REVISION OF NYMPHAEA CANDIDA RANGE – NEW DATA ON THE DISTRIBUTION AND HABITAT PREFERENCES OF THE SPECIES IN SOUTHERN POLAND

The paper presents results of geobotanical and taxonomic studies on the distribution and habitat requirements of Nymphaea candida in southern Poland. The researches were conducted in southern Poland in 2003-2009, in the provinces of Lower Silesia, Lublin, Małopolska, Opole, Silesian province as well as, in southern parts of Mazowieckie and Lubuskie. Flowers, leaves and fruits of Nymphaea species were collected from 27 locations. Altogether pollens from 73 populations of N. candida and 18 of N. alba from all the researched area were measured. The trophic level of an ecosystem was evaluated according to the results of the total nitrogen, total phosphorus, chlorophyll a, transparency and biological parameters. As the result of the studies of more than 200 water bodies, 57 localities of N. candida were documented within the investigation area. The populations of N. candida occupy mid-forest water bodies and river ox-bow lakes. A 998nowak_kopia:ASBP310W.qxd 2010-12-19 23:26 Strona 333


INTRODUCTION
Nymphaea candida C. Presl. is an Euro-Siberian element of aquatic flora with the western range limit on the line of the Rhine (Hegi 1965;Meusel et al. 1965;Hulten and Fries 1986;Muntendam et al. 1996).According to Wayda (2000), N. candida occurs in Poland only in the northern part of the country reaching there the southern limit of its general continuous range.However, the species was also reported from the Czech Republic (Hejný and Slavík 1997), southern regions of Germany (Benkert et al. 1996), eastern France, Switzerland, south-western Romania, Austria, Hungary and former Yugoslavia (Tutin et al. 2002;Muntendam et al. 1996).The general distribution map of the species in Europe is presented in Fig. 1.Considering the fact that aquatic plants are distributed widely and limited mostly by climatic zones, a distribution gap in southern Poland is hardly possible to really exist.Moreover, N. candida was reported from southern Poland by Polish and German botanists in the 19th and early 20th centuries (detailed list of papers cited in Results).Unfortunately, the herbarium materials documented these localities and deposited in WRSL were destroyed during the Second World War, so they could not be re-examined.It is worth mentioning that the data published by German botanists were based on careful plant examinations.Moreover, most of their floristic reports have been later confirmed, so they are regarded as a credible source of botanical information.Nevertheless, these data were questioned by Wayda (2000), and in result not included in the "Atlas of Vascular Plants of Poland" (Zając and Zając 2001).On the other hand, the occurrence of N. candida in the area of Silesia and the Pojezierze Łęczyńsko-Włodawskie Lakeland was mentioned by Kłosowski (2001) in the "Polish Red Data Book of Plants".Localities of the species were also reported from the southern part of Lubuskie province by Kujawa-Pawlaczyk and Pawlaczyk (2003) and from Lower Silesia by Bobrowicz and Konieczny (2000).Authors of the present paper have found the individuals of water lily in southern Poland, identified in the field as N. candida, based on the botanical key of Rutkowski (1998) or Kubát (2002).Further investigations proved, that N. candida occurs also in other regions of central and southern Poland (Nobis 2007;Nowak and Nowak 2007).RECOGNITION N. candida is closely related to N. alba L., with many varieties and subspecies described (Glück 1924;Heukels and Van der Meijden 1990).Despite the fact that the species has been extensively investigated (Glück 1924;Neuhäusl and Tomšovic 1957;Radics 1967;Casper and Krausch 1981;Jones and Clarke 1981;Muntendam et al. 1996), its identification still brings confusions.Most useful for identification both of the species during the field studies is the number of stigma rays.In the case of N. candida, the stigma consists of 9-14 rays, whereas in the case of N. alba between 15 and 25 (Muntendam et al. 1996).Several specimens with 12-13 stigma rays were hardly possible to determine according to pollen diameter or exine shape.Surelly these cases are rather hybrids of both species.The individuals collected by the authors of this paper in southern Poland have usually had 9 or even 7 carpellary teeth in stigma.Also the equatorial diameter of pollen grains seems to be a comfortable feature in some cases, however these analyses are possible only in the laboratory.The highest values of the equatorial length of N. alba pollen do not exceed 42 µm.The pollen of N. candida are in general larger than those of N. alba (Muntendam et al. 1996).
The morphologically closest to N. candida is N. alba var.minor DC.It is a starvation form differing from typical specimens of Nymphaea alba only in size.Recently, this form has been reported from Opole Silesia (Spałek 2007), but with no taxonomical evidences and herbarium documentation.According to Oberdorfer (1994) and Muntendam et al. (1996), the occurrence of N. alba var.minor is restricted to colder, northern regions of Europe.
A separate taxonomical problem related with Nymphaea candida is the possible hybridisation with N. alba.Morphologically intermediate forms between these two species are known for a quite long time (e.g.Conard 1905;Glück 1924).Also during N. candida survey in southern Poland such intermediate forms were collected.Molecular studies on hybrid specimens of N. alba and N. candida are planed in the future.
The Nymphaea genus is also of considerable importance from the conservation point of view.Both species are legally protected in Poland (Rozporządzenie Ministra Środowiska 2004).As a taxon with restricted range in Poland N. candida was categorised as a vulnerable species in the Polish Red Data Book of Vascular Plants (Kłosowski 2001).So, it is important to have precise information about the distribution of both species to conduct the effective management and conservation.
The successive aims of the present study were: 1) to provide old, published floristic data on the distribution of N. candida in southern Poland; 2) to present the current distribution of the species in the discussed area; 3) to stimulate further molecular researches on infraspecific hybridisation in the genus in the area of co-occurrence of the two species representing the genus Nymphaea; 4) to provide data on habitat preferences of N. candida from selected localities, and finally 5) to discuss the present threat status of N. candida in Poland.

MATERIALS AND METHODS
The field studies were conducted in southern Poland in 2003-2009 (Figs 1 and 2).Flowers, leaves and fruits of the Nymphaea species were collected from 27 locations, mainly in Opole Silesia in July and September 2008 on the allowance DKFOPPogiz-4211/I-62/1887/08/ep issued by the Polish Minister of Environment.The plants were collected during warm days between 10 am. and 6 pm.The fresh material was identified and described in the field using the botanical key of Rutkowski (1998).
On the basis of botanical keys by Rutkowski (1998), Kubát (2002), Oberdorfer (1994), Hejný and Slavík (1997), as well as regarding the research works of Muntendam et al. (1996) and Wayda (2000), the following fifteen features were chosen to describe the specimens: the diameter of the stigma (column 1 in Table 1), the number of carpellary teeth (2), the position of the flower on water (3), the shape of the flower (4), the side view of the flowerbase (5), the underside view of the flowerbase (6), the stigma surface structure and colour (7, 8), the diameter of the flower (9) vertical/erect sepals (10), the colour of the underside leafblade (11), the nervation of the leafblade (12), the direction of the main nerves leafslips (13) and the proportion of the stigma to the width of the ovary ( 14) and for some specimens also the pollen diameter and pollen exine morphology (values given in the distribution list).All collected individuals were determined in the field with careful measurement of the chosen features.Altogether, 64 flowers, 70 leaves and 15 ovaries belonging to 62 specimens were collected and checked.The average stigma diameters, the number of carpellary teeth and the flower diameter were compared to the values of N. alba.Pollen diameter was measured using the Olympus BX41 microscope with objective UPlan SA 100× 00/017/FN 26.5 and photographed by the Olympus Camedia camera C3040 200M.Altogether pollens from 73 populations of N. candida and 18 of N. alba from all the researched areas were measured.Half of them was sampled from fresh material, another half from herbarium collections (WRSL, KRA).The collected material was stored in the herbarium of the Division of Geobotany and Plant Conservation of the Opole University (OPUN).
The trophic level of an ecosystem is evaluated according to the results of the total nitrogen, total phosphorus, chlorophyll a, water transparency and biological parameters (Dodds et al. 1998).However, it is generally admitted that phosphorus plays an important role in the development of aquatic plants and, in most cases, is a limiting factor of eutrophication in temperate lakes (Parinet et al. 2004).In order to estimate the status of investigated water bodies, the Trophic State Index (TSI) was calculated according to Carlson's equations (Carlson 1977):

TSI =
The TSI values, constructed on a scale of 0 to 100, indicate the trophic level of ecosystems from oligotrophic (<40) to eutrophic (60-100).To minimize the sampling mistake the mean value of three indices (TSI TP , TSI Chl and TSI SD ) were taken while determining the trophy level (Pełechaty et al. 2007).Also the reference classification adopted by OECD was taken into account (OECD 1982;Solheim 2005).
The endanger status of N. candida in southern Poland was evaluated according to the IUCN categorization method (IUCN 2001).All doubtful specimens, probably hybrids, were excluded from the analyses.Only evident individuals of Nymphaea candida were taken into account when generating map and listing the location of the species.
The average values of the pollen diameter of both species were compared using the t test for independent variables.

THE DISTRIBUTION OF NYMPHAEA CANDIDA IN SOUTHERN POLAND
All localities of N. candida from southern Poland published before 2001 are listed below.Additionally, new records as well as old localities which have been confirmed recently (after 2001) in the field are also presented (Fig. 2).

MORPHOLOGICAL FEATURES OF THE COLLECTED SPECIMENS
The field and laboratory measurements of the collected 62 specimens are presented in Table 1.Altogether 64 flowers, 70 leaves and 15 ovaries were collected and determined.The three most diagnostic characters were compared in Table 2 with values known for Nymphaea alba (Nowak and Nowak 2007).
The mean value of the stigma diameter for the collected flowers and ovaries is ca.5.87 mm ranging from 4 to 13 (Table 2, Fig. 3).The average number of carpellary teeth meets perfectly the strict N. candida range of this features and counts ca.9.29, ranging from 7 to 12 (Table 2, Fig. 4).Also the flowers of the verified specimens were small with the diameter restricted to the N. candida profile.The average value of this characteristic was 5.95 cm ranging from 4 to 8.5 (Table 2, Fig. 5).
The pollen diameter was measured for 98 specimens representing 73 populations of N. candida and for 31 specimens representing 18 populations of N. alba.The average value of the parameter was for N. candida 43.95 µm and for N. alba 33.63 µm (Table 2,.The pollen diameter for both species has statistically different values (p<0.001;Fig. 8).

HABITAT PREFERENCES OF NYMPHAEA CANDIDA C. PRESL IN SOUTHERN POLAND
The populations of N. candida in southern Poland occupy mainly water bodies especially surrounded by forests, but in some cases also river ox-bow lakes within the meadow-field landscapes.The most suitable habitat conditions for this species occur in shallow waters in the shore zone with the amplitude of the water column fluctuating from 0.5 to 2 m.Populations of N. candida develop on open water or close to the rushes with high insolation rate.Higher shading influences directly on the decrease of density and cover rate of the species.In thick reeds or high rushes the species occurs sporadically with a low population size.
The significant number of these populations were found in artificial reservoirs -fish ponds or forest ponds.Regarding the trophy level N. candida occupies different habitats, mainly mesotrophic but also eutrophic with high content of organic matters (Table 3).

THE STATE OF THREAT OF NYMPHAEA CANDIDA IN SOUTHERN POLAND
To show the conservation state of N. candida in southern Poland, the number of localities for separate regions were compared (Table 4).

DISCUSSION
During the presented studies the extent area of southern Poland was surveyed aiming to solve the distribution problem of N. candida and N. alba.Both species of water lily are closely related and many morphological features overlap without any distinct differences.However, the number of carpellary teeth and the pollen diameter seems to be the most useful characteristic features for recognition of the two species as it hardly overlaps (Muntendam et al. 1996).The most useful diagnostic feature, even during the field studies, is the number of carpellary teeth, which is countable in flower as well as in the ovary.
Using the current botanical keys we come to the conclusion that, with no doubts, Nymphaea candida occurs in south Poland.Moreover, the preliminary analyses of the rarity of both species show, that N. candida is quite frequent.The number of carpellary teeth, which should not exceed 14, was often below 10.The mean value for the examined specimens was 9.29 (from 7 to 12).The mean value of pollen diameter was 43.95 µm (the maximum value for N. alba doesn't exceed 42 µm) and in some cases even more than 50 µm (see the distribution list and Figures 6 and 7).Thus, all the individuals suited the range designated for N. candida by Muntendam et al. (1996), Wayda (2000), Rutkowski (1998) and others.Also the other countable features, like the diameter of the flowers (5.95 in average) or the proportion of the stigma diameter to the ovary one (mean value 0.6) evidently indicated N. candida (Table 1 and 2).Almost all plants had cup-shaped flowers, the   square-shaped flowerbase (sometimes with a rough edge), the orange stigma, and the flower partially submerged in the water with erected sepals.Leaves were in the majority red or red-green on the bottom side with converging nerves of leafslips.
The results of the study in comparison to the recent researches on N. alba (Nowak and Nowak 2007) revealed the considerable differentiation among Nymphaea species.Remarkable differences were found within some diagnostic features, such as the stigma diameter (5.95 mm for N. candida and 13.4 mm for N. alba) and the number of carpellary teeth (respectively 9.29 and 16.1).At the same time, the flower diameters also differ significantly reaching 5.95 for N. candida and 11.2 for N. alba (Table 1).The most important difference is within the value of pollen diameter, which has the average 43.Former habitat analyses show that N. candida prefers mesotrophic, clear waters with mineral-organic substrates (Szańkowski and Kłosowski 1999;Kłosowski 2001).Some reports conclude that also eutrophic water reservoirs could give suitable conditions for development of N. candida populations (Tomaszewicz 1979).In the Czech Republic N. candida was found in varied types of water, among others in meso-and oligotrophic fish ponds, oxbow lakes and bog ponds (Tomšovic 1997).Within the researched area the species were noted in waters of intermediate character in terms of trophy level, between the values for meso-and eutrophy.Only in three sites populations of N. candida were found in evidently mesotrophic waters and in two cases in eutrophic.A slight shifting of the trophy level in the mesotrophy direction for the analysed samples is detectable when investigating the chlorophyll a.This analyse classifies the waters generally to mesotrophy and in the case of the Staw Nowokuźnicki pond even to oligotrophy (OECD 1982).Thus, we found the species able to thrive in poor waters of small, mid-forest ponds or bog ponds as well as in rich with mineral substances waters of fish ponds or ox-bow lakes.Such adaptation potential significantly broadened the habitat amplitude of N. candida in southern Poland.
However, in comparison to N. alba habitats, it seems to be sure that N. candida occurs in waters of slightly lower trophy level, what was suggested earlier by Tomaszewicz (1979).
The outcomes of the conducted researches have considerable meanings for the phytogeography of the Nymphaea genus in Central Europe.They fill up the distribution gap in the southern part of N. candida range in Europe and definitely explain and prove the results of former researches.The continuous distribution of N.candida in southern Poland is also well-founded in climatic and ecological conditions (see Kłosowski 2001).With no doubts the south part of Poland is the transition area where the distribution ranges of both species overlap.Existence of hybrids within the genus could cause the morphological characters very unstable and labile making the easy determination of the species, its hybrids or introgressive forms hardly possible.Further cytological and genetic researches of different specimens of Nymphaea genus in southern Poland should be conducted to solve the problem of genetic flow between the populations of both relatives and their hybrids.
The taxonomic and chorological surveys in neighbouring areas should be attentively monitored.Some interesting new data on the distribution of N. candida were published for the Saxony region in south-east Germany.The revision of the collected specimens and new field studies changed the regional range of the species, considerably diminishing the distribution acreage and splitting it into some small patches (Otto et al. 2000;Otto 2004).Thus, there is a question whether the natural south-western range of N. canidida in Germany, Austria, the Czech Republic, Switzerland and Poland is continuous or patchy due to uncomfortable ecological conditions, e.g.too high elevation or a lack of suitable waters.This situation is possible for some areas in south Poland where we found scarce number of the N. candida population (e.g.Małopolska).

THE STATE OF THREAT OF NYMPHAEA CANDIDA IN SOUTHERN POLAND
The detailed study on the taxonomy and distribution of the species within the Nymphaea genus is obviously indispensable tool in proper management and effective conservation of both taxa (comp.Nowak and Nowak 2004).Without precise data relating to chorology, dynamic and range changes of these plants the legal protection of water lilies could be ineffective.
In the neighbouring countries N. candida is considered as a taxon with different dynamic and endangerment status.In the Czech Republic it was categorized as a critically endangered (Procházka 2001).Generally, in the area of Germany it is regarded as an endangered but in Bavaria as a critically endangered (Korneck et al. 1996).In the last edition of the Polish Red Data Book of Plant, Kłosowski (2001) treats N. candida as a vulnerable.Surprisingly, the species has not been red listed in Poland (Zarzycki and Szeląg 2006).In separate regions of southern Poland, N. candida was evaluated against criteria of IUCN and was classified as a vulnerable taxon in Opole Province (Nowak et al. 2008).
It is hardly possible to precisely determine the real degree of endangerment in the whole country using only informa- tion from southern regions of Poland.Because of the taxonomical discrepancies there is also a lack of credible data in some regions.So, it seems to be indispensable to verify the synanthropodynamic situation of N. cadida as well as N. alba in whole Poland.
The comparative analyses within the different time periods for separate regions of southern Poland show, that for some provinces N. candida increases the number of locations.Inconsiderable decrease is observed in case of Lower Silesia, much worse is the situation in Upper Silesia (Table 4).Even worse is the situation in Lubelskie voivodeship, however, there are some additional information relating to pollen findings of N. candida in several lakes, which were not included in presented assessment (see Bałaga et al. 2002;Bałaga 2007a, b).In some regions (e.g.Podkarpacie) the species was found for the first time.However it does not prove its expansive properties, but rather reflects the intensification of floristic surveys and shows the potential of N. candida to settled in mountainous areas.It is also worth to mention that in some regions (north-eastern part of the Małopolska Uppland or Opole Silesia) the species is more frequent than N. alba (comp. Nobis 2007).
According to the IUCN guidelines to species assessment (IUCN 2001) the data gathered during the presented study do not allow to consider N. candida as a vulnerable species in Poland.Still existing populations known from the XIX century, numerous stable locations and abundant populations in some regions at present suggest that the species should be regarded as a least concern (LC).Also the analyze of the habitat accessibility, a biotope extent, an ecological amplitude against the trophy level and direct human impacts support this categorization.Nevertheless it is probable, that in some regions the species could have the considerable withdrawing tendency and has to be red listed in higher categories.

Fig. 1 .
Fig. 1.The distribution range of Nymphaea candida in Europe with the investigation area.

Fig. 2 .
Fig. 2. Distribution of Nymphaea candida in southern Poland on the background of ATPOL grid. 1 -locations known before the year 2001; 2 -locations known before the year 2001, documented with herbarium specimens verified according to methods described in the paper; 3 -locations noted after 2001.
Fig. 3. Values of stigma diameter of N. candida specimens.
Fig. 7. Pollen diameter of N. candida specimens.Fig. 8. Results of comparison of pollen diameters for N. candida and N. alba checked by t test.

TABLE 1 .
Sampled specimens with their location and morphological characteristic.

TABLE 2 .
Mean values of quantitative features for Nymphaea candida and N. alba.

TABLE 3 .
Habitat characterisation of the surveyed population of Nymphaea candida Explanations: E -conductivity; T -temperature; TP -total phosphorus content; Chl -chlorophyll A content; TSI -Carlson Trophy Index

TABLE 4 .
The endangerment status of N. candida in south Poland.