"%/5#P*-P1%))"L2-*(#$*3".-'%/(*4"%$,"1%5$(*2,#" -/" :()2%''43" (0" +-2$*($5" 9%)" (1.-))(&'#" ,2#" *-" *"#" -0"*"#").-/#"+"%/5#3"%$,"*"#"#1())(-$"/%*#"-0"+"%/5#," C782:=4BB
>5
C74
34?>B8C
<>D=C
>5
B?>A4B
?4A
B05
B?>AD;0C8>=
>5
C74
C74
7H<4=80;
BDA5024
0=3
5A><

70
>5
C74
5>A4BC43
&%)(,(-1#3" &#($5" ($" *"#" /%$5#" -0" ?SF\?S" ).-/#)" .#/" C4AA8C>AH
&8<8;0A;H
C>
C74
?A4B4=C
?0?4A
C74B4
A424=C;H
)%1.'#6" !"#$" *"#" $21&#/" -0" ).-/#)" ($" %" )%1.'#" .2&'()"#,".%.#/)"($:-':#,"*"#"'#:#'"-0")(1('%/(*4"($"*"#" #W+##,#," ?SV3" *"#$" 2)2%''4" %$" -:#/'-%," -0" ).-/#)" 270A64
?>;0A8CH
1DC
383
C78B
0C
;>F4A
;4E4;B
10B838><4
-++2//#," -$" *"#" +-''#+*($5" )2/0%+#" %$," *"#" ).-/#" ).#+(#)"%$,")(&'($5").#+(#)"5/-2.B6"O"#)#"/#+#$*".%.#/)" 2>D=C8=6
F0B
8BB81;4
'74
1D;:
>5
B?>A4
B0?R@3" *"#")%1.'#)"9"#/#"*"#").-/#"+"%/5#P*-P1%))"L2-*(#$*" +-$)()*#,"-0"?SQ\?SV
B?>A4B
E4A064
E0;D4
>5
270A64B
9%)"1#%)2/#,"%$,"*"#"1%5$(*2,#"-0"*"#").-/#"+"%/5#" -0" *"#" ).-/#)" ($" %" ).-/#" )%1.'#" 9%)" ($" *"#" /%$5#" -0" 0BB4BB43
#DA
?0?4A
8=2>A?>A0C4B
0;B>
C74
B?>A4
1%5$(*2,#"-0"?SP?SSS"#">?R@6" )%1.'#)"9"#/#"-$'4"*"#"+"%/5#".-'%/(*4"9%)",#*#/1($#,6" +"+# 324$5 D-'%/(*4" -0" ).-/#" +"%/5#)" 9%)" )*2,(#," ($" FQV" ).-/#" +"#93$4'0$16!)#342"$#74$5 )%1.'#)" -0" ?QV" &%)(,(-1#)3" +-:#/($5" VS" ).#+(#)3" 
64=4A0

50<8;84B
0=3
4867C
>A34AB
'74
1D;:
+"!#93$4'0$16!)#5$6873 >5
B0;;42C43
8=
  
C74
;0C4A
O-" )*2,4" *"#" ./(1%/4" #'#+*/-)*%*(+" +"%/5#)" -0"  


0=3

2>;;42C8>=
F0B
20AA843
&%)(,(-).-/#)" ($" $%*2/%'" +-$,(*(-$)3" %$" #W.#/(1#$*%'" -2*" *-"5#*"1-/#"/#./#)#$*%*(:#)"0-/")-1#"*%W-$-1(+%'" )#*P2." 9%)" ,#)(5$#," %$," &2('*" %)" %" .-/*%&'#" ,#:(+#" 6A>D?B
D=68
6A>F8=6
8=
C74
=>AC7F4BC4A=
?0AC
>5
C74
> @6" O"#" ,#:(+#" +-$)()*#," -0" *"/##" .%/*)M" %" +"%1&#/" 0BCDA>?40=
$;08=
8=
BC>=80
C74
B>DC740BC4A=
C>
2>;;42C
C74
B?>A4B
0
BC0=3
C>
7>;3
C74
270<14A
D?
?0AC
>5
C74
0D20BDB
!>D=C08=B
I4A10890=
0=3
C74
D=34A
0
10B838><4
0=3
0
10CC4AH
0
2>=BC0=C
E>;C064
=>AC7F4BC4A=
?0AC
>5
C74
0D20BDB
!>D=C08=
3H640
B>DA24
D4
C>
C74
B<0;;
B8I4
0=3
;867C
F4867C
>5
C74
%DBB80
F4A4
DB43
C>
BCD3H
'01;4
?
&?0C80;;H
0=3
+"%1&#/" %$," *"#" 12'*(P)*#." 1-&('(*4" -0" *"#" )*%$,3" *"#" *#1.-/%''4" *"#" 1-)*P)*2,(#," ).#+(#)" 9#/#" ;/+#*( 34E824
F0B
DB401;4
8=
0;;
CH?4B
>5
C74
2>=Z6DA0C8>=
>5

B?>A4140A8=6
BCAD2CDA4
>5
C74
10B838><4
<8=8A8I>=C0;
?A>942C8>=
38<4=B8>=B
F4A4

G

2<
0=3
($" *"#" Y%2+%)2)3" *"/##" 4#%/)B6" O"#" %1-2$*" -0" ).-/#" C74
<8=85

)%1.'#)" %$," &%)(,(-1#)" #W%1($#," 9%)" *"#" "(5"#)*" +16" N$" *"#" +"%1&#/3" *9-" :#/*(+%'" .%/%''#'" 1#*%'" .'%*#)" ($" ;/+!-/,*!*( ,!4!(/0&( &F
$
0ABC

B0BC0C82
Z4;3
85
2>==42C43
C>
C74

10B838><4B
5>;;>F43
1H
;/+#*(
C74
4;42CA>BC0C82
Z4;3
<08=C08=43

#=4
B?>A4
B0
>A34AB
&4" *"#" +"%/5#," .'%*#)3" */%<#+*-/(#)" -0" *"#" +"%/5#," Y%$*"%/#''%'#)" %$," Y-/*(+(%'#)6" O"#" &(55#)*" $21&#/" ).-/#)",#+'($#,"0/-1"*"#":#/*(+%'6"O"#").-/#)"+%//4($5" -0" 5#$#/%" %$," 0%1('(#)" #W%1($#," 9%)" ($" *"#" -/,#/"

765 !"#$%&'"%()#%'* +,-".,#* +#%' / 0122123 4563 78

101 !7&&""#(&)7&+"#,"*%-&

)'*#+,'( !"#$ %"#&'( 1#$2 %,+"'#(-'*'.(+$/''''''''''''''''''''''''''''''0''$-,(#+"&

3456 %.77"$8'#.+.7( 9''$"7##8':&+'(,# ;<ƒ'=>€'?8''6@ƒ'=;€':' 9 345> %A$,(B8'&.77"$8'#.+.7( Cw$D&"EF#8':&+'(,# ;<ƒ'3@€'?8''65ƒ'3<€':C

345= %A$,(B G#77,&+.8':&+'(,# *
 )


 *' )
 G

 %.77"$ H#2#+#E#8'IJ"$K#,F#( *
 )


 *' )
 H

 %.77"$ L.J"$,AE8'I-/B"#''SM.&&,#T *
)


*' )
 L

 %.77"$8'#.+.7( )#N"7##8':&+'(,# *
 )


 *' )
 )

345; I.+.7( G#$+.8':&+'(,# *
)


 *')
 G$ 345<' I.+.7( P'2"8':&+'(,# *' )'

 *' )' P 34

 I.+.7( 9"-.8':&+'(,# *
 )


 *' )
 9"

8$7+"(5@((%+.-/''('A'E#$,+/''Q'+N"'A$,7#$/'"E"*+$'&+#+,*'*N#$B"&''Q'K#EE,&+,*'K#&,-,'&A'$"&',('+N"'N/7"('7/*"+'.&'IB#$,*'7/*"+"&R'+,7"'#(-'E'*#+,'(' 'Q'*'EE"*+,'(''Q'&A'$"'AE"&S'T('#EE'E'*#+,'(&8'"U*"A+'G#$+.8'+N"'&A'$"&'V"$"'*'EE"*+"-',('(#+.$#E'Q'$"&+'*'(-,+,'(&S'T('+N"'+'V(''Q'G#$+.8'' +N"'&A'$"&'V"$"'*'EE"*+"-',('#'*"EE#$''Q'#'N'.&"S

60A820;4B

B?4284B

50<8;84B
=

10B838><4B
5"#$57)65# +-:#/($5"F?").#+(#)3"+"%/5#)"9#/#")*2,(#,"/#.#%*#,'43" +-''#+*($5" *9-" *-" #(5"*" ).-/#" )%1.'#)" .#/" &%)(,(-1#6" 5"!#!5'#'20$ N$" )#:#/%'" ).#+(#)3" ).-/#" )%1.'#)" 9#/#" +-''#+*#," ($" #5
C74

10B838><4B
4G0<8=43
A4?40C43;H
'01;4
FB3" 38554A4=C
B40B>=0;
E4A=0;
04BC8E0;
0=3
0DCD<=0;
8=

10B838><4B
C74
?>;0A8CH
CH?4
>5
B?>A4
270A64B
28A20380=
=>2CDA=0;
<>A=8=6
38DA=0;
0=3
4E4=8=6
9%)"(,#$*(+%'"($"%''")%1.'#)"-0"*"#")%1#"0/2(*($5"&-,43" ?4A8>3B
0=3
8=
38554A4=C
08A
2>=38C8>=B
86DA4
?%3"?&3"?+B6 %$," ,(00#/#$*" ($" Q" &%)(,(-1#)" >>164#00)+( <#'')%!4#)+"

9*:3%"(5$@((%A"*,"&''+"7A'$#$,E/'7'&+'"U#7,("-',('+N"'&+.-/''('A'E#$,+/''Q'+N"'A$,7#$/'"E"*+$'&+#+,*'*N#$B"&''Q'K#EE,&+,*'K#&,-,'&A'$"&',('+N"' N/7"('7/*"+'.&'IB#$,*'7/*"+"&R'-,.$(#E'-,&+$,K.+,'(''Q'+N"'*'EE"*+,'('+,7"''Q'&A'$"'AE"&S'W,B.$"'&#(('075#'%0'4'75G'&N'V&' &.77#$/'Q'$'+N$""'&A"*,"&R'&#(('075#(0'#SX'(-#$+&"DT'Y#$7#&+'8', #0'%4'!05'SW$ST'YS 'P#$&+S'#(-', #2127('!1(#?,"7"EwS

766 !"#$%&'"%()#%'* +,-".,#* +#%' / 0122123 4563 78

102 )#%).
*##'*(')
")!
&
+-'&'%.
'
!)#'%. + *

9*:3%"(57@((%A"*,"&''+"7A'$#$,E/'7'&+'"U#7,("-',('+N"'&+.-/''('A'E#$,+/''Q'+N"'A$,7#$/'"E"*+$'&+#+,*'*N#$B"&''Q'K#EE,&+,*'K#&,-,'&A'$"&',('+N"' N/7"('7/*"+'.&'IB#$,*'7/*"+"&R'#((.#E'-,&+$,K.+,'(''Q'+N"'*'EE"*+,'('+,7"''Q'&A'$"'AE"&S'W,B.$"'&#(('075#'%0'4'75G'&N'V&' &.77#$/'Q'$'+N$""'&A"*,"&R'&#(('075#(0'#SX'(-#$+&"DT'Y#$7#&+'8', #0'%4'!05'SW$ST'YS 'P#$&+S'#(-', #2127('!1(#?,"7"EwS'

9*:3%"(5&@((%A"*,"&''+"7A'$#$,E/'7'&+'"U#7,("-',('+N"'&+.-/''('A'E#$,+/''Q'+N"'A$,7#$/'"E"*+$'&+#+,*'*N#$B"&''Q'K#EE,&+,*'K#&,-,'&A'$"&',('+N"' N/7"('7/*"+'.&' IB#$,*'7/*"+"&R' 7"#(' #,$' +"7A"$#+.$"'S+ƒT' #(-' 7"#(' #,$' $"E#+,D"' N.7,-,+/'S
T' -.$,(B' +N"' *'EE"*+,'(&' 'Q' &A'$"' AE"&S'W,B.$"'&#(('075#'%0'4'75G'&N'V&'&.77#$/'Q'$'+N$""'&A"*,"&R'&#(('075#(0'#SX'(-#$+&"DT'Y#$7#&+'8', #0'%4'!05'SW$ST'YS 'P#$&+S' #(-', #2127('!1(#?,"7"EwS

767 !"#$%&'"%()#%'* +,-".,#* +#%' / 0122123 4563 78

103 !7&&""#(&)7&+"#,"*%-&

?.7K"$''Q'$"A"#+"-E/'"U#7,("-' ?.7K"$''Q'&A'$"'AE"& %A"*,"& K#&,-,'7"& 'A"$'K#&,-,'7"

0(')!1!H56'5# 14#('5#SW$ST'P'+ES'Z'Y'.J#$ 3 6 010'12&14#276#0'S%*N.7#*NST'YS 'P#$&+S 3 6

0146'04'75'&AS 3 6 6'K#&,-,'7"8'<'AE"&'Q'$'"#*N[' 41)#5#$1)#064'75'S)ST'CS'P,*2U'QS = 3'K#&,-,'7"8'5'AE"&[' 3'K#&,-,'7"&8'@'AE"& 3'K#&,-,'7"8'<'AE"&[' 41)'6125'5#2'0'!1(#S%VST'YS 'P#$&+S 33 3'K#&,-,'7"8'@'AE"&[' 4'K#&,-,'7"&8'6'AE"&'Q'$'"#*N''' 41)'6125'5#415#'SIEKS'Z'%*NV",(ST'YS 'P#$&+S > 6'AE"&'A"$'K#&,-,'7" 3'K#&,-,'7"8';'AE"&[' 601"#4)#22(067)'SY"$&ST'Y#+S = >'K#&,-,'7"&8'='AE"&'Q'$'"#*N' 8H"0#((7)#$#447%'0#7)'SW$ST'YS 'P#$&+S 3\ 6

!64'75#64'8'('5'SW$ST'W$S 3 6

!64'75#6742'5'S]",(7ST'W$S >^6\ 6'AE"&'A"$'K#&,-,'7"

#!!'07)#74'06'!7)'SX.EEST'L$#/ 3\ 6

I'((75#'081(7675'SX#+&*NT'W$S > 6'AE"&'A"$'K#&,-,'7"

#(('075#64#)7(##SX'(-#$+&"DT'X'(-#$+&"D'Z'YS?S'X'$,&'D = 6'AE"&'A"$'K#&,-,'7"

&#(('075#(0'#SX'(-#$+&"DT'Y#$7#&+' 3 >'AE"& 3'K#&,-,'7"8'@'AE"&[' &#(('075#2127('!1(#?,"7"Ew' = 3'K#&,-,'7"8';'AE"&[' 6'K#&,-,'7"&8'='AE"&'Q'$'"#*N &1('16'&AS 3 6

141"#"(##2'0'#SX$'+ST'1.$$,EE 3 6

6412&4'#&140#)00''#SW$ST'%S').(-"EE'Z'?#((QS 3 6

2'0#((#64161)#0615'SX#+&*NT'u.+#$# 3\ 6

4'!&1(1)#') 4'!67)#SW$ST'YS 'P.77S 3 6

#5'!7(1)H!#5#!'64'075#SY"$&ST':S'_#B&+$S 3 6

8$7+"(4@('T(+$#K#&,-,'7"'&+.-/''('+N"'A'E#$,+/''Q'+N"'A$,7#$/'"E"*+$'&+#+,*'*N#$B"&''Q'K#EE,&+,*'K#&,-,'&A'$"&',('N/7"('7/*"+'.&'IB#$,*'7/*"+"&R' (.7K"$''Q'K#&,-,'7"&'A"$'&A"*,"&'#(-'(.7K"$''Q'AE"&'A"$'K#&,-,'7"''"U#7,("-S'X'+N'+.$B"&*"(+'#(-'('(`+.B"&*"(+'S7#$2"-'' K/'\T'K#&,-,'7"&'#$"'A$"&"(+"-S

A
$
0ABC
?&4@#'&(#&3"O"#'#."-/%'#)B3" =#((!4)+( ).-/#)"+%//(#,"#'#+*/-)*%*(+"+"%/5#)3"%$,")#+-$,'43"*"%*" &)'!&4-!&()+
D;;
A0H
?/0#-&(#&3" 8-'#*%'#)B3" %$," *"#"*4.#"-0".-'%/(*4",()*/(&2*(-$"-0").-/#"+"%/5#)"9%)"*"#" A&,!4#00&(&-'/-/+#4-/*&
0CB27
_DC0A0
A&,!4#00&(#&3" )%1#"9(*"($"%").#+(#)6 8-'#*%'#)B@6"O"#"&%)(,(-1#)"9(*"",(00#/#$*".-'%/(*4"*4.#)" #1B4AE0C8>=B
01>DC
B?0C80;
0=3
C4A0;
E0A80C8>=B
9#/#"+-''%.)($5"-/"+#%)($5"*-").-/2'%*#"($"*"#"+-''#+*($5" -0".-'%/(*4"-0"*"#"./(1%/4"#'#+*/-)*%*(+"+"%/5#)"($";/+#*( 30H
>A
C74
30H
05C4A
'01;4
QB6" DC
8=
CF>
A468>=B

:<
0?0AC
5A><
4027
O9-"-/"1-/#"&%)(,(-1#)"9#/#"#W%1($#,"($"?V").#+(#)" >C74A
8=
BC>=80
8=
CF>
>A
C7A44
B8C4B
0?0AC
5A><
4027
'01;4
B6"N$"?Q").#+(#)3"*"#".-'%/(*4"*4.#"-0").-/#"+"%/5#)" >C74A

C>

:<
0=3
8=
C74
0D20BDB
!>D=C08=B
9%)"(,#$*(+%'"($"%''"&%)(,(-1#)"-0"*"#")%1#").#+(#)3"%$," CF>
B8C4B

0?0AC
5A><
4027
>C74A

:<
'01;4
B6" ,(00#/#$*" ($" F" ).#+(#)6" N$" 8-'/,5&'!&( 5/'4#+&44!!( A
&4E4A0;
?74=>;>6820;
B40B>=B
0=3
<>BC
>5
38DA=0;
&
D=34;;

"0==5
>=4
10B838><4
5A><
C7A44
0=3
CF>
.#/(-,)"%)"9#''"%)",(00#/#$*"%(/"+-$,(*(-$)"9#/#"+-:#/#," &%)(,(-1#)"0/-1"??"($"=&(-&'!)*(-)',!*
*48=<
A
38554A43
86DA4B
?%3" ?&3" %$," ?+B6" O"#" .-'%/(*4" -0" *"#" ./(1%/4" 5A><
C74
>C74AB
'01;4
QB6" O"#)#" ,(00#/($5" &%)(,(-1#)" #'#+*/-)*%*(+"+"%/5#)"9%)")%1#"($"%''").-/#")%1.'#)"-0" 9#/#"+#%)($5"*"#").-/2'%*(-$"-/",/4($5"2."($"*"#"+-''#+*($5" *"#")%1#").#+(#)6" 30H
>A
C74
30H
05C4A
'01;4
QB6"O"#"*2/5#)+#$*"&%)(,(-1#)" =
0;;
18?>;0A
B05

B?4284B
B7>F43
ZABC;H
C70C
8=
0;;
B?>A4
B0

768 !"#$%&'"%()#%'* +,-".,#* +#%' / 0122123 4563 78

104 )#%).
*##'*(')
")!
&
+-'&'%.
'
!)#'%. + *

%A"*,"&'#(-'&A'$"'*'EE"*+,'('-#+#R' Y'E#$,+/'-,&+$,K.+,'(''Q' SK#&,-,'7"'(.7K"$'Z'#7'.(+''Q'&A'$"' %+#+"''Q'K#&,-,'7" :&&"(*"''Q'"U*"A+,'(#E,+/ &A'$"'*N#$B"& AE"&T'SE'*#+,'('Z'/"#$'Z'-#+"'Z'N'.$&T'

!64'75#&#(875'SW$ST'W$S' 1'-"$#+"E/'-$/',('+N"'-#/'K"Q'$"8' # 

$"($
ST8'3T'SCw$D&"EF#8'345>8'%"A+`3@`358'34`4T -$/,(B'.A'-.$,(B'+N"'AE,(BS' 'Q'&A'$"&R'+N"'AE"S&T' ' "(+,$"E/'*'(&,&+"-''Q'('(` !64'75#6742'5'S]",(7ST'W$S' )''2"-'+.$B"&*"(+',('+N"'-#/'K"Q'$"8' *N#$B"-'&A'$"&S STTT8'6T'SCw$D&"EF#8'345>8'%"A+`3;8'4`34'#(-' 7'-"$#+"E/'-$/'-.$,(B'+N"'AE,(BS'  %"A+`3@8'35`6OT b$,"-'.A',('+N"'-#/'#Q+"$S :U+$"7"E/'V"#2'&A'$.E#+,'(',('+N"' 8H"0#((7)#$#447%'0#7)'SW$ST'YS 'P#$&+S c(,A'E#$`A'&,+,D"',('+N"' -#/'K"Q'$"'#(-',('+N"'AE,(B'-#/&S' ST8'6T'S)#N"7##8'345=8'%"A+`=8'3O`33'#(-' ("#$
#!
! "

$
%A'$.E#+,'('*"#&"-',('+N"'&"*'(-' %"A+`;8'3<`6OT &"*'(-'AE"S AE,(B'-#/S )''2"-'+.$B"&*"(+',('+N"'-#/'K"Q'$"' T(+$#K#&,-,'7"'-,QQ"$"(*"&' #!!'07)#74'06'!7)'SX.EEST'L$#/ c(,A'E#$`A'&,+,D"',('+N"' #(-',('+N"'AE,(B'-#/&S'0'EE#A&"-' ,('A'E#$,+/'-,&+$,K.+,'( ST8'6T'S)#N"7##8'345=8'I.B`6<8'3@`34'#(-' ("#$
#!
! "

$
,('+N"'-#/'#Q+"$S'%A'$.E#+,'('*"#&"-',('  I.B`648'35`34T' &"*'(-'AE"S +N"'&"*'(-'AE,(B'-#/S  ]"#2'&A'$.E#+,'(',('+N"'-#/'K"Q'$"' 2'0#((#64161)#0615#SX#+&*NT'u.+#$#' ! "

$
("#$
#!
 

$
("#$
#! 
&
ST8'6T'S)#N"7##8'345=8'%"A+`358'3<`6O'#(-' .(,A'E#$`("B#+,D"',('+N"' %A'$.E#+,'('*"#&"-',('+N"'&"*'(-' %"A+`3<8'3<`6OT &"*'(-'AE"S AE,(B'-#/S 
#! "%$

$
("#$
#! 
X,A'E#$',('K'+N'AE"&S' !64'75#6742'5'S]",(7ST'W$S -#/8'"U+$"7"E/'V"#2',('+N"'&"*'(-' T('36'AE"&''Q'+N"''+N"$' SdT8'6T'SP'2"8'34'AE"&''Q'+N"''+N"$' ?#((QS AE,(B'-#/S'W$#,E',('+N"'-#/'#Q+"$S 6'K#&,-,'7"&'&+.-,"-8' STTT8'3T'SCw$D&"EF#8'345>8'%"A+`>O8'3@`34T K,A'E#$S

8$7+"(9@((%+.-/''('A'E#$,+/''Q'+N"'A$,7#$/'"E"*+$'&+#+,*'*N#$B"&''Q'K#EE,&+,*'K#&,-,'&A'$"&',('+N"'N/7"('7/*"+'.&'IB#$,*'7/*"+"&R'('(`+.$B"&*"(+' K#&,-,'7"&'#(-'+N",$'&A'$"'AE"&S'

?.7K"$''Q' ).-/#)B3"#W+#.*"($"*"/##3"*"#"$21&#/"-0").-/#)"($"-$#" %A"*,"& K#&,-,'7"& D=8?>;0A
?0AC
!"#"" *"#" ).-/#)" +%//4($5" +"%/5#)" -0" *"#" )%1#" )(5$B" 9%)" ?6?P0-'," *-" ?XP0-'," "(5"#/" *"%$" *"#" 0('61!H #'&AS 6 =D<14A
>5
B?>A4B
F8C7
C74
>??>B8C4
?>;0A8CH
86DA4
?+" 0146'04'75'&AS 6 ($">?R@B6"O"#"./#:%'#$+#"9%)"1-/#"*"%$"FFSP0-',"($"*"#" 41)#5#$1)#064'75'S)ST'CS'P,*2U'QS 4 )%1.'#"-0"3&4-5&'#00)*((!:&'!)*").-/#)3"($"*"#")%1.'#" -0"A5#0#,5/'&(-#''#*-'!*").-/#)3"%$,"($"-$#"-0"*"#"*9-" 41)'6125'5#2'0'!1(#S%VST'YS 'P#$&+S =O .!,-/,/')*( :#-)0!4)*( ).-/#" )%1.'#)"( #E4A;>>:8=6
C74
41)'6125'5#415#'SIEKS'Z'%*NV",(ST'YS 'P#$&+S =

*($4" .-)(*(:#" .%/*)3" 9#" */#%*#," *"#)#" *"/##" )%1.'#)" 601"#4)#22(067)'SY"$&ST'Y#+S @ 0B
D=8?>;0A=460C8E4
!"#"3" *"#" )%1.'#)" "%:($5" -$'4" $#5%*(:#'4"+"%/5#,").-/#)B6" !64'75#6742'5'S]",(7ST'W$S 4 !#"#W+'2,#,"0/-1"*"#"*%W-$-1(+%'"%$%'4)()"%''"$-$P I'((75#'081(7675'SX#+&*NT'W$S = CDA64B24=C
10B838><4B
'01;4
Q
DAC74A
0=0;HB8B
0C
&#(('075#(0'#SX'(-#$+&"DT'Y#$7#&+' 6 *"#").#+(#)"%$,""(5"#/"'#:#')"9%)"&%)#,"-$"*2/5#)+#$*" &#(('075#2127('!1(#?,"7"Ew' = 10B838><4B
 
10B838><4B
0=3

B?>A4
B0E4A8=6

B?4284B
'01;4
V" %$," 

64=4A0

X'(-#$+&"D'Z'YS?S'X'$,&'D 50<8;84B
0=3

>A34AB
'74
?>;0A8CH
>5
?A8<0AH
4;42CA82
'26121475# #67('075'SX.EEST'YS 'P#$&+S 6 270A64B
8B
?A4B4=C43
>=
86DA4
F
#5
C74

B?4284B
)*2,(#,3" ??" ).#+(#)" "%," 2$(.-'%/P.-)(*(:#3" F?" ).#+(#)" (#741675#1564#675'SC#*aST'YS 'P.77S 6 "%," 2$(.-'%/P$#5%*(:#3" %$," ?V" ).#+(#)" "%," &(.-'%/" #427(#(!4H)05'S].EQ"(T'CS'%*N$x+S 6

,()*/(&2*(-$"-0").-/#"+"%/5#)6" 6412&4'#&140#)00''#SW$ST'%S').(-"EE'Z'?#((QS 6 5"5#$175 =
0;;
B4E4=
64=4A0
F8C7

B?4284B
BCD3843
C74
8$7+"(C@((T(+"$K#&,-,'7"'&+.-/''('A'E#$,+/''Q'+N"'A$,7#$/'"E"*+$'&+#+,*' *N#$B"&' 'Q' K#EE,&+,*' K#&,-,'&A'$"&' ,(' N/7"('7/*"+'.&' ).#+(#)"-0"*"#")%1#"5#$2)""%,"*"#")%1#"+"%/5#"*4.#6" IB#$,*'7/*"+"&R' (.7K"$' 'Q' +.$B"&*"(+' K#&,-,'7"&'' [$(.-'%/P.-)(*(:#",()*/(&2*(-$"-0").-/#"+"%/5#)"-++2//#," "U#7,("-'A"$'&A"*,"&S

771 !"#$%&'"%()#%'* +,-".,#* +#%' / 0122123 4563 78

105 !7&&""#(&)7&+"#,"*%-&

?.7K"$''Q 0'EE"*+,'('-"+#,E& %A"*,"& &A'$"' K#&,-,'7"& %,+" !"#$ b#+"'#(-'N'.$& AE"& >)0'6#)75!4''S)ST')#7S 3 3 &""'e34f

>)0'6#8%'06'SX.EEST')#7S 3 3 ) 345= %"A+`638'3

006&4#((75#!' 4'75'W$S 3 3 &""'e34f I.B`>38'4R=5`33R35'#(-'3;R;O` 0(')!1!H56'5# 14#('5#SW$ST'P'+ES'Z'Y'.J#$ 3 6 ) 345= 3@R>; 0('61!H #'&AS 6 6 &""'e34f

01(64'!'#2#4#00'5#S)ST'1.$$,EE 3 3 ) 345= I.B`6<8'+,7"'3@R=;`3

010'12&14#276#0'S%*N.7#*NST'YS 'P#$&+S 3 6 &""'e34f

0146'04'75'&AS 6 > &""'e34f

0H('0"41 5'"'7)##81(8#05'SW$ST'CyE,*N 3 3 P 345< %"A+`34`6O8'3OROO`3

?#0"416&#(##%4'5#1!0'X$"&ST'X'.$-'+'Z'L#EJ,( 3 3 &""'e34f

4())7('0#8#(76'2#5'S0.$+,&T'%,(B"$' 3 3 9" 34<@ g*+`63`668'3;R;O`3;R6O

41)#5#$1)#064'75'S)ST'CS'P,*2U'QS 4 >= G#KE"'@8'#E&''&""'e34f'Q'$'6@'AE"&

41)'6125'5#2'0'!1(#S%VST'YS 'P#$&+S =O @O G#KE"'@8'#E&''&""'e34f'Q'$'6<'AE"&

&""'e34f'Q'$'='AE"&['>'AE"&'"#*N'C8'345>8'C.E/`>8' 41)'6125'5#415#'SIEKS'Z'%*NV",(ST'YS 'P#$&+S' =5

601"#4)#22(067)'SY"$&ST'Y#+S' @ 3< G#KE"'@8'#E&''&""'e34f'Q'$'3>'AE"&

601"#4)#(7!'"7)'S0.$+,&T'YS 'P#$&+S 3 3 H 345= I.B`338'3@R;;`35R;;

70%&7&0'#0'6'"'SY"$&ST'M/D#$-"( 3 3 &""'e34f

!64'75#&#(875'SW$ST'W$S 3 3 C 345> %"A+`3@`358'3

!64'75#614)'01575'S%*N#"QQST'L$#/ 3 3 &""'e34f %"A+`3@8'35RO4`34ROO'#(-'%"A+`358' !64'75#64'8'('5'SW$ST'W$S 3 6 ) 345= 3

!64'75#6742'5'S]",(7ST'W$S &""'e34f'Q'$'3O'AE"&['6'AE"&'C8'345>8'%"A+`3;8'4ROO` 4 36 34R3;'#(-'%"A+`668'35R=;`34R=;

#2'56#07"#SX.EEST'0''2" 3 3 &""'e34f

#%(1!H56'"'7)#(#7!1I06&7)'SX$"&ST'CyE,*N 3 3 9" 34<@ ?'D`5`3O8'6OROO`3@R>O

#057(4'#4"'6#S%'V"$K/T')vJ#$''TK,J# 3 3 ) 345= %"A+`>8'3@RO;`35RO;

I'((75#'081(7675'SX#+&*NT'W$S = 5 &""'e34f

&#(('075#(0'#SX'(-#$+&"DT'Y#$7#&+' 6 = &""'e3

&#(('075#0'%4'!05'SW$ST'YS 'P#$&+S' 3 3 &""'e3

&#(('075#2127('!1(#?,"7"Ew = 3< &""'e3

&""'e34f'Q'$'3'AE"['3'AE"'98 '34568'g*+`33`368'3=R;5` '26121475# #67('075'SX.EEST'YS 'P#$&+S 6 6 3OR;5

&""'e34f'Q'$'3'AE"['3'AE"')8'345=8'%"A+`3>8'3

1(H21475# "'75'SY"$&ST'%*NV",(S 3 3 &""'e34f

1(H21475#537)1575'S_.-&ST'W$S 3 3 9# 34<@ I.B`3<8'3ORO4`6OR>O

8$7+"(0@('%A"*,"&''&+.-,"-'Q'$'+N"'A'E#$,+/''Q'+N"'A$,7#$/'"E"*+$'&+#+,*'*N#$B"&''Q'K#EE,&+,*'K#&,-,'&A'$"&',('+N"'N/7"('7/*"+'.&'IB#$,*'7/*"+"&R' (.7K"$''Q'+.$B"&*"(+''K#&,-,'7"&'#(-'&A'$"'AE"&8'#(-'-"+#,E&''Q'+N"'*'EE"*+,'(''Q'&A'$"&'AE"&S'1#$2&''Q'E'*#+,'('#&',('G#KE"'3S

770 !"#$%&'"%()#%'* +,-".,#* +#%' / 0122123 4563 78

106 )#%).
*##'*(')
")!
&
+-'&'%.
'
!)#'%. + *

?.7K"$''Q 0'EE"*+,'('-"+#,E& %A"*,"& &A'$"' K#&,-,'7"& %,+" !"#$ b#+"'#(-'N'.$& AE"&

%"A+`3>8'34R3>`6OR=;'#(-'%"A+`3=8' 141"#"(##2'0'#SX$'+ST'1.$$,EE 3 6 ) 345= 3

156'#(#7!1)((#(('S1.$$,EET'CyE,*N 3 3 ) 345= I.B`648'3=R3;`6OR3O

7557(#8'015'),(-KE#- 3 3 &""'e34f

4!1"10#') 4'!675#SPST'YS 'P#$&+S 3 3 ) 345= %"A+`3;8'3>`34R6;

g*+`3=8'3O'#(-'g*+`3;8' #427(#(!4H)05'S].EQ"(T'CS'%*N$x+S 6 6 G$ 345; 3@R6O`35R6O

6#4#7)#47%157)'Y"$&S 3 3 &""'e34f

6412&4'#&140#)00''#SW$ST'%S').(-"EE'Z'?#((QS 6 6 &""'e34f

7'((75#(76#75'S)ST'M'.&&"E 3 3 &""'e34f

&#(#2&14#6#44#564'5#:N$NS 3 3 &""'e34f

4'!&1(1)##37#564#'S)ST'YS 'P.77S 3 3 &""'e34f

4'!&1(1)#') 4'!67)#SW$ST'YS 'P.77S 3 6 &""'e34f

#5'!7(1)H!#5#!'64'075#SY"$&ST':S'_#B&+$S 3 6 &""'e34f

#41!1)75'&AS 3 3 9# 34<@ %"A+`>8'34R;O`63R>;

&52)*23"-8$7+"(0@('%A"*,"&''&+.-,"-' Q'$' +N"' A'E#$,+/' 'Q' +N"' A$,7#$/' "E"*+$'&+#+,*' *N#$B"&' 'Q' K#EE,&+,*' K#&,-,'&A'$"&' ,(' +N"' N/7"('7/*"+'.&' IB#$,*'7/*"+"&R'(.7K"$''Q'+.$B"&*"(+''K#&,-,'7"&'#(-'&A'$"'AE"&8'#(-'-"+#,E&''Q'+N"'*'EE"*+,'(''Q'&A'$"&'AE"&S'1#$2&' 'Q'E'*#+,'('#&',('G#KE"'3S

?.7K"$''Q )'*#+,'( !"#$ b#+" _'.$& %.K&+$#+" K#&,-,'7"& AE"&

41)'6125'5#2'0'!1( :&+'(,#8'9 3456 %"A+'3O`33 34`4 3 3 '!## '#58'&+.7A

:&+'(,#8'C 345> 1#/'63`'668'6<`64 <`368'6O`; 5 63 '!## '#58'&+.7A '!## '#58'&+.7A'#(-'-"#-' :&+'(,#8'G 345= C.("'3`=8'5`48'33 4`368'3<`63 >O >@ +$.(2['>(075#'0!08'&+.7A GN"'0#.*#&.&8'L 345= I.B'6O 35`34 6 6 > '#5'&AS8'-"#-'+$.(2

41)'6125'5#$1)#064'75

:&+'(,#8'9 3456 %"A+'3@ 3=`3< 3 3 E#67('&AS8'-"#-'+$.(2

:&+'(,#8'C 345> C.("'3`68'= 35`66 = 64 E#67('&AS8'-"#-'+$.(2

GN"'0#.*#&.&8'H 345= I.B'33`36 5`3O8'3@`34 6 6 &+.7A'#(-'-"#-'+$.(2

GN"'0#.*#&.&8'L 345= I.B'34 35`34 6 6 4%75#14'#06('58'-"#-'+$.(2

601"#4)#22(067)

:&+'(,#8'9 3456 C.E/'3;`358'%"A+'36 3O`348'3@`= 3 > -'V("-'E'B -'V("-'-"*,-.'.&'E'B[''('# :&+'(,#8'C 345> C.E/';`5 3O`338'34`6= = 3= !14"68'-"#-'+$.(2 042'075#!7!5'!75''$' GN"'0#.*#&.&8'L 345= I.B'6O 3<`34 3 3 4%75#14'#06('58'&+.7A

8$7+"(;@((%A#+,#E'#(-'+"7A'$#E'D#$,#+,'(&''Q'A'E#$,+/''Q'+N"'A$,7#$/'"E"*+$'&+#+,*'*N#$B"&R'E'*#+,'('#(-'+,7"''Q'*'EE"*+,'(''Q'&A'$"'AE"&8'&.K&+$#+"' 'Q'K#&,-,'7"S'

' 1#$2&''Q'E'*#+,'(R'L‚L.J"$,AE8'C'‚'Cw$D&"EF#8'G'‚'G#77,&+.8'9‚9''$"7##8'H‚H#2#+#E#S'GN"'B"'B$#AN,*'*''$-,(#+"&',('G#KE"'3S

772 !"#$%&'"%()#%'* +,-".,#* +#%' / 0122123 4563 78

107 !7&&""#(&)7&+"#,"*%-&


  

   

 '   '     

                   
  
  

        
   % 
       
                 
       
 
    

                     
   
       ! !   

!                        "    

  
 #    $  $    $ 
$ 

 

 
 
 
 % 
  &    
 (     

                                    
  

  

   

  (   (  
   
  
  
  
   
    

9*:3%"(4@((Y'E#$,+/' 'Q' +N"' A$,7#$/' "E"*+$'&+#+,*' *N#$B"&' 'Q' K#EE,&+,*' K#&,-,'&A'$"&' ,(' N/7"('7/*"+'.&' IB#$,*'7/*"+"&' #(-' AN/E'B"("+,*' $"E#+,'(&N,A&'K"+V""('+N"'&A"*,"&''"U#7,("-',('+N"'A$"&"(+'&+.-/S'GN"''$-"$&'V"$"'E,("-'.A'#&',('AN/E'B"("+,*'$"E#+,'(&N,A'+$""''(' W,B'6''Q'+N"'_,KK"++€&'AN/E'B"("+,*''D"$D,"V''Q'+N"'IB#$,*'7/*'+,(#'e6Of8'+N"'Q#7,E,"&''Q'IB#$,*#E"&'V"$"'#$$#(B"-'#**'$-,(B'+''e63fS' GN"'+$""',&'K#&"-''('7.E+,AE"'#(#E/&"&''Q'$,K'&'7#E'M?I'B"("'&"a."(*"&S'X$#(*N'E"(B+N&'#$"'('+'A$'A'$+,'(#E'+''B"("+,*'-,&+#(*"&S' 1#$2&'Q'$'&A'$"'*N#$B"&',('&A'$"'A'A.E#+,'(&R'‚'("B#+,D"'*N#$B"&''(E/'S.(,A'E#$`("B#+,D"'-,&+$,K.+,'(''Q'A'E#$,+/T8'^'A'&,+,D"'*N#$B"&' '(E/'S.(,A'E#$`A'&,+,D"'-,&+$,K.+,'(''Q'A'E#$,+/T8'^Z‚'‚'("B#+,D"'*N#$B"&'7'$"'(.7"$'.&'SK,A'E#$'-,&+$,K.+,'(''Q'A'E#$,+/8'("B#+,D"'A#$+' A$"D#,E,(BT8'^^Z‚'A'&,+,D"'*N#$B"&'7'$"'(.7"$'.&'SK,A'E#$'-,&+$,K.+,'(''Q'A'E#$,+/8'A'&,+,D"'A#$+'A$"D#,E,(BTS

($" .5#00!4)*" ]2^'6" %$,( 7&4/6#'+&
$
0ABC"" [$(.-'%/P ($" 8-'/,5&'!&(#&#

64=4A0

B?4284B
'74
50<8;84B
$#5%*(:#" ,()*/(&2*(-$" -++2//#," ($" ;/+!-/,*!*( $
0ABC( 9(*"",(00#/#$*"+"%/5#"*4.#)"($"5#$#/%"-0"*"#")%1#"0%1('4" =&(-&'!)*( D#/)6" %$,( $+&4!-&" _(''6" #W" 8-#"16" 8(.-'%/" %/#" %)" 0-''-9)M" 8-#'#&(#&#C( A'!(5/0/+&-&(#&#( %$,( 38BCA81DC8>=
8=
F7827
C74
=460C8E4
270A64B
0A4
<>A4
7&4/6#'+&-&(#" O9-" 0%1('(#)" 9#/#" -0" *"#" /#'%*#," $21#/-2)B"-++2//#,"($"./01,/')*($
!8274;8
4G
30=B
*4.#M"*"#4""%,").-/#)"9(*""#(*"#/"$#5%*(:#"+"%/5#)"-$'43" %$,(A'!(5/0/+&(A
&C0D34
#5
C74

64=4A0
BCD3843
-/" &(.-'%/3" 9(*"" 1-/#" $21#/-2)" $#5%*(:#" +"%/5#)Z B4E4=
703
D=8?>;0A?>B8C8E4

703
D=8?>;0A=460C8E4
8-#'#&(#&#( 
64=4A0

B?4284B
A'!(5/0/+&-&(#&#( %$,"?Q"5#$#/%""%,"&(.-'%/",()*/(&2*(-$"-0").-/#"+"%/5#)6" 
64=4A0

B?4284B
=
7&4/6#'+&-&(#&#

64=4A0

B?4284B
>??>B8C4
CH?4B
>5
B?>A4
270A64B
D=8?>;0A 5"9#!0')@ .-)(*(:#"%$,"2$(.-'%/P$#5%*(:#3"9#/#"-&)#/:#,6" #5
C74
C4=
50<8;84B
F8C7
CF>
>A
<>A4
64=4A0
BCD3843
)#:#$""%,"*"#")%1#"+"%/5#"*4.#"($"5#$#/%"%$,").#+(#)"-0" 5"<#4#$4 C74
B0<4
50<8;H
86DA4
FB6"[$(.-'%/P.-)(*(:#",()*/(&2*(-$" #5
C74
5>DA
>A34AB
F8C7
CF>
>A
<>A4
50<8;84B
=>=4
-0" ).-/#" +"%/5#)" -++2//#," ($" >1+#4/(5&#-&(#&#" "%,"*"#")%1#"+"%/5#"*4.#"($"#(*"#/"5#$#/%"-/").#+(#)"" 
64=4A0

B?4284B
(=8?>;0A=460C8E4
38BCA81DC8>=
>5
>5
C74
B0<4
>A34A
86DA4
F
=
$>;H?>A0;4B

64=4A0
).-/#"+"%/5#)"-++2//#,"($"A5#0#,5/'&(#&#

64=4A0

B?4284B
0;;
C7A44
CH?4B
>5
B?>A4
270A64B
18?>;0A
F" ).#+(#)B3" ;/+!-/,*!6&(#&#



B)**)0&(#&#" 2$(.-'%/P.-)(*(:#"%$,"2$(.-'%/P$#5%*(:#B"9#/#"-&)#/:#,6" 


.51*&0&('!&(#&#



0=3
3/4!/,5/'&(#&#" 'F>
CH?4B
>5
B?>A4
270A64B
18?>;0A
0=3
D=8?>;0A 


8?>;0A
38BCA81DC8>=
>5
B?>A4
270A64B
>22DAA43
=460C8E4
F4A4
>1B4AE43
8=
%DBBD;0;4B

64=4A0

773 !"#$%&'"%()#%'* +,-".,#* +#%' / 0122123 4563 78

108 )#%).
*##'*(')
")!
&
+-'&'%.
'
!)#'%. + *


B?4284B
60A820;4B

64=4A0

B?4284B
0=3
$
D<<
-F.
06A443
F8C7
>DA
Z=38=6BWC74H
70E4
C74
>;4C0;4B

64=4A0

B?4284B B0<4
270A64
CH?4
86DA4
QB6

9"5#!0')@ 9"#'5"755'21 O"#)#" /#)2'*)" )"-9" *"%*" +"%/5#" *4.#" ($" %" .-.2'%*(-$" -0"&%''()*(+"&%)(,(-).-/#)"()"$-*"%'9%4)"*"#")%1#"($"%''" 9"!# 3$"'$5 5#$#/%" 9(*"($" %" 0%1('46" N$" %$4" +%)#3" .-))(&'4" %''" *%W%" O"#)#" /#)2'*)" )"-9" *"%*" +"%/5#" *4.#" ($" %" ).-/#" 5A><
B?4284B
C>
<>=>?7H;4C82
50<8;84B
0A4
270A02C4A8I43
?>?D;0C8>=
!"#"3"*4.#"-0"*"#",()*/(&2*(-$"-0"*"#".-'%/(*4"-0" 1H
B?428Z2
CH?4
>5
?>;0A8CH
>5
C74
?A8<0AH
4;42CA82
270A64
).-/#"+"%/5#)"($"%".-.2'%*(-$"-0"&%''()*(+"&%)(,(-).-/#)" -0"&%)(,(-).-/#)6 '(&#/%*($5" 0/-1" *2/5#)+#$*" &%)(,(-1#)B" ()" %" +-$)()*#$*" 0#%*2/#"-0"%").#+(#)6"O"#"+"%/5#"*4.#",-#)"$-*",#.#$," 9"9#4#$4 -$" ,(00#/#$*" 4#%/)3" ,(00#/#$*" ,%4)3" ,(00#/#$*" *(1#)"" &?>A4
270A64
30C0
0A4
8=BD5Z284=C
C>
270A02C4A8I4
-0"%",%43":%/(-2)"9#%*"#/"+-$,(*(-$)3"%$,"-$"&%)(,(-1#)" -/,#/)" -/" 1%<-/" +'%,#)" -0" *"#" ."4'-5#$#*(+" */##" -0" 6A>F8=6
>=
38554A4=C
BD1BCA0C0
;;
C7A44

270A64
CH?4B
$%&'!(/+1(#-#*"
"48C74A
3>
C74H
4=01;4
C>
B44
0=H
D=8?>;0A?>B8C8E4
D=8?>;0A=460C8E4
0=3
18?>;0A
20=
."4'-5#$#*(+"*/#$,)"-0"*"#",#:#'-.1#$*"-0").-/#"+"%/5#" >22DA
8=
C74
B?4284B
>5
60A82>F
0=3
?A4E8>DB;H
7&4/6#'+&( 9"<#&!4%'1%#0$"&!1'50 &,,0&4&-)+
$4AB
$0CVV?>B8C8E4;H
270A643
B?>A4B
O"#" /#)2'*)" -0" *"()" )*2,4" ($,(+%*#" *"%*" *"#" ./(1%/4" >F@3" ./01,/')*( *2)&+/*)*
D3B
AVV?>B8C8E4;H
0=3
#'#+*/-)*%*(+" +"%/5#" -0" &%)(,(-).-/#)" -/(5($%*#)" 0/-1" $#5%*(:#'4" +"%/5#," ).-/#)" >?3F@3" 8#',)0&( 0&('1+&4*" ?A>24BB4B
>22DAA8=6
8=
C74
5D=6DB
C
70B
144=
*D;54=

&27APCVV=460C8E4;H
270A643
B?>A4B
-Q@B6" )*%*#," #%/'(#/" %'/#%,4" *"%*" *"#" .-'%/(*4" ,()*/(&2*(-$" -0" +"%/5#)" 12)*" &#" %++-2$*#," 0-/" ($" %$4" *"#-/4" -0" *"#" 9"+#$175 1#+"%$()1" -0" ).-/#" ,()+"%/5#" >Q.
#DA
7H?>C74B8B
O"#)#" /#)2'*)" )"-9" *"%*" +"%/5#" *4.#" ($" %" ).-/#" ()" %)" 0-''-9)6" O"#" ./(1%/4" +"%/5#3" !"#"3" %" +"%/5#" ?>?D;0C8>=
8B
0
2>=B8BC4=C
540CDA4
>5
0
64=DB
;;
C7A44
+%//(#,"-$"*"#"%(/&-/$#").-/#"%0*#/"*"#")#'0P./-.2')(-$" >5
C74
270A64
CH?4B
D=8?>;0A?>B8C8E4
D=8?>;0A=460C8E4
9D55
5A><
C74
BC4A86<0
8B
0
BD?4A8B8C8>=
>5
0=3
18?>;0A
20=
>22DA
8=
C74
64=4A0
>5
60A82>C7
3/,'!4#00)*
$
0ABC
0=3
.5/0!/-&
A
&%)(,(%6" N$" %" ).#+(#)" 9(*"" &(.-'%/" ./(1%/4" +"%/5#)3"

   

   

     

  

       
    
         
      

    
      
                                 

                  

9*:3%"(9@((Y'E#$,+/''Q'+N"'A$,7#$/'"E"*+$'&+#+,*'*N#$B"&''Q'K#&,-,'&A'$"&',('IB#$,*#E"&'#(-'AN/E'B"("+,*'$"E#+,'(&N,A&'K"+V""('+N"'&A"*,"&''"U#7,("-' ,('+N"'"#$E,"$'&+.-,"&'#(-',('+N"'A$"&"(+'&+.-/S'GN"'Q#7,E,"&''Q'IB#$,*#E"&'V"$"'#$$#(B"-'#**'$-,(B'+''AN/E'B"("+,*'$"E#+,'(&N,A'+$""''(' W,B'3''Q'+N"'AN/E'B"("+,*''D"$D,"V''Q'+N"'IB#$,*#E"&'B,D"('K/'1#+N"("/'#6#(,'e63fS'GN"'+$""',&'K#&"-''('7.E+,AE"'#(#E/&"&''Q'$,K'&'7#E' M?I' B"("' &"a."(*"&S' X$#(*N' E"(B+N&' #$"' ('+' A$'A'$+,'(#E' +'' B"("+,*' -,&+#(*"&S' :E"*+$,*' -#+#' 'Q' >%4'!75# !)2#564'5' )S' #$"' Q$'7' e386f8'#(-'0124'0#((75#&'5!#05'SW$ST'M"-N"#-8'9,EB#E/&'Z'1'(*#ED'8'0,#)'!!#75'SX.EEST'9,EB#E/&8'_'AAE"'Z'C#*aS'C'N(&'(8'&1('16# &'%&(0"#05'5'SY"*2T'IS_S'%7S'Z'_"&E"$8'#(-', #5374415'S9#NET'YS 'P.77S'Q$'7'e6fS'1#$2&'Q'$'&A'$"'*N#$B"&',('&A'$"'A'A.E#+,'(&R'‚' ("B#+,D"'*N#$B"&''(E/'S.(,A'E#$`("B#+,D"'-,&+$,K.+,'(''Q'A'E#$,+/T8'^'A'&,+,D"'*N#$B"&''(E/'S.(,A'E#$`A'&,+,D"'-,&+$,K.+,'(''Q'A'E#$,+/T8'^Z‚' ‚'("B#+,D"'*N#$B"&'7'$"'(.7"$'.&'SK,A'E#$'-,&+$,K.+,'(''Q'A'E#$,+/8'("B#+,D"'A#$+'A$"D#,E,(BT8'^^Z‚'A'&,+,D"'*N#$B"&'7'$"'(.7"$'.&' SK,A'E#$'-,&+$,K.+,'(''Q'A'E#$,+/8'A'&,+,D"'A#$+'A$"D#,E,(BTS'

77/ !"#$%&'"%()#%'* +,-".,#* +#%' / 0122123 4563 78

109 !7&&""#(&)7&+"#,"*%-&

-$#"-0"*"#)#"./-+#))#)"5#$#/%*#)"%"$#5%*(:#"+"%/5#3" ($*#/%+*(-$)" ,2#" *-" %" $-$P2$(0-/1" ,()*/(&2*(-$" -0" (-$(+" %$,"*"#"-*"#/"5#$#/%*#)"%".-)(*(:#"+"%/5#"-$"%").-/#6" .21.)"%$,")#+-$,%/4"*/%$).-/*")4)*#1)"($"*"#"%.(+%'" N$"*"-)#"&%)(,(%3"9"(+""+-$*/(&2*#"*"#".-)(*(:#".%/*"-0"*"#" %$,"./-W(1%'""4."%'"+#'')"> PQS.
B
<>BC
10B8380
0A4
&(.-'%/"./(1%/4"+"%/5#)3"*"#"./-+#))"5#$#/%*($5".-)(*(:#" "4."%'"#$,"+#'')">@3"*"#"&(-#'#+*/(+%'"%+*(:(*4"1(5"*"&#" 270A64
>=
C74
B?>A4
H84;3B
BCA>=64A
270A64
F8C7
18664A
($:-':#,"($"*"#"0-/1%*(-$"-0"&-*""*"#")*#/(51%*%"%$,"*"#" 1%5$(*2,#B" *"%$" *"#" +"%/5#" 4(#',#," &4" *"#" ./-+#))" &%)(,(-).-/#)6"N0")-3"*"#$"%$"#W+#))"$#5%*(:#"+"%/5#"-/" 5#$#/%*($5" $#5%*(:#" +"%/5#`" %$," D!(#( D#'*&" ($" *"#" %$" #W+#))" .-)(*(:#" +"%/5#3" 9"(+"" %/()#)" -$" *"#" ).-/#" &%)(,(%3"9"(+""+-$*/(&2*#"*"#"$#5%*(:#".%/*"-0"*"#"&(.-'%/" %$,"/#1%($)"-$"(*"9"#$"*"#").-/#"<21.)"-00"*"#")*#/(51%3" ./(1%/4" +"%/5#)6" N$" *"#" &%)(,(%3" 9"(+"" +-$*/(&2*#" *"#" 9(''"+-$*/(&2*#"*-"*"#").-/#"./(1%/4"+"%/5#6" $-$P+"%/5#," ).-/#)3" *"#" ./-+#))#)" 4(#'," *"#" +"%/5#)" -0" *"#" )%1#" 1%5$(*2,#6" N$" %" ).#+(#)" 9(*"" 2$(.-'%/" 9"B#'2)2%'"!)#'03246!1"$ ./(1%/4" +"%/5#)3" *"#" ./-+#))#)" 5#$#/%*#" *"#" +"%/5#)" O"#"./(1%/4"#'#+*/-)*%*(+"+"%/5#)"-0"&%)(,(-).-/#)"%/#" -0"*"#")%1#".-'%/(*43"-/"-$#"./-+#))"5#$#/%*#)".-)(*(:#" ./-&%&'4" *--" )1%''" *-" )#/:#" 0-/" ).-/#" '(&#/%*(-$" >Q3V@" +"%/5#)" %$," *"#" -*"#/" 5#$#/%*#)" $#5%*(:#" +"%/5#)" &2*" *"#4" %/#" $-*" $#+#))%/('4" 02$+*(-$'#))" > @6"O"#)#" 9"#/#%)"-$#"-0"*"#1"5#$#/%*#)"12+"")*/-$5#/"+"%/5#" 270A64B
20=
4=70=24
C74
2>;;8B8>=
45Z284=2H
14CF44=
C74
<06=8CD34
38554A8=6
1H
>=4
>A34A
>A
<>A4
C70=
C74
B<0;;
10B838>B?>A4B
0=3
Z=4
3A>?;4CB
-Q?@3" *"2)" >C74A
&4?0A0C8>=
>5
270A64B
C7A>D67
?D;;8=6
0?0AC
C74
($+/#%)($5"*"#"./-&%&('(*4"0-/").-/#)"*-"&#"9%)"#,"-2*" 38554A4=C
BDA5024B
5A><
4027
>C74A
8B
:=>F=
8=
?7HB82B
5A><
C74
0C<>B?74A4
1H
<8BCB
2;>D3B
>A
3A8II;4B
0B
CA81>
4;42CA8Z20C8>=
>A
CA81>4;42CA82
270A68=6
-FF@6" #=
0
10B838D<
C74
BC4A86<0C0
0B
F4;;
0B
C74
B?>A4B
O"()" 1#+"%$()1Z1#+"%$(+%'" )#.%/%*(-$" -0" *"#" ).-/#" %/#" 0-/1#," )(12'*%$#-2)'43" &2*" *"#" ).-/#)" '(&#/%*#" 0/-1"*"#")*#/(51%Z"%)"&##$"%'/#%,4")255#)*#,">Q@"*-" )2++#))(:#'43" $-*" )(12'*%$#-2)'4" >QF@6" O"#" )*%5#" #W.'%($"*"#"-/(5($"-0"./(1%/4"+"%/5#)"-0"&%)(,(-).-/#)6" -0" ,#:#'-.1#$*" %$," 1%*2/%*(-$" -0" ).-/#)" ($+'2,#)"" >=B834A8=6
C74
B<0;;=4BB
>5
C74
2>=C02C
0A40
380<4C4A
*"#" 0-/1%*(-$" -0" *"#" 82''#/U)" ,/-." ($(*(%')" %$," *"#(/" -0" *"#" )+%/" -$" *"#" ).-/#3" "('213" %$," *"#" )*#/(51%" *-." ,#:#'-.1#$*" 2$*('" *"#" ,()%..#%/%$+#" -0" *"#" *"($" 9%''" ()" ($" *"#" /%$5#" -0" S6?" *-" S6R" a1" >FQ3 @3" &#($5" 1-)*'4" '%4#/")2//-2$,($5"*"#",/-.">.
C
C74
BC064
>5
;814A0C8>=
S6V"a1">@B"%$,"*"#"5/#%*$#))"-0"*"#"+"%/5#"1%5$(*2,#" -0" ).-/#)3" %" ).-/#" %0*#/" ).-/#" #W.#/(#$+#)" *"#" /%.(,"  U
4
8=
C74


>5
0;;
B?4284B
4G0<8=43
#W.%$)(-$" -0" 82''#/U)" ,/-." %$," *"#" )#'0P./-.2')(-$" >@6" >Q3?R@B3"-$#"$##,)"*-"L2#)*(-$"9"#*"#/"*"#"1%5$(*2,#" G,/#,"Y-/$#/">FQ@3"9"-"1%,#"*"#"5#-1#*/(+%'"%$%'4)()" -0" *"#" +"%/5#" -&*%($#," &4" */(&-#'#+*/(+" +"%/5($5" +-2'," >5
C74
?>B8C8>=
>5
B?>A4B
>=
C74
10B838D<
7H?>C74B8I43
&#" ($" *"#" )%1#" -/,#/" %)" *"%*" 1#%)2/#," ($" $%*2/%'" *"%*"%$"%+*(-$"%*"%",()*%$+#"12)*"&#"($:-':#,"*-")#+2/#" +-$,(*(-$)b"O"#")2/0%+#"+"%/5#"0-/"*"#"+-$*%+*"%/#%"-0" *"#"/#52'%/").%+($5"-0").-/#)3"%$,"*"%*"*"#"0-/+#"%..#%/)" S6F" a1F
2>AA4B?>=3B
C>
C74
380<4C4A
>5

T<
F0B
*-" &#" #'#+*/-)*%*(+" /#.2')(-$6" N0" *"#" ).-/#P).%+($5" ()" 20;2D;0C43
5>A
C74
?A>24BB
;8<8C8=6
C74
270A68=6
!"#""0-/" ,#*#/1($#," &4" )*#/(51%*(+" ).%+($5" -$" *"#" &%)(,(21P 0E0;0=274
1A40:3>F=
0=3 >A
Z4;3
4<8BB8>=
DB8=6
C74
%.#W3" *"#$" *"#" +-2/)#" -0" *"#" ,#*#/1($%*(-$" 12)*" &#" <>38Z43
$0B274=
2DAE4
-FV@B3" %$," (*" 9%)" 0-2$," *"%*" +-$$#+*#,"9(*""*"#""4%'($#"+%."9"(+""0-/1)"%*"*"#"%.#W" J  U U
4;4<4=C0AH
270A64B
20=
14
B4?0A0C43
-0" *"#" &%)(,(21" )"-/*'4" �-/#" *"#" )*#/(51%*%" #1#/5#" F74=
B?>A4
38B270A64
38BC0=24B
0A4
 U U
T<
>FQ@6" O"#" )2&)*%$+#" -0" *"()" +%." 0-/1)3" #:(,#$*'43" *"#" >A38=0AH
38BC0=24B
8=
60A82><0B

9%'')"-0"*"#")*#/(51%*%"%$,"*"-)#"-0"*"#").-/#"/2,(1#$*)`" K-$#)3".#/)-$%'"+-112$(+%*(-$B6 ./#)21%&'4" (*" &#+-1#)" #'#+*/(+%''4" +"%/5#," %*" *"#" O"()" #)*(1%*#" )"-9)" *"%*3" ,#).(*#" $#%/'4" BDA5024
0=3
142><4B
?0AC8C8>=43
8=C>
1;>2:B
F7827
BD1<82A>B2>?82
B8I4
>5
C74
2>=C02C
BDA5024B
CA81>4;42CA82
2$,#/'(#" *"#" &%)#)" -0" )*#/(51%*%" >FQ@6"O"#" "4.-*"#)()" 270A68=6
<0H
2>=CA81DC4
A4<0A:01;H
C>
C74
?A8<0AH
1%,#"&4"Y-/$#/"%&-2*"%$"#'#+*/-)*%*(+"9%4"*-")#+2/#"*"#" 270A64B
>5
C74
10B838>B?>A4B
#22DAA4=24
>A
01B4=24
/#52'%/").%+($5"-0").-/#)"-$"*"#"&%)(,(21")2..-/*)"*"#" -0"*"#"*/(&-#'#+*/(+"+"%/5($5",#.#$,)"-$"*"#".'25")#%'($5" 8340
C70C
C74
ZABC
8==4A
?A>24BB
2>=CA81DC8=6
C>
C74
B?>A4
*"#"*-."-0")*#/(51%"%$,"*"#""('21"%..#$,(W3"%$,"-$"*"#" ./(1%/4"+"%/5#)"-++2/)"'-$5"�-/#"*"#").-/#"'(&#/%*(-$6" 3478B24=24
I>=4
B8CD0C43
14CF44=
C74
?;D6B N0"0-/1%*(-$"-0"*"#").-/#"./(1%/4"+"%/5#)"($)"#%/'4"%*" G'#+*/-."4)(-'-5(+%'" )*2,(#)" 1%,#" &4" O%*(%$%" *"#").-/#",#:#'-.1#$*%'")*%5#3"*"#$"*"#").-/#)""%:($5" 4;>I4AB:0H0
F8C7
2>F>A:4AB
B7>F
C70C
8=
D?;43
0/-1"#%+""-*"#/"0-/"*"#"#$*(/#"*(1#"-0",#:#'-.1#$*"%$," 24;;B
E80
8>=82
[DG4B
C70C
C74B4
8>=82
[DG4B
?A><>C4
1%*2/%*(-$3"*"2)")#+2/($5"*"#"/#52'%/").%+($5"-0").-/#)" ;>20;
<4C01>;82
B?4280;8I0C8>=
>5
8=38E83D0;
7H?70;
-$"*"#"&%)(,(216 )#51#$*)"%$,"0%:-/"*"#(/"02$+*(-$%'""#*#/-5#$#(*43"%$," D-))(&'4" %''" *%W%" 0/-1" ).#+(#)" *-" 1-$-."4'#*(+" C70C
8=C46A0C8>=
>5
24;;B
C0:4B
?;024
E80
;>20;
8=C4A24;;D;0A
50<8;84B
0A4
270A02C4A8I43
1H
B?428582
CH?4
>5
?>;0A8CH

774 "#$%&'"%()#%'* +,-".,#* +#%' / 0122123 4563 78

110 )#%).
*##'*(')
")!
&
+-'&'%.
'
!)#'%. + *

-0" *"#" ./(1%/4" #'#+*/(+" +"%/5#" -0" &%)(,(-).-/#)6" "(128)$#%$0$165 O"#)#" ,(00#/#$*" *4.#)" %/#" -&:(-2)'4" +-//#'%*#," 9(*""" D=:=>F=
?7H;>64=4C820;;H
38554A4=C80C43
CH?4B
>5
'70=:B
0A4
3D4
C>
'7><0B

>=4B
(=8E4AB8CH
>5
&(-#'#+*/(+" %+*(:(*4" ($" ).-/#" ,#:#'-.1#$*" *"%*" "%:#" %>274BC4A
(&
5>A
74;?
8=
C74
38B2DBB8>=
01>DC
C74
-/(5($%*#," ($" .%/%''#'6" A4.-*"#*(+%''43" *"#" ,(00#/#$*" CA81>4;42CA820;
270A68=6
C>
47>
'434AB>>
(=8E4AB8CH
+"%/5#" *4.#)" 1%4" &#" +-//#'%*#," 9(*"" ,(00#/#$*" -0" O%/*23" G)*-$(%B" 0-/" +-11#$*)" -$" *"#" 1%$2)+/(.*3" BCA0C4684B
>5
B?>A4
B?A4038=6
0=3
38BCA81DC8>=
>5
0=3
C>
!0AC8=
0B4<00
5>A
;8=6D8BC82
74;?
'F>
A454A44B
).#+(#)B6 +-$)*/2+*(:#'4"+-11#$*#,"-$"*"#"1%$2)+/(.*6

!"<"%"2&"#

- .
D;;4A
%
%4B40A274B
>=
5D=68

=
022>D=C
>?Q@" !#&)*#/" K63" N$*/-,2+*(-$" *-" 02$5(3" Y%1&/(,5#" -0" *"#" ./-,2+*(-$3" '(&#/%*(-$3" %$," ,().#/)(-$" -0" [$(:#/)(*4"D/#))3"?RXS *"#").-/#)"-0""41#$-14+#*#)"*/#%*#,"&-*%$(+%''4" - .
A46>AH
$
&?42D;0C8>=B
>=
10B838>B?>A4
0=3
?7HB820;;H
;B>
B><4
>1B4AE0C8>=B
D?>=
'(&#/%*(-$6" 82''#*($" -0" *"#" 8/(*()"" J4+-'-5(+%'" *"#" ,()+"%/5#" %$," ,().#/)(-$" -0" *"#" ).-/#)" -0" &>284CH

 
 
 %)+-14+#*#)"%$,"-0"D('-&-'2)3"C-$51%$)3"E/##$" - .
&48=54;3


$0=38B
&
"
C<>B?74A82
274<8BCAH
I"Y-63"C-$,-$3"?RSR %$,"."4)(+)M"0/-1"%(/".-''2*(-$"*-"+'(1%*#"+"%$5#3" >F@" E/#5-/4" D6A63" G'#+*/-)*%*(+" +"%/5#)" -$" ).-/#)" -0" =3
43
>1>:4=
"
>7=
*8;4H

&>=B

5D=68
"0CDA4


 - .
&C>;I4%H12IH=B:8
 
8><4270=82B
>5
B?>A4
- .
&F8=10=:
$
'0660AC

DC278=B>=
&
'74
,()+"%/5#"($"*"#"8%)(,(-14+-*%3"D"_"*"#)()3"J(%1(" 1#%)2/#1#$*" -0" *"#" #'#+*/-)*%*(+" +"%/5#)" -$" (=8E4AB8CH
(&
 B?>A4B
>5
!4AD;8DB
;02AH<0=B
*D;5
A
==
>C - .
!>=4H
"$
8B274A
!*
8><4270=82B
>5
>=3>=

 
 ).-/#" /#'#%)#" ($" ."4*-.%*"-5#$)3" N$M" _#()($5" A6" -.
A46>AH
$
!82A>18>;>6H
>5
C74
0C<>B?74A4
=3
3
$;0=C
A4;0C8>=B78?B
=3
43
'74
!H2>C0
)
43
>7=
*8;4H

&>=B
"4F
+>A:V'>A>=C>
 &?A8=64A)4A;06
4A;8=
4834;14A6
 -.
*41BC4A

$A>2C>=
!
0E4H
%
D;;4A
- .
&00A
!
=E4BC860C8>=
>5
C74
4;42CA>BC0C82
270A64

!40BDA4<4=C
>5
C74
4;42CA820;
270A64
>=
-0" &%)(,(-).-/#)" -0" *"#" D"#''($2)" (5$(%/(2)" 5/-2.3" )-1#" &%)(,(-).-/#)" %$," %$" %))#))1#$*" -0" *9-" 4=CA0;
DA>?40=
>DA=0;
>5
8>;>6H

  .-))(&'#" 1#+"%$()1)" -0" &%''()*-).-/#" ./-.2')(-$3"  
'A0=B
A
!H2
&>2
 
 V 
 - .
&00A
!
&0;<

<8BB8>=
A0C4
>5
270A643
B?>A4B
-.
;N<4=M>=

HC>;>6H
0=3
?;42C>;>6H
>5
C74
($"&%)(,(-14+#*-2)"02$5("%$,"*"#"/#'%W%*(-$"*(1#"-0" A41#$-14+#*#)6" F$," /#:()#," #,63" K6" Y/%1#/3" C748A
4;42CA82
270A64B
4A>18>;>680
 
 
&CDCC60AC

-.
*41BC4A

0E4H
%
D;;4A

=6>;3
'
- .
8114CC
&

?7H;>64=4C82
>E4AE84F
>5
C74
8%''()*-).-/#",()+"%/5#"($"N*#/)-$('(%".#/.'#W%$+#3" 60A82>

C8=0
!H2>;>680


 V 
'A0=B
A
!H2
&>2
 
 
 FSSX -.
!2 0D67;8=

42:4CC

+>>=
&
>F?@" J%*"#$4"D6863"Y2/*()"K6J63"A-0)*#**#/"H6"#*"%'63"J%<-/" ['*/%)*/2+*2/#" %$," #:-'2*(-$" -0" &%''()*-).-/(+" 2;034B
>5
60A820;4B
0
2DB
?7H;>64=4C82
10B838>B?>A4B
>C

8=
&>2

  
   
>E4AE84F
!H2>;>680


 V

 - .
>41

&C0C82
4;42CA8Z20C8>=
&?A8=64A)4A;06
-.
+>>=
&
!2 0D67;8=

0B838>B?>A>64=4B8B
4A;8=
 ($"8-'#*2)"/2&($#''2)3"NN3"C%*#").-/#",#:#'-.1#$*3" - .
>A=4A

&CD384B
8=
C74
10B838DA4 !H2>;>680

 
 
 ).%+($5" %$," *"#" 8-'#*2)" ).-/#)3" O"#" E%/,#$U)" - .
*41BC4A

0E4H
%
=6>;3
'
#A868=
>5
C74
D;;4C8=
&8=60?>A4

 
 ;8@D83
8=
D;;4AYB
3A>?
'A0=B
A
!H2
&>2
 
  - .
4;;4A

>=CA81DC8>=
K
;0
2>==08BB0=24
 
 ,#" 'U($0/%)*/2+*2/#" ,#" '%" .-/-(" ).-/(L2#" ,#)" - .
>0C4B

>55<0==

>A<0C8>=
0=3
?7H;;>?7>A0;4B
'7OB4
34
3>2C>A0C
(=8E4AB8CN
34
,()+"%/5#"-0")#+-$,%/4").-/(,(%"-0"*"#"&2$*"02$52)3" "4D27LC4;
&D8BB4
 '8;;4C80
5>4C830
!H2>;>680

 
  
 - .
&270554AC
%!
;42CA>?7>C>6A0?7H
*8;4H

&>=B
- .
*41BC4A

0E4H
%
'DA=4A
%
)0?>A
0B
0
"4F
+>A:

B>DA24
>5
F0C4A
8=
D;;4AYB
3A>?
!H2>;
%4B

 
- .
$>C0?>E0
')
B;0=838

4;>I4AB:0H0
 
 '
4E8=0
""
'A0=B24;;D;0A
8>=82
BCD384B
1H

775 !"#$%&'"%()#%'* +,-".,#* +#%' / 0122123 4563 78

111 !7&&""#(&)7&+"#,"*%-&

8=CA024;;D;0A
?>C4=C80;
A42>A38=6B
8=
"4DA>B?>A0
- .
4;>I4AB:0H0
'
4BB;4A
""
B0:>E0
$
+/%))%" "4."%#M"O/%$)0#/" -0" #$#/54" 0/-1" ./-W(1%'" 4AH018=0
+D
"4DA>B?>A0
2A0BB0
;867C
B86=0;
C>
0?820;
24;;B
&
4CC4AB

  
  
CA0=B3D2C8>=
8B
05542C43
1H
%#&
>DA=0;
>5
&86=0;
 'A0=B3D2C8>=
)>;

AC

  

?064B
- .
4;>I4AB:0H0
'
$>C0?>E0
')
=CA07H?70;
FS?F +-112$(+%*(-$" ($" )#51#$*#," 14+#'(213" - .
&00A
!
">??4;
!
&0;<

=70=24<4=C
>5
G?4A8<4=C0;
!H2>;>6H

 

C74
2>;;8B8>=
45Z284=2H
14CF44=
10B838>B?>A4B
0=3
- .
4;>I4AB:0H0
'
A8CB:88
!&
&>:>;>EB:88
)+D
+'-2,",/-.'#*)"&4"#'#+*/-)*%*(+"+"%/5#)"+%//(#,"-$" J#1&/%$#"#'#+*/-5#$#)()"%$,"*"#"./-&'#1)"-0"+#''" 5A4B7;H
4<8CC43
10B838>B?>A4B
DA>?40=
4A>B>;
38554A4=C80C8>=
8=
18>;>68H0

>=54A4=24
$A06D4
BCC7
&4?C4<14A

 
  

8=
%DBB80=
F8C7
BD<<0AH
8=
FS?Q G$5'()"B - .
D;;4A
%
%4B40A274B
>=
5D=68

DAC74A
- .
4;>I4AB:0H0
'
4;;C>24;;
2><=
8=
($:#)*(5%*(-$)" 2.-$" *"#" ./-,2+*(-$" %$," '(&#/%*(-$" ,(00#/#$*(%*(-$"-0"14+#'(%'"02$5(3"J#1&/%$#"I"Y#''" -0").-/#)"($""41#$-14+#*#)3"C-$51%$)3"E/##$"I" 8>;>6H 8>;>68274B:84
!4<1A0=H

 
Y-63"C-$,-$3"?RFF 

$!
 

776 "#$%&'"%()#%'* +,-".,#* +#%' / 0122123 4563 78

112 113 CURRICULUM VITAE

Maret Saar

Date and place of birth: 20.09.1946, Tabivere, Estonia Nationality: Estonian Address: Estonian University of Life Sciences, Institute of Agricultural and Environmental Sciences, Department of Botany, Fr. R. Kreutzwaldi 5, 51014, Tartu Phone: (+372) 53 963 787 Fax: (+372) 731 3988

Education 1964 Lähte Secondary School, Diploma 1964-1969 Tartu State University, Dept. of Physics and Chemistry, student of physics (optics). Diploma of higher education 1969 (eq. to MSc in physics) 1971-1974 Institute of Zoology and Botany of the Estonian Academy of Sciences, Dept. of Mycology, postgraduate student (aeromycology) 2007-2015 Doctoral School of Estonian University of Life Sciences, programme of Environmental Sciences and Applied Biology

Professional experience 1969-1971 Tartu University, Electroluminescence and Semiconductors Laboratory, Air Electricity Laboratory, technician 1975-2004 Institute of Zoology and Botany, Dept. of Mycology, senior engineer, researcher 2005-... Estonian University of Life Sciences, Institute of Agricultural and Environmental Sciences, Dept. of Botany, researcher, senior technician 2014-2015 Tartu University, Institute of Physics, Dept. of Bio and Environmental Physics, specialist (12 months)

Research Interests Aerobiology (pollen and fungal spores in outdoor air) Phenology (higher plants, fungi) Mycology (sporulation and spores of basidiomycetes)

114 Research Projects 1989-2006 various research projects including: to performing the routine aerobiological monitoring of pollen and fungal spores in Estonia, to provideing the population with forecasts of allergenic pollen and spores, and to studying the seasonality of the composition of the pollen and fungal spectrum in Estonia.

Publications Kupias R, Helander M L, Saar M, Mäkinen Y. 1989. Comparison of some pollen concentrations in Finland and Estonian SSR. Aerobiologia, 4, 94 - 103. Saar M, Saan T. 1993. Puud näitavad, et kevaded on varasemaks muutunud. Eesti Loodus 1993, 65-66. Ekebom A., Nilsson S, Saar M, Hage-Hamsten van M. 1997. Comparative study of airborne pollen concentrations in Tartu, Roma/Gotland and Stokholm (1990-1996). Grana, 36, 366-372. Saar M, Gudžinskas Z, Ploompuu T, Linno E, Minkiene Z, Motekaityte V. 2000. Ragweed plants and airborne pollen in the Baltic states. Aerobiologia, 16, 101-106. Saar M. 2001. Aerobioloogiline kalender. – Kogumikus: Ahas R (toim), Publicationes Instituti Geographici Universitatis Tartuensis, 90, Eesti looduse kalender, lk 164-186. Saar M. 2005. Nothing interesting but the origin: annual dynamics of airborne pollen composition in Estonia. Interdisziplinäre Zeitschrift für Allergologie und Umweltmedizin Allergo Journal, 14, 276-277. Saar, M. 2007. Seasonality in quantity of atmospheric fungal aerosol in Tartu (Estonia). Folia Cryptogamica Estonica, 43, 57-67. Meltsov V, Poska A, Saar M. 2008. Pollen size in Carex: The effect of different chemical treatments and mounting media. Grana, 47, 220 - 233. Saar M, Meltsov V. 2011. Passports of sampling sites in routine aerobiological monitoring of outdoor air. Clot B, Comtois P, Escamilla-Garcia B. (eds). Aerobiological Monographs, Towards a comprehensive vision. Montreal, Canada: MeteoSwiss (CH) and University of Montreal (CA), pp. 215 – 231.

Presentation of work Saar M, Gudžinskas Z, Ploompuu T, Linno E, Minkiene Z, Motekaityte

115 V. 1998. Ragweed plants and airborne pollen in the Baltic states. The 6th International Congress on Aerobiology, 31 August – 5 September, Perugia, Italy. Saar M, Meltsov V. 2006. Passports of sampling sites in routine aerobiological monitoring of outdoor air. The 8th International Congress on Aerobiology, 21-25 August, Neuchatel, Switzerland. Meltsov V, Saar M. 2007. The vegetation of surroundings of aerobiological sampling site. Pollen Monitoring Programme (PMP), 6th International Meeting, 3-9 June, Jurmala, Latvia. Saar M. 2009. Flowering and airborne pollen of Poaceae in Tartu (Estonia). The COST725 Final Conference on Phenology, 10-11 March, Geisenheim, Germany. Saar M. 2010. Electric charges of ballistic basidiospores immediately after release in Agaricomycetes. The 9th International Congress on Aerobiology, 23-27 August, Buenos Aires, Argentina. Saar M. 2013. Natural emission of electrostatically charged basidiospores into the atmosphere. 14th NAF Symposium on Aerobiology, 19- 22 August, Riga, Latvia. Saar M, Noppel M, Salm J. 2013. Enhancement of the collision effi ciency between basidiospores and cloud droplets by electrostatic charges carried on freshly emitted basidiospores.The European Aerosol Conference, 1-6 September, Prague, Czech.

Memberships 1976- Estonian Naturalists’ Society 1989- European Aeroallergen Network 1990- International Association for Aerobiology 1996-2005 International Society of Biometeorolology

VIIS VIIMAST KAITSMIST

PRIIT PÕLLUMÄE ASSESSMENT OF PRIVATE FOREST OWNERS’ COOPERATION IN ESTONIA EESTI ERAMETSAOMANIKE KOOSTÖÖ ANALÜÜS Dotsent Henn Korjus 21. august 2015

PRIIT-KALEV PARTS SUSTAINABLE COMMUNITY MANAGEMENT IN ESTONIA: REFLECTIONS ON HERITAGE PROJECTS ON KIHNU ISLAND, IN VILJANDI COUNTY, AND IN VARIOUS PROTECTED AREAS KESTLIK KOGUKONNAKORRALDUS: KULTUURIPÄRANDIGA SEOSTUVATE ARENDUSTEGEVUSTE PEEGELDUSI KIHNU SAARELT, VILJANDI MAAKONNAST JA EESTI KAITSEALADELT Professor Kalev Sepp 21.august 2015

BERT HOLM CULTIVATION OF WILLOWS (SALIX SP.) USING RESIDUES OF THE WASTEWATER PURIFICATION PROCESS AND BIOGAS PRODUCTION AS FERTILISERS PAJUDE (SALIX SP.) KASVATAMINE KASUTADES VÄETISTENA REOVEEPUHASTUSPROTSESSI- JA BIOGAASI TOOTMISE JÄÄKE Vanemteadur Katrin Heinsoo 21. august 2015

MARTI TUTT FACTORS AFFECTING BIOCHEMICAL COMPOSITION OF LIGNOCELLULOSIC BIOMASS AND ITS EFFECT ON SELECTION OF PRETREATMENT METHOD AND ON BIOETHANOL PRODUCTION POTENTIAL LIGNOTSELLULOOSSE BIOMASSI BIOKEEMILIST KOOSTIST MÕJUTAVAD TEGURID NING BIOKEEMILISE KOOSTISE MÕJU EELTÖÖTLUSMEETODI VALIKULE JA BIOETANOOLI TOOTLIKKUSELE Professor Jüri Olt, vanemteadur Timo Kikas 28. august 2015

KADRI MAIKOV ANALYSIS OF GREEN ROOMS AND LANDSCAPE CHARACTERISTICS ROHEALADE MAASTIKUKARAKTERISTIKUTE JA RUUMIDE ANALÜÜS Professor Kalev Sepp 31.august 2015

ISSN 2382-7076 ISBN 978-9949-536-94-8 (trükis) ISBN 978-9949-536-95-5 (pdf)

Trükitud taastoodetud paberile looduslike trükivärvidega © Ecoprint