THE ESTIMATION OF ADONIS VERNALIS POPULATIONS IN CHOSEN PATCHES OF LUBLIN UPLAND

The number of Adonis vernalis populations, their size and abundance in xerothermic communities of the Lublin Upland have decreased signifi cantly in the last 40–50 years, which indicates that there is a real risk of species extinction in the studied area. The paper is an attempt to indicate the possible factors which infl uence the decrease of Adonis vernalis populations. Detailed studies were carried out in six sites differing habitat conditions, land use and the size of Adonis vernalis populations. Observations were made of the abundance of blooming and the number of anthers per fl ower. The ability for reproduction was estimated indirectly by the mass of pollen delivered and its biological value. The populations of Adonis vernalis differ in number of anthers per fl ower (94.4 – 131.5, on average), the mass of pollen delivered (0.83 mg – 3.18 mg per 100 anthers, on average), the biological value of pollen and the size of pollen grains. Generally, stronger populations were characterized by better quality and a higher quantity of pollen. Potential energy of pollen in fl owers from Pliszczyn and Łabunie was 69–72%, compared to 36%–47% in the remaining populations. Grass burning did not cause a reduction of potential biological value of Adonis vernalis pollen. Xerothermic swards in Pliszczyn and Kąty regularly burnt developed strong, most abundant populations of Adonis vernalis with favorable pollen. The development of the shrub layer, if it is out of control, stands in the way of satisfactory reproduction of halophilous Adonis vernalis.

swards of continental-submediterranean type, belonging to the order Festucetalia valesiaceae.In Poland it is a relict species of steppe zone fl ora.Its distribution is disjunct with some isolated growth places of extrazonal xerothermic habitats in the Lublin Upland, Małopolska, and the lower Warta or Wisła rivers (G a w ł o w s k a , 1956; Z a j ą c and Z a j ą c , 2001).Because of the small size of its populations, the disappearance of their habitats or a small number of locations, Adonis vernalis is a threatened element of the fl ora and is protected by law (R o z p o r z ą d z e n i e … 2004 ).
The stands of Adonis vernalis in the Lublin Upland are usually found on loose loam calcareous soils rich in CaCO 3 .The taxon occurs in patches of the Inuletum ensifoliae association, in community with Brachypodium pinnatum and Teucrium chamaedrys as well as on the edges of light pine-oak forests.F i j a ł k o w s k i (1961) described in Lubelszczyzna (the Lublin region) 32 stations, including 8 with abundant populations composed of over one thousand individuals.On the next eight stations, the populations were slightly weaker and comprised 100-500 plants.The remaining populations were found in decline with approx.5 individuals.The detailed biology of Adonis vernalis with an established model of vegetative organs was described by J a n k o w s k a -B ł a s z c z u k (1995).Entomophilous, pollen yielding fl owers of Adonis vernalis has been found valuable for bee-like hymenoptera because of the early blooming period (D e n i s o w and W r z e s i e ń , 2006).
The aim of the study was to estimate chosen populations of Adonis vernalis in the Lublin Upland.Biological features of pollen grains were also analyzed.An attempt was made to indicate the possible factor causing variance in pollen grain features as well as in population size.and Pliszczyn (51 o 18'N, 23 o 41' E -FE28) (Fig. 1) -(Z a j ą c , 1978).Habitat conditions were estimated separately for each station (slope inclination, soil, patch size) (Tab.1).

Study
The mean annual air temperature in the study region is 7.5 o C with seasonal changes between monthly mean minimum -4.5 o C in January and monthly mean maximum temperature of 18.9 o C in July.Annual average precipitation is 600-650 mm with humidity of 70%.The period of vegetation totals between 200 and 210 days (Z i n k i e w i c z and Z i n k i e w i c z , 1973; K a s z e w s k i et al. 1995).

Study methods
The vegetation on each site was described according to Matuszkiewicz (2001).Special attention was paid to the land use in the area.Phenological observations concerning the duration of blooming were made (inter alios J a b ł o ń s k i and S z k l a n o w s k a , 1997).Additional detailed investigations of individuals were carried out.The number of individuals per square unit, abundance of blooming, number of anthers per fl ower, mass of pollen delivered by anthers and fl owers were analyzed.The mass of pollen delivered was estimated according to S z k l a n o w s k a method (1995).The anther size was determined on the basis of fresh and dry weight of 100 anthers.During the time of full blooming, closed, fully grown anthers from randomly chosen fl ower buds were isolated in a laboratory and separated from fi laments.The anthers were placed in special glass containers (4 replications with 100 anthers for each side and year of study).The value of pollen was evaluated in terms of its viability in standard acetocarmin slides and germination after 24 hours in an agar medium with saccharose.The size of pollen, based on the length of polar and equatorial axis, was determined in glycerin jelly slides.

Statistical analysis
The relationship between the dry mass of anthers and the pure mass of delivered pollen was established using correlation coeffi cient (r).Statistical analyses concerning features of the androecium and pollen features were made.Data were subjected to one-way ANOVA.The signifi cance of differences between means was tested by Duncan's test at α = 0.05.

Characteristics of xerotermic patches with
Adonis vernalis.Variations of chosen populations between the stations and a comparison to previous studies are shown in Fig. 2. Unfortunately, on most stations the populations of Adonis vernalis declined substantially.
The population of Adonis vernalis in Łabunie, which had been estimated as large as 6 thousands individuals back in 1961, had decreased signifi cantly since then and was estimated at only 70 indiviluals with 1-5 fl owering stamens each.Adonis vernalis grows on the edge of a loose light pine-oak forest situated on shallow rendzina soil (Fig. 3A).Conservation management is undertaken and branches of Cornus sanguinea, strongly developing in the undergrowth layer, are regularly hewn off.Other species sharing the patch are Primula veris, Brachypodium pinnatum, Carex michelii, Anemone sylvestris, Hepatica nobilis, Viola reichenbachiana, Peucedanum cervaria.
The xerothermic sward in Chomęciska Małe was located in an old stone pit.The population of Ado-nis vernalis is vanishing and in the period 2002-2007 it was estimated at 15 individuals, on average, that is three times less compared to literature data.Adonis vernalis is a component of the community developing on shallow rendzina soil in the old stone pit.Nowadays, the area is an illegal dumping ground.Brachypodium pinnatum, Teucrium chamaedrys, Peucedanum cervaria predominated on fragments of the patch.The process of overgrowing with Prunus spinosa and Rosa canina is far advanced.
The xerotermic grassland with Adonis vernalis in Wólka Leszczańska is limited to 400 m 2 and located on ravine slopes neighboring fallow land (Fig. 3B).The shape and form of Adonis vernalis is similar to the population in Chomęciska Małe.Adult individuals form small clumps with only one up to a few fl owers.The patch is composed of Brachypodium pinnatum, Teucrium chamaedrys, Peucedanum cervaria, Anemone sylvestris, Gallium boreale, Salvia pratensis, S. verticillata.The succession process is taking place and the growth of numerous Prunus spinosa bushes as well as Juniperus communis and Viburnum opulus is observed.
The xerotermic grasslands in the Stawska Góra nature preserve, near the town of Chełm, are connected with eroded chalky slopes of different inclination and covered with shallow rendzina.Apart from Adonis vernalis, the patch is composed of other typical xerothermic taxa such as Primula offi cinalis, Euphorbia cyparissias, Potentilla arenaria and it is clearly predominated by Brachypodium pinnatum, Anemone sylvestris and Aster amellus.The population of Adonis vernalis numbers ca. 100 individuals and has declined signifi cantly comparing to literature data, as it was estimated at 3000 in 1961.The expansion of Prunus spinosa shrubs is well advanced.
The xerothermic patches in Kąty near Zamość occur on slopes of roads adjoining fi elds or on the south facing hill slopes covered with chalk and situated in the vicinity of light pine-oak forests edges.The population of Adonis vernalis, which numbered ca.4000 individuals in 1961, is now estimated at ca. 100 clumps with a few to tens of fl owers.Beside typical xerotermic species, such as Brachypodium pinnatum, Peucedanum cervaria, Salvia pratensis, Thalictrum minus, a few rare taxa were found, e.g.Linum fl avum, Cerasus fruticosa, Peucedanum alsatica, Orchis purpurea and Orchis militaris.Year after year the vegetation was burnt down, especially in the spring time.
At the Pliszczyn site near Lublin, Adonis vernalis is a component of the Adonido-Brachypodietum pinnati community situated on a loess-covered slope of the Bystrzyca River valley with south inclination (Fig. 3C).The Adonis vernalis population, compared to literature data, is nearly constant and is estimated at ca. 1000 individuals.Shapely clumps of adult individuals produce tens of fl owers.The xerothermic patch is predominated by grass and meadow species (Medicago falcata, Brachypodium pinnatum, Gallium verum, Euphorbia cyparissias, Veronica spicata, Salvia pratensis, Phleum phleoides, Chamaecytisus ratisbonensis).The thermophilous vegetation in Pliszczyn is a fragmented island surrounded by arable fi elds and fallows.The area has been regularly burnt, usually in early spring.
The time and period of blooming were similar for the populations of Adonis vernalis under consideration (Fig. 4).The blooming of Adonis vernalis in the conditions of Lublin Upland occurred between the middle and the end of April.Every year, the blooming of the populations in Chomęciska Małe and Wólka Leszczańska was the shortest.The populations in Pliszczyn and Stawska Góra bloomed almost simultaneously and their blooming was the longest, lasting six weeks.
Pollen characteristics.The size and abundance of the Adonis vernalis populations under consideration were in close relation to the studied features of fl owers and the biological value of pollen grains.Generally, stronger populations were characterized by better quality and a higher quantity of pollen.The anthers of Adonis vernalis are spirally arranged in the fl ower receptacle and their ripening is gradual.Great variability is characteristic of the number of stamens in the fl owers of plants across the studied populations.The most abundant and vital populations in Pliszczyn and Kąty develop fl owers with the highest number of stamens (Tab.2).Statistical-ly lower numbers of anthers were noticed in the fl owers of Adonis vernalis from the sites at Wólka Leszczańska and Stawska Góra where the populations were found to be vanishing.At the same time, the decline in the number of individuals in both the abovementioned populations was substantial, compared to literature data.Signifi cant differences in the fresh and dry mass of 100 anthers as well as in the amount of delivered mass of dry pollen were recorded between the populations (Fig. 5).The biggest anthers were found in the fl owers from Kąty, Łabunie and Pliszczyn.The fresh weight of 100 anthers, including pollen, was 48.9 mg, 46.3 and 42.9, respectively.Also, an analysis of the relation of dry weight of anthers to fresh weight indicated that tissues of the anthers from the Kąty and Pliszczyn sites were substantially better hydrated than of those from Łabunie.Interestingly, the dry weight of 100 anthers from Łabunie was 18.4 mg, on average, which was the highest, but at the same time the effi ciency of the pollen producing archespore was the lowest, thus the mass of delivered pollen was only 2.6 mg per 100 anthers.The mass of dry pollen delivered by anthers positively correlated with anther size (r = 0.64571) as well as with archespore effi ciency.The worst developed anthers in the fl owers at Wólka Leszczańska and Chomęciska Małe yielded 0.83 mg and 1.12 mg of pollen, respectively.Signifi cantly better developed anthers from the remaining sites delivered two-three times more pollen.The biological value of pollen was estimated on the basis of viability and germination (Tab.3).The quality of pollen and the population sizes were interdependent.The observed differences between the populations, as far as the biological value of pollen is concerned, were with high probability caused by external factors.The strongest populations at Pliszczyn and Kąty were characterized by the highest viability together with the highest pollen germination.The average viability was 91.7% and 97.8%, respectively, while germination ability over 65%.The relatively weak population at the Łabunie station with high viability of pollen grains had very low germination ability, only 36%.The remaining studied populations of Adonis vernalis delivered pollen with signifi cantly lower viability and potential for effective fertilization with pollen tube formation.The germination of pollen grains was as low as 16.2 -27%.
The lowest size of pollen grains was found in the Łabunie, Chomęciska Małe and Wólka Leszczańska populations (Tab.4).An average P diameter ranged 20.5 -21.5 μm and E diameter 17.8 -19.0 μm.The biggest pollen grains were found at the Pliszczyn site (P = 25.6 μm, E = 22.9 μm).A lot of pollen grains from Wólka Leszczańska and Stawska Góra were mis-shapen, degenerate, which was probably associated with their high sterility.

DISCUSSION
The paper undertakes an attempt to fi nd the possible factor which caused the signifi cant limitation in abundance of Adonis vernalis plant populations at the chosen stations from the Lublin Upland.The decrease in individuals, compared to data published by F i j a łk o w s k i (1961), was found to be noticeably important in the case of four out of six studied populations.Thus, it is highly important to emphasize the necessity of Adonis vernalis conservation.The relatively stable structure of individuals in the populations from Pliszczyn and Kąty shows that they found favorable conditions for development within the covered areas.
In principle, a correlation between the external factors and land management at the stations with Adonis vernalis, as well as the quality and quantity of pollen delivered and the population size and abundance, was confi rmed.
J a n k o w s k a -B ł a s z c z u k (1995) reported that Adonis vernalis is a perennial that reproduces mainly generatively.In our study we estimated only the potential for generative reproduction on the basis of pollen grain features.It is widely accepted that biological features of pollen infl uence sexual reproduction (e.g.Ś n i e ż k o , 1991).Our results corroborate the opinion of other authors (e.g.M u l c a h y and M u l c a h y , 1987) that external factors infl uence the functionality of pollen.We found the biological value of pollen and its abundant mass delivered by anthers to be satisfactory only in fl owers from the vital populations at Pliszczyn and Kąty.Additionally, both populations had fl owers with the most satisfactorily developed androecium with high effi ciency of archesporial tissues.The xerothermic swards there were regularly burnt and represent good fragments of thermophilous vegetation.Unfortunately, the other populations at Chomęciska and Wólka Leszczańska with spring burning and shrub succession under control had individuals with the androecium delivering pollen of low quality and quantity.Possibly the differences found are more in connection with some external factors than grass burning practices.
The station at Stawska Góra was an example of degraded grasslands, invaded by the expansion of different shrubs, mainly Prunus spinosa, Frangula alnus, Rhamnus cathartica and Cornus sanguinea.Adonis vernalis there is in strong competition for light and probably water supplies which may infl uence the pollen production of halophilous taxa.Similar conditions in the Łabunie reserve with secondary succession changed the xerothermic sward into a light pine forest.The lack of light seems to be a limiting factor for Adonis vernalis development there.

Fig. 4 .
Fig. 4. The average time of Adonis vernalis L. blooming depending on the location in Lublin Upland.Stages of blooming beginning full end

Table 1
Characteristics of chosen stations with Adonis vernalis L. in Lublin Upland.
The features of androecium and the mass of pollen delivered by different populations of Adonis vernalis L. means within columns referring to the same feature indicated with the same index are not signifi cantly different at α = 0.05 The size of pollen grains of Adonis vernalis L. from different stations in Lublin Upland.
** pollen germination in relation to viability in %

Table 3
The biological value of pollen in fl owers from different populations of Adonis vernalis L.
). CONCLUSIONS 1.The abundance and size of Adonis vernalis populations in the xerothermic patches of the Lublin Upland have decreased signifi cantly in the last 40 -50 years.The threat of their extinction in the studied area is real.2. The populations of Adonis vernalis differ in androecium development, the mass of pollen delivered by anthers and fl owers, the biological value of pollen and the size of pollen grains.3. Grass burning did not cause a reduction of potential biological value of Adonis vernalis pollen.The xerothermic swards in Pliszczyn and Kąty regularly burnt developed strong, most abundant populations of Adonis vernalis with favorable pollen.4. The development of the shrub layer, if it is out of control, stands in the way of satisfactory reproduction of halophilous Adonis vernalis.The biological value of pollen decreased signifi cantly in every population with a considerable light shortage.5. Thermophilous vegetation of the Lublin Upland deserves conservation as a source of rare and protected taxa.