ECOLOGICAL FEATURES OF AMBROSIA ARTEMISIIFOLIA L . FLOWERS AND CHARACTERISTICS OF AMBROSIA L . POLLEN SEASONS IN THE CONDITION OF LUBLIN ( POLAND ) IN THE YEARS 2001-2008

In the study, the biology of fl owering of Ambrosia artemisiifolia L. was investigated and the pattern of the Ambrosia pollen seasons in Lublin in the years 2001-2008 was characterised. The structure of male and female A. artemisiifolia flowers was observed in cultivated plants under controlled conditions in the 2000 vegetative season. The number of pollen grains produced by the stamen, flower, inflorescence and plant was determined. It was shown that in A. artemisiifolia flowers nonfunctional pistils occurred with a reduced ovary, performing the role of a pollen presenter. The pistils found in female flowers differed significantly in their morphological features from the pistils in male flowers. It was calculated that one stamrn produced an average of 3 375 pollen grains, whereas one flower 16 875. A plant which produces 20 racemes may release over 420 million pollen grains into the atmosphere. The Ambrosia pollen seasons in particular years had different patterns. In some years, the days of maximum concentration were in the second half of August, in other years in the first half of September. Over the 8-year period, the maximum daily concentration had a mean value of 116 pollen grains in m 3 (31-311). Annual total concentrations of Ambrosia pollen grains ranged between 194 and 1200 grains, and it was 523 grains on the average. The pollen seasons were characterised by the occurrence of several-day-long interruptions in the presence of airborne pollen in the atmosphere of Lublin, which may indicate the long-distance transport of Ambrosia pollen.


INTRODUCTION
Ambrosia artemisiifolia L. (Asteraceae) comes from North America.It was transferred to Europe, including Poland, together with a shipment of cereals at the end of 19 th century (H o l z f u s s , 1937).In Poland the occurrence of several species of the genus Am-brosia is recorded in different regions of the country: Ambrosia artemisiifolia L., A. psilostachya DC. and A. trifi da L. (Ta c i k , 1971; R u t k o w s k i , 1998; Z a j ą c and Z a j ą c , 2001). A. artemisiifolia has been noted, inter alia, in the Lublin region (F i j a ł k o w s k i 1994), in Wrocław (M a l k i e w i c z and W ą s o w i c z , 2003), in the Upper Silesia region (C h ł o p e k and To k a r s k a -G u z i k , 2006).Ambrosia psilostachya sites have been described, among others, in Szczecin (Ć w i k l i ń s k i , 1968; P u c , 2004) and the Lublin region (Ś w i ę s and W r z e s i e ń , 2002).
Plants of the genus Ambrosia brought to Poland are most frequently encountered near railway tracks or at ruderal sites.They also grow on dry swards and at roadsides, and they sometimes occur as weeds in crops.In some locations, they are treated as species occurring temporarily (F i j a ł k o w s k i , 1994; Ś w i ę s and W r z e s i e ń , 2002).They belong to quarantined plants.
Ambrosia pollen grains are the most frequent cause of pollen allergy in North America (B o u s q u e t et al. 2001).C o m t o i s (1998) defi nes representatives of the genus Ambrosia as allergophytes.
In A. artemisiifolia pollen grains, one of allergens with the strongest effect on humans (Amb a 1) is found, which causes allergies in many countries in an ever increasing number of people (C o m t o i s , 1998; J ä g e r , 2000).
In Warsaw the frequency of positive skin tests to the ragweed pollen allergen is found to have increased in patients from 0. The aim of the study was to investigate the biology of fl owering of A. artemisiifolia growing in experimental plots in Lublin in order to determine the number of pollen grains produced by the stamen and the fl ower as well as to compare the pattern of Ambrosia pollen seasons in the years 2001-2008.

MATERIALS AND METHODS
Ambrosia artemisiifolia L. seeds were obtained from the Botanical Garden in Dijon (France).Plants grew in experimental plots in Lublin under controlled conditions during the 2000 growing season.Over a dozen plants were subjected to observations.Details of the structure of male and female fl owers of the investigated Ambrosia artemisiifolia plants and their development were observed.
The number of male fl owers in fl ower heads, the number of fl ower heads borne on one stem and on the plant as well as the diameter of fl ower heads were determined in ten plants.The number of pollen grains produced in the stamen was evaluated as a mean from 10 anthers from which separate smear slides stained with basic fuchsine were made.The number of pollen grains produced by a single fl ower, fl ower head and plant was determined.
The investigations of pollen concentrations were carried out by the volumetric method using a VPPS 2000 Lanzoni pollen trap.The sampler was placed in Lublin's city centre (Śródmieście) at a height of 18 m.Daily Ambrosia pollen concentrations and annual total concentrations over the 8-year study period, 2001-2008, were calculated from the obtained aerial plankton samples.

Floral morphology
Ambrosia artemisiifolia is a herbaceous plant.In the conditions of Lublin, in 2000 the plants reached a height of 100 cm.They fl owered from 20 August till the end of October, producing on their stems numerous anemophilous fl owers occurring in tiny fl ower heads (Figs 1 A, B).
Flowers of this species are unisexual.The downward pointed fl ower heads (anthodia), with an average of 25 staminate fl owers clustered in them (Tab.1, Figs 1 C, E, F), are 4.0 -6.5 mm in diameter.They are grouped in loose, terminal racemes composed of 50 fl ower heads, on the average (Tab.1).The anthodium is protected by a cup-shaped involucre, made up of several fused bracts, which has a toothed edge or is provided with upward bent fl aps (Fig. 1 D).
The fi rst male fl owers develop in the circumference of the anthodium.The fl ower height reaches 1.7 mm, on the average, and its diameter 0.6 mm.The yellow-green semi-transparent corolla is composed of 5 fused petals which form in their upper part free lobes opening only slightly at anthesis (Figs 2 A, B).Anthers are the fi rst to come out through an opening formed at the apex of the corolla in some male fl owers.In other male fl owers, the apical portion of the non-functional pistil, which forms a brush-shaped pollen presenter, was observed to appear fi rst between the corolla lobes (Figs 2 E, F, G).The pistil with a reduced ovary has a cylinder-shaped style (Figs 2 H, 3 E).In its upper portion, the style is provided with brush-shaped outgrowths which sweep out and raise pollen, released in the fl ower inwardly from the bursting anthers already inside the closed corolla.This pistil in the bud reaches the half-height of the corolla (Fig. 2 C) and gradually elongates as the fl ower develops (Fig. 2 D).
The stamens (5) observed in the buds of male fl owers were fused by their heads, but at anthesis they separated (Figs 3 A, B).The fi laments were very short in the bud (Fig. 3 F).There was an outgrowth at the apex of the elongated anther (Figs 3 B, F).At anthesis, the anthers stuck out much above the corolla (Fig. 3 A).
Female fl owers are gathered in single-fl owered anthodia subtended by small bracts.These anthodia grow in clusters beneath the male infl orescences (Figs 4 A, C).They are subtended by several hairy bracts.The female fl ower is composed of one pistil with an inferior ovary and two fi liform red-coloured stigmas (Fig. 4 B) which elongate signifi cantly during fl owering (Fig. 4 C).In the upper portion of the receptacle fused with the ovary, the calyx is visible, forming a ring of tiny nodules which remain later on the fruit.In the basal part, the pistil's stigmas are enclosed by strongly reduced, hairy petals of the corolla (Fig. 4 B).The share of female fl ower heads in the total number of infl orescences in the investigated plants was small.
It was found that one stamen produced 3 375 pollen grains, on the average (Tab.1).The calculations show that one fl ower produces an average of 16 875 grains, one fl ower head 421 875 grains, whereas a raceme 21 093 750 pollen grains.A plant which produces 20 racemes releases on the average over 420 million pollen grains into the atmosphere.These grains are tricolporate.They reach an average size of 18 x 20 μm.The exine surface is covered with spinules with a length of about 1 μm (Figs 3 G,H).
In some years, the days of maximum Ambrosia pollen concentrations were in the second half of August (2001,2004,2005,2007), in other years, in the fi rst half of September (2002,2003,2008).But in 2006 two days with the same, and at the same time, the highest pollen concentration were distinguished (Figs 5,8).
The pollen season pattern covering mean concentrations from the eight-year study is characterised by the presence of two peaks.The fi rst peak is seen on 20 August, the other one on 4 September (Fig. 7).It results from the occurrence of maximum concentrations at different dates in the study years.In particular years, the maximum pollen concentrations ranged between   31 and 311 pollen grains in m 3 per day.The lowest concentration was in 2006, and the highest one in 2002.
Very high concentrations also occurred in the years 2001 and 2008 (Fig. 8).The 8-year mean maximum concentration was 116 pollen grains in m 3 per day.
A comparison of pollen sums in particular months: August, September and October, is presented in Tab. 2. The comparison shows that in some years higher pollen sums were recorded in August, whereas in others in September.The mean number of pollen grains from the study years was the highest in September.The seasonal pollen index (SPI), that is, the annual total concentration of Ambrosia pollen grains, ranged between 194 and 1200 grains (Fig. 9).The largest amount of pollen grains was recorded in 2002, whereas the lowest in 2007.Relatively high annual sums were also obtained in the years 2008 (712 pollen grains) and 2005 (646 pollen grains).The 8-year mean SPI reached 523 pollen grains.

Ambrosia fl ower morphology
On one stem of the investigated A. artemisiifolia plants, we found the occurrence of 10-90 (50 on the average) male fl ower heads.But B a s s e t and C r o m p t o n (1975) report that 10-200 of such fl ower heads may occur at the top of stems of this species.The diameter of the investigated male fl ower heads was 4.0-6.5 mm.Ta c i k (1971) mentions similar dimensions (a diameter of 5-7 mm) of male fl ower heads of this taxon.
Our calculations show that 12-60, on the average 25, male fl owers formed in one anthodium.Ta c i k (1971) reports that from several up to 20 fl owers are found in the anthodium of Ambrosia genus representatives.
Male fl owers have a yellow and green coloured fused corolla with fi ve free lobes at the apex.In our study, we found that there were 5 stamens in the male fl ower and 1 non-functional pistil with a reduced ovary as well as a well-developed style and a stigma.The brush-shaped outgrowths on the stigma, forming the pollen presenter, support the of the male elements in the fl ower, raising pollen grains released from the anthers above the level of the corolla lobes.It seems that such a structure of the fl ower is a remnant of this plant's adaptation to entomophily, and the reduction of the ovary, with the pollen presenter left/formed at the apex of the style, is one of ecological features facilitating external pollen exposition under the conditions of wind pollination.Attention is attracted to a completely different structure of the pistil in functionally male and female fl owers.In female fl owers, a well-developed ovary and the presence of two long stigmas of the pistil, typical for anemophilous plants, are noticeable.
In Ambrosia, female fl owers occur in single-fl owered heads (Ta c i k , 1971; B a s s e t and C r o m p t o n , 1975) which form small clusters in the axils of the upper bracts, beneath the male fl ower heads.Ta c i k (1971) reports that clusters of female fl owers are most frequently made up of 5-6 fl owers, whereas in the investigated individuals we observed 2-7 fl owers subtended by several involucral bracts.In the conditions of Lublin, clusters of female fl owers were most frequently composed of 3 fl ower heads.
The reduced calyx occurring in female fl owers forms a ring of nodulelike outgrowths which remain on the fruit, developing into a circle of tiny prickles.Ta c i k (1971) fi nds that the number of differentshaped prickles formed on the achene in A. artemisiifolia is within the range of 4-7.According to data reported by B a s s e t and C r o m p t o n (1975), achenes in this species are 3.5 mm in length and 2.5 mm in width.They form a terminally located beak with a length of 2 mm surrounded by a circle of thorns reaching a length of up to 1 mm.The fruit coat is lignifi ed.It seems that the beak located at the apex of the achene is formed from reduced corolla elements, and the thorns from the corolla outgrowths.
In our study, we found that the number of pollen grains produced by the stamen was 3375, on the average.But the number of pollen grains released by a plant producing 20 infl orescence stems may reach 421 875 000.C o m t o i s (1998) also reports that one Ambrosia plant can produce tens up to hundreds of millions of pollen grains.

A comparison of Ambrosia pollen seasons
Lublin belongs to these Poland's cities in which high and very high Ambrosia pollen concentrations have been noted over the period of the last eight years.
A comparison of annual sums and daily maximum concentrations of Ambrosia pollen grains in 6 Poland's cities in the years 2001-2005 demonstrates that both the highest annual sums and the highest daily pollen concentrations occurred in Lublin.The mean annual sums from the abovementioned 5-year period were as follows for the particular cities: Lublin 602, Rzeszów 332, Sosnowiec 331, Szczecin 196, Kraków 179 and Poznań 121.In 2002 the highest annual pollen sums were recorded in most of the abovementioned cities, however, the value obtained for Lublin (1200 grains) was several times higher than in Kraków It can be concluded from the analysis of the pattern of the Ambrosia pollen seasons that large quantities of airborne pollen of this taxon found in the air of Lublin may come from the long-distance transport.It is indicated by the pollen seasons, distributed over all the years of study, with the alternate occurrence of days with Ambrosia pollen content in the air and days when there were no such airborne pollen.In the pattern of the pollen seasons from the years 2001-2008, one can observe several-day-long interruptions in the infl ow of Ambrosia pollen, which is probably associated with meteorological conditions.The effect of meteorological conditions on the presence of airborne Ambrosia pollen in the air of Lublin has been demonstrated in our previous paper (P i o t r o w s k a and We r y s z k o -C h m i e l e w s k a , 2006).
Our observations and consultations with other botanists show that no Ambrosia plant individuals have been found in Lublin.The nearest locations of this plant have been found over 60 km away from Lublin ( Ś w i ę s and W r z e s i e ń 2002), which confi rms the long-distance transport of this pollen.
In our previous paper we have shown that maximum intradiurnal Ambrosia pollen concentrations occur at different times of the day: before noon, in the afternoon and at night.On most days with high Ambrosia pollen concentrations, the largest amount of pollen grains was recorded between 4 and 8 pm (P i o t r o w sk a and We r y s z k o -C h m i e l e w s k a , 2006).
3 % in 1998 to 1.5 % in 2003 (R a p i e j k o , 2004).Ambrosia pollen has been found in the air of different Poland's cities for many years (K a s p r z y k , 1996, 2008; S t a c h and S i l n y, 1999; S t ę p a l s k a et al. 2002; M a l k i e w i c z and W ą s o w i c z , 2003; We r y s z k o -C h m i e l e w s k a et.al. 2003; P u c , 2004, 2006; P i o t r o w s k a and We r y s z k o -C h m i e l e w s k a , 2006).

Fig. 1 .
Fig. 1.Ambrosia artemisiifolia A -Flowering plant, B -Plant before the start of fl owering with a terminal raceme formed from numerous male fl ower heads, C -Portion of the raceme with male fl ower heads (x 7), D -Upper side of the anthodium with the visible involucral bract (x 14), E -Male fl ower head before the start of fl owering (x 14), F -Anthodium with blooming male fl owers (x 14).

Fig. 2 .
Fig. 2. Portions of Ambrosia artemisiifolia male infl orescences.A, B -Portions of anthodia with blooming fl owers in which pollen-shedding stamens grow above the corolla (A -x 30, B -x 40), C, D -Male fl owers with the non-functional pistil performing the role of the pollen presenter (asterisk) (C -x 40, D -x 60), E, F, G -Male fl owers, in which the pollen presenter (asterisk) is the fi rst to appear during fl owering (E, F -x 30, G -x 100), H -The pistil's style with a brush-shaped pollen presenter (x 150).

Fig. 3 .
Fig. 3. Male fl owers, their portions and Ambrosia artemisiifolia pollen grains.A -Different growth stages of male fl owers coming from one anthodium (x 20), B, C -Portions of male fl owers with the involucre, stamens and the pollen presenter (asterisk) (B -x 80, C -x 100), D -Burst anthers releasing pollen (x 100), E -Stamen and the style of the non-functional pistil from a male fl ower (asterisk), (x 50), F -Stamen excised from a fl ower bud (x 200), G -Pollen grain in polar view (x 1300), H -Pollen grain in equatorial view (x 1300).

Fig. 7 .
Fig. 7. Average concentrations of Ambrosia pollen grains in the air of Lublin on the basis of eight-year-long investigations (2001-2008).
(200 grains) (C h ł o p e k and D ą b r o w s k a , 2006; K a s p r z y k , 2006; M y s z k o w s k a , 2006; P u c , 2006; S t a c h , 2006).In the abovementioned 5-year period, the highest daily Ambrosia pollen concentrations were also recorded in 2002.In four of the abovementioned six cities, located at a distance of several hundred kilometres from one another, the peak day was recorded on 4 September 2002 (C h ł o p e k and D ą b r o w s k a , 2006; K a s p r z y k , 2006; P u c , 2006).The years 2006 and 2007 were characterised by low annual sums of Ambrosia pollen grains both in Lublin and in several other cities in Poland (R ap i e j k o et al. 2006; L i p i e c et al. 2008).In 2006 the highest annual totals occurred in Sosnowiec (534 grains), Wrocław (207 grains) and Lublin (193 grains), whereas in 2007 in Sosnowiec (234 grains), Lublin (172 grains) and Warsaw (159 grains).The calculation of the mean annual sum of Ambrosia pollen for the years 2001-2007 for several Poland's cities shows that the highest number of pollen grains was recorded in Lublin (482), and then in Sosnowiec (346).