Morphological variation in genus Nymphaea L. (Nymphaeaceae Juss.) of the European Russia

Polina A. Volkova, Alexey B. Shipunov

Volkova P.A., Shipunov A.B. Morphological variation in genus Nymphaea L. (Nymphaeaceae Juss.) of the European Russia // The materials of the White Sea Expedition of Moscow South-West High School. Vol. 6 [Electronic resourse]. 2006. Mode of access: http://herba.msu.ru/shipunov/belomor/english/2006/nymph.htm

Abstract

Detailed investigation of the morphological variability of Nymphaea species was developed on 48 populations in the European Russia. We studied both macromorphological characters in field (including analysis of leaf shape with the geometrical morphometry) and palynology. Only one polymorphic species, N. candida, growths in the most part of European Russia. Nymphaea tetragona is absent on the investigated territory, and N. alba have been found only in Astrakhan region (Volga river mouth). These three species are separated by several morphological characters of fresh plants. Nymphaea tetragona differs from N. alba and N. candida on the sculpture of exine of proximal part of pollen grains. Size characters of Nymphaea leaves and flowers do not depend on organic content in water.

Introduction

Genus Nymphaea (white water-lilies) is the taxonomically difficult group; different taxonomists distinguish from two (Uotila 2000) up to 12 species (Heslop-Harrison 1955, Papchenkov 2003) of white water-lilies on the territory of the European Russia. In addition, many species are believed to have numerous subspecies, chromosomal races, forms of hybrid and artificial origin (Heslop-Harrison 1955, Uotila 2000, Papchenkov 2003). This ambiguous situation is caused by high level of interspecific polymorphism (Komarov 1937, Heslop-Harrison 1955, Kupriyanova 1976) with poorly investigated nature and intensive interspecific hybridization, which is pointed by many authors (Heslop-Harrison 1955, Uotila 2000, Papchenkov 2003) and, however, remains lacking of strong evidence.

Nymphaea species have high morphological plasticity. Size of leaves and flowers and also some qualitative characters of flowers are thought as strongly depended on hydrological (especially temperature) and edaphic conditions (Heslop-Harrison 1955, Kupriyanova 1976, Dubyna 1982). However, quantitative estimation of edaphic conditions was not performed in the abovementioned studies. Size of different parts of Nymphaea species also depends on age of the plant (Gluck 1924 as cited in Muntendam et al. 1996, Dubyna 1982).

The most common opinion is that there are three Nymphaea species in Russia: N. alba L., N. candida Presl. and N. tetragona Georgi. Nymphaea alba is spreaded across all European Russia, N. candida growths both in the European Russia and Siberia, and N. tetragona occurs in Siberia, in the Russian Far East and in the Kola peninsula (Komarov 1937; Muntendam et al. 1996). Similar values of qualitative diagnostic characters are pointed out in all analyzed sources, so in theory, these characters are able to distinguish these three species (Table 1). Unfortunately, many main diagnostic characters (e.g. color and shape of the stigma, shape of cup base etc.) change or disappear even after very careful pressing in herbaria (Lisitsyna 2003). Moreover, the differences in the leaf blade shape, which appears to be a frequently used diagnostic character (Komarov 1937, Lisitsyna et al. 1993, Uotila 2000), before and after herbarization are very similar with interspecific differences, i.e. leaves with leaf blade shape which is typical for N. alba obtain more typical for N. tetragona shape after the herbarization (Volkova 2007). That is why it is so important to investigate morphology of white water-lilies on the fresh material (Uotila 2000).

Table 1. Main diagnostic characters for distinguishing Nymphaea species in Russia (data from literature)

Character N. alba N. candida N. tetragona
Shape of cup base round rounded-quadrangular quadrangular with prominent ribs
Shape of filaments of inner stamens linear lanceolate oval
Sculpture of pollen grains exine baculums verrucas granular
Number of stigma lobes (7) 8-20 (23) 6-14 (20) (4) 5-10 (16)
Shape of stigma disc almost flat (slightly concave) strongly concave strongly concave
Color of stigma disc yellow yellow, orange, red yellow, red, purple
Shape of the central stigma projection short spherical long conical long conical
Flower diameter (cm) (3) 5-15 (20) (3) 5-11 (16) 3 (and less) –6 (10)
Ovary appearance does not become narrower near stigma, covered up to the top with scars of fallen stamens become narrower near stigma, is not covered up to the top with scars of fallen stamens become narrower near stigma, is not covered up to the top with scars of fallen stamens
Bud shape oblong-ovoid with obtuse top oblong-ovoid with acute top four-sided pyramid
Leaf shape widely-elliptic either rounded-ovate or rounded widely-elliptic either rounded-ovate or rounded elliptic either rounded-ovate or rounded
Shape of main leaf veins almost straight bent along the full length bent only in the first third of the length
Length of the leaf (cm) (10) 15-30 (35) (6) 12-26 (30) (4) 5-9 (20)
Width of the leaf (cm) (8) 14-27 (35) (8) 12-24 (30) (3) 4-10 (16)

However, there is the opinion that N. candida and N. alba can be distinguished with certainty only basing on size, shape and exine sculpture of pollen grains (Kupriyanova 1976, Muntendam et al. 1996, Uotila 2000). L.A. Kupriyanova (1976) showed that Nymphaea pollen grains in the European part of the USSR are characterized by high morphological stability and can be used for distinguishing species; this opinion based mainly on the exine sculpture. Interspecific hybrids of Nymphaea are characterized by lower fertility (Komarov 1937, Heslop-Harrison 1955) and various morphology of pollen grains (Kupriyanova 1976). One should note that these investigations were conducted on the small samples (1-3 flowers per species), while there is large variance of the palinomorphology even within one Nymphaea population (Volkova 2007), which let us consider these studies as preliminary ones. Investigations of J.B. Muntendam with colleagues (1996) were carried out on the SEM-micrographs; this method does not let estimate correctly the shape and the sizes of intact pollen grains because of their deformation during scanning microscopy in the high vacuum (Volkova 2007). This is almost all existing literature on diagnostic palinomorphology of European Nymphaea species, as far as we know, although pollen characters are mentioned in many wide-spread keys and proceedings as diagnostic ones (e.g. Komarov 1937, Dubyhna 1982, Tzvelev 2000, Uotila 2000). In contrast to the previous investigations, V.A. Poddubnaya-Arnoldy (1967) noticed that structure, size and shape of pollen grains can vary significantly within one species, although these are diagnostic characters.

Materials and methods

Materials

Topographically separated group of white water-lily plants were treated as a population; and distinct group of leaves and flowers were treated as one plant (recognizing of water-lily plants is often damaged by the active branching of the underwater rhizomes and their frequent defragmentation).

We investigated 48 populations of white water-lily from 47 reservoirs and rivers in Karelia republic, Moscow, Tver, Kaluga, Chelyabinsk, Lipetsk and Astrakhan regions of European Russia (Fig. 1). We also investigated three populations of N. tetragona on the shore of lake Bajkal (Western Siberia), situated close to the type location of this species (Krupkina 2001), and two populations of N. alba s.l. from there for comparison with populations from European Russia. Data were collected from June to September in 2003-2005.

Figure 1. Collection sites for the investigated populations. Population numbers refer to the Table 2. Investigated populations in Western Siberia (shore of the lake Bajkal) are not shown. 1—Karelia republic (populations number 120, 130-138, 206-211); 2—Tver region (101-116); 3—Moscow region (117, 118); 4—Moscow region (119); 5—Kaluga region (128, 129); 6—Lipetsk region (127); 7—Chelyabinsk region (121); 8—Astrakhan region (213-217).

Table 2. Investigated populations of Nymphaea spp.

population number name of stream or reservoir saprobity index1 proportion of fertile pollen grains (%)2 class of water purity1 type of the exine sculpture3
14. Karelia republic, Loukhi region
120 Lake Indigo 0.68 … 2 4
130 Lake Tikhoe 0.8 80 2 1
131 Lake Gamarbiya 0.75 … 2 1
132 Peatbog near Lake Gremyakha … 89 … 2
133 Lake Sennoe 0.85 … 2 2
134 Lake Evrika 0.7 100 2 2
135 Lake Speloe 0.7 93 2 4
136 Lake Kh 0.65 38 2 2
137 Lake I 0.75 98; 88 2 2
138 Lake La 0.78 94 2 1
206 Riv. Sinyaya … 97 … 2
207 Lake Verkhnyaya Pazhma, western part … 98; 93 … 2
208 Lake Verkhnyaya Pazhma, eastern part … 82; 88; 99 … 4
209 Mouth of riv. Nol'ozyorskaya … 99 … 2
210 Head of riv. Nol'ozyorskaya … … … …
211 Lake Tajnoe … 98 … 1
2. Tver region, Udomlya environs
101 Lake Zaverkhov'e 1.7 100 3 4
102 Lake Matras 1.4 100 2 2
103 Lake Klin 1.24 … 2 …
104 Lake Borovno 1.4 … 2 2
105 Lake Belen'koe 1.4 100 2 4
106 Lake Golovets 1.87 95 3 3
107 Lake Perkhovo 1.4 … 2 2
108 Lake Moldino, eastern part 1.6 … 3 1
109 Lake Moldino, north-eastern part 1.6 … 3 4
110 Lake Moldino, central part … 99 … 2
111 Lake Rogozno … 33 … 4
112 Lake Turishino … 99 … 2
113 Lake Soroka 2 5 3 4
114 Lake Volkovo 1.5 97 3 4
115 Lake Pisoshno 1.4 95 2 4
116 Lake Pochaevo 1.5 63 3 2
3-4. Moscow region
117 Pond Sterlyazhij near Zvenigorod 1.8 77; 87 3 4
118 Lake Sima near Zvenigorod 0.95 … 2 2
119 Pond near tourists base near Mozhajsk 1.4 95 2 2 and 3
5. Kaluga region, Mosal'sk district
128 Riv. Ressa, upper stream 1.5 100 3 1
129 Riv. Ressa, lower stream 1.7 100 3 1
6. Lipetsk region
127 Lake Mokhovoe, near Lipetsk … 91 … 2
7. Chelyabinsk region, Bredy district
121 Riv. Karaganka 1.9 93 3 2
8. Astrakhan region, delta of the Volga river
213 Erik Finogenov … … … …
214 Erik Volodarovskij … 98 … 1
215 Kultuk Pryamoj Lotosnyj, northern part … 95 … 1
216 Kultuk Pryamoj Lotosnyj, central part … 97 … 1
217 Krep' Blinovskaya … 98; 100 … 1
9. Western Siberia, near the Lake Bajkal
201 Lake near road Irkutsk—Ulan-Ude, 905 km … … … 3
202 Ponds of Bajkal pulp and paper plant … 80 … 2
203 Lake near road Irkutsk—Ulan-Ude, 202,5 km … … … 3
204 Peatbog Leshkovskoe … 96 … 3
205 Ponds near railway station Slyudyanka … 93 … 2

1 -- according to Sladechek, 1967
2 -- data for several flowers from the same population are separated with semicolon
3 -- proximal part of the pollen grain was investigated, for exine types description see Results
4 -- number of region corresponds to the Fig. 1

We tried to investigate not less then 15 plants per population; however, there were populations with fewer plants, so 344 plants were investigated in total. Six qualitative and six quantitative characters (which are mentioned as diagnostic in most keys and proceedings) were observed for each plant (Table 1, Fig. 2). The amount of organic in the water were estimated via saprobity index, which was determined from list of indicator species of diatoms and their abundance in the water sample (Sladechek 1967).

Figure 2. Investigated quantitative characters of Nymphaea leaves and flowers

 
A — Leaf: AC—width, DE—length, BD—position of the maximum width. B — Flower: A—diameter of the circle of outer stamens, B—length of the outer petal

Analysis of the leaf shape

Thin-plate spline (TPS) method of geometrical morphometry (Bookstein 1991, Adams et al. 2004; Shipunov & Bateman 2005) was used for investigations of the variability of leave shape in Nymphaea. This method lets us directly explore shapes, excluding size factor, with usage of landmarks situated on the contours. In general, contour of one fresh leaf (maximum leaf) from one plant per population was outlined. In addition, contours of all leaves from 11 plants from different populations were also obtained to investigate whether leaf shape depends on leaf position on the rhizome. Contours of N. candida and N. tetragona leaves from “Flora Nordica” (Uotila 2000) and from “Illustrated Flora of Northern US and Canada” (Britton & Brown 1913) were used for the reference as “anchors”.

To digitize contours, we used 100 equally spaced landmarks (first landmark located on the base of leaf). This algorithm of producing (pseudo)landmarks let us compare objects shapes without getting any information about biological sense of observed differences (Kores et al. 1993), which entirely fits our aim. The coordinates of the landmarks were written in the data file with help of screen digitizer tpsDig (Rohlf 2006). The coordinates of consensus configuration and also the values of main, relative and partial warps, characterizing the degree of differences between the specimen and consensus configuration were calculated with help of program tpsRelw (Rohlf 2007), which realizes the idea of geometrical morphometry in prototype of primary component analysis. Original coordinates were normalized by Procrustes fit method (alpha = 0).

We investigated the dependence of leaf shape on its position on the rhizome with help of tpsRegr (Rohlf 2005) software; the fit to the regression model was tested using a generalization of Goodall’s (1991) F-test. Averaging of leaves blade shape was done with help of program tpsSuper (Rohlf 2003). Data files were edited and converted with help of auxiliary program tpsUtil (Rohlf 2000).

Pollen morphology

Our preliminary studies have shown that acetolysis treatment of pollen grains changes significantly neither pollen size, nor character of exine sculpture (Volkova 2007). That is why all the investigations of pollen with light microscopy were carried out on intact (unacetolysed) pollen from herbarized flowers. Pollen was measured using MIKMED-1 light microscope (magnification 1.5×15×40) with ocular micrometer to within 1 mkm. We measured not less then 10 pollen grains in polar view per flower (564 pollen grains were measured in total). Light microscopy does not let us place most of investigated samples into one of the traditionally distinguished exine-based pollen types (Kupriyanova 1976, see also Table 1). That is why we continued investigation of exine sculpture on the scanning electron microscope (Camscan S-2, accelerating voltage of 20 kV, hereafter SEM). Pollen of several specimens from herbarium (MW) from typical localities was also investigated under SEM for the reference. Pollen grains were coated with gold-palladium arroy (approx. 25 nm thick). We made photos of first 3-10 pollen grains that were seen in the appropriate position (polar view or equatorial view) using software Scan Microcapture 2.20119, designed by A.V. Grigor'ev (421 pollen grains in total).

Pollen fertility was assessed on herbarized flowers by the acetocarmin staining method (Radford et al. 1974) on 100 pollen grains per flower. All unstained or faintly stained pollen grains were considered sterile (Table 2).

Statistical data analysis

Four morphotypes (hereafter “anchor specimens”) which correspond to theoretical plants of N. alba s. str., N. candida, N. tetragona and N. × sundvickii (N. candida × N. tetragona hybrid), were “simulated”, based on the common conceptions about morphology of these taxonomical units (Table 1).

We used multivariate analysis of variance, nonparametric correlation analysis, nonparametric Wilcoxon test and parametric Student test for detection differences and connections between variables. Shapiro-Wilks test was performed to test normality. Kruskal non-metric multidimensional scaling (hereafter MDS: Ripley 1996) of similarity matrices computed with “daisy” (Kaufman & Rousseeuw 1990) metrics was used for the classification based on the morphology; last method was specially developed for data with mixed type of variables (both continuous and discrete) such as our data. Principal component analysis (hereafter PCA) was used for classification of the leaves shapes (because in this case we deal with continuous variables of relative warps matrix, Pavlinov 2001). Dichotomous recursive classifications (Ripley 1996) were used for calculations of the typical character values during the preparation of the diagnostic key. All calculations and graphs creation were made in R environment for statistical computing (R Development Core Team 2005).

Results

Macromorphology: metric characters

Classification of investigated plants by MDS basing on their morphology put of N. candida “anchor” between of N. alba and N. tetragona “anchors”, and N. × sundvickii between N. candida and N. tetragona. This result corresponds completely to the common conception of the morphology of these taxonomical units and serves as evidence of adequacy of our classification (Fig. 3a). One can see three fuzzy “clouds” of plants on the plot of two first MDS dimensions. All plants from Astrakhan region (populations 213-217) and plants from three seaside populations from Karelia republic (populations 130, 131 and 211: Table 2) are agglomerated around N. alba “anchor”. These plants have typical for N. alba macromorphology (Table 1) with the exception for the rounded-quadrangular shape of flower base of Karelian plants. All plants of N. tetragona from the shore of the lake Bajkal (populations 201, 203 and 204) are concentrated around N. tetragona “anchor”. Other investigated plants are concentrated around N. candida “anchor” (Fig. 3a).

Figure 3. Multidimensional scaling of morphometric data for Nymphaea in European Russia. “Anchor specimens” are marked with black circles.

A. Each plant is marked by the number of the region (see Fig. 1, Table 2).

B. Each plant is marked by the symbol, according to the size of pollen grains:
filled circles—“small” pollen, filled triangles—“large pollen”, open circles—unknown pollen size.

Values of morphological characters which were typical for each of distinguished groups were calculated and used for the diagnostic key of three European Russian Nymphaea species. We used such simple and reliable characters as shape of the flower base, shape of filaments of inner stamens, number of stigma lobes, size of the leaf and flower size. This key is appropriate for distinguishing Nymphaea species in nature but not in the herbarium, which can be considered as weakness of the key, caused, however, by the peculiarities of our object.

Diagnostic key for the distinguishing of Nymphaea species in the European Russia (qualitative characters are illustrated on Fig. 4)

1. Cup base is square, with clear ribs (view from the peduncle). Filaments of inner stamens are rounded. Plants are small: length of outer petals does not exceed 3 cm, width of leaf does not exceed 9 cm ..... N. tetragona Georgi
--- Cup base is rounded or rounded-quadrangular, without clear ribs. Filaments of inner stamens are oblong. Plants are larger ..... 2

2. Cup base is rounded (view from peduncle). Filaments of inner stamens are linear. Leafs cover each other, raising themselves over the water. Number of stigma lobes exceeds 13. Length of the leaf exceeds 15 cm ..... N. alba L.
--- Cup base is rounded-quadrangulate, clear edges are never seen. Filaments of inner stamens are linear or lanceolate. Leafs are floating on the water. Number of stigma lobes is less then 13 cm, sometimes more but in this case length of the leaf does not exceed 15 cm ..... N. candida Presl

Figure 4. Qualitative characters, used for separation of three Nymphaea species in European Russia.

A-C — shape of flowers base (view from peduncle, not in one scale):

A.Nymphaea alba

B. Nymphaea candida

C.Nymphaea tetragona

D-F — shape of the filament of inner stamen (in one scale):

D. Nymphaea alba

E. Nymphaea candida

F. Nymphaea tetragona

Macromorphology: shape of the leaf

We can distinguish three poorly isolated plot zones as the result of classification of leaves with PCA, based on their shape (relative warps matrix, GM data) (Fig. 5, 6). Zone A consists only of plants from Astrakhan region whereas zone B consists of N. tetragona plants from lake Bajkal shore. Nymphaea tetragona “anchor” is situated in the central area of zone B. All other plants including N. candida “anchor” are situated between zones A and B.

Figure 5. Primary component analysis of relative warps matrix (geometrical morphometry data) for leaf contours of Nymphaea in European Russia. Each plant is marked by the number of the region (see Fig. 1, Table 2). "Anchor specimens" are marked with black circles. Plants from Astrakhan region ("A zone") and from Siberia ("B zone") are separated.

Figure 6. Averaged contours of leafs for three groups of leaves with different shape.

A — plants from Astrakhan region (Fig. 5, Zone A),

B — group of N. candida “anchor” (Fig. 5, central region),

C — plants from Siberia (Fig. 5, "B zone").

We discovered significant dependence (generalized Goodall F-test: p<0.05) of leaf shape on the leaf position on the rhizome (distance from the apex) for 6 of 11 investigated plants. However, the character of this dependence was not same for different plants. Leaves of two plants rounded and their lobes became more divergent, while moving in the acropetal direction, whereas leaves of two plants elongated and their lobes became less divergent, and leaves of the other two plants did not change their shape.

Dependence of sizes of leaves and flowers on the organic content in the water

Differences in organic content between all investigated reservoirs and streams are not high. Values of saprobity index vary from 0.65 to 2.00, which corresponds to the second and the third class of water purity (Table 2). Most of investigated reservoirs and streams are oligo- or betamesosaprobic (saprobity index 1.4-2.0) and all Karelian lakes with floating mats and one lake of this type in the Moscow region are oligokseno- or xenobetasaprobic (saprobity index 0.65-1.00) according to V. Sladechek (1967). We did not revealed any significant correlations between size characteristics of plants and saprobity index or any significant differences in sizes of plants.

Pollen morphology

Values of maximum and minimum equatorial diameters have bimodal distribution. Almost all investigated populations clearly separate into the groups with “small” (maximum equatorial diameter is 32-40 µm, minimum—28-37 µm) and “large” (44-52 µm è 38-50 µm, correspondingly) pollen grains. Values of maximum to minimum equatorial diameter ratio have unimodal distribution with median 1.08 and quartile range 1.04–1.1.

Plants of N. alba and N. tetragona morphotypes have “small” pollen. Plants with N. candida morphotype have both “small” and “large” pollen (Fig. 3b); it is not possible to distinguish separate morphotypes in N. candida according to the pollen size.

Pollen fertility in different plants from one population did not vary more then for 15%. Plants from vast majority of the populations have highly fertile pollen (more then 75% of fertile pollen grains), whereas one population (116) from Tver region have deferred pollen fertility (63%), two populations from Karelia (136) and from Tver region (111) have low pollen fertility (33-38%) and one population from Tver region (113) has almost sterile pollen (5% of fertile pollen grains), see Table 2. Pollen grains from plants of this last population were very small in number (10-20 pollen grains per anther) and deformed. Macromorphology of plants from this population was also quite original and combines characters of N. alba (linear filaments of inner stamens, yellow and flat stigma) and N. candida (conic central projection of stigma).

Visual analysis of SEM-micrographs revealed that exine sculpture is not homogenous on the whole surface of pollen grain. Sculpture of distal part of pollen grain does not demonstrate any discrete patterns, being consisted of different in size verrucas that become larger near equator. The exine sculpture of the proximal part of pollen grains is very diverse. Hereafter we will describe the exine sculpture of proximal part of pollen grain as “exine sculpture” since this part is the most perspective for distinguishing different pollen morphotypes in Nymphaea. Four main types of exine sculpture can be distinguished with some transitions between them:

  1. rare (7-9 sculpture elements per 100 µm2 of pollen grain surface) dense groups and single verrucas combined with single baculums of 2-4 µm length (Fig. 7à);
  2. quite dense (22-38 sculpture elements per 100 µm2 of pollen grain surface) verrucas and baculums of 1-5 µm length (Fig. 7b);
  3. very dense frequently merging different sized verrucas varying from almost flat to evidently prominent ones (Fig. 7c);
  4. combined type, presented by different combinations of verrucas and baculums of various density (13-33 sculpture elements per 100 µm2 of pollen grain surface) (Fig. 7d).

Figure 7. Scanning electron micrographs of Nymphaea pollen grains, view from the proximal pole. Different types of the exine are shown. Population numbers refer to Table 2.

A — exine sculpture of the first type (population number 128);

B — exine sculpture of the second type, variant with long baculums (population number 137);

C — exine sculpture of the third type, variant with prominent verrucas (population number 106);

D — exine sculpture of the fourth type (population number 105)

Exine sculpture frequently varies essentially within one population. The population from one pond in the Moscow region (population 119) seems especially interesting to us. Near the southern shore of the pond there are plants with N. candida morphotype (Table 1) and exine sculpture of the second type. In the middle of the pond there are plants with some morphological characters of N. tetragona (bent along the full length main veins of the leaf, strongly invaginated stigma—see Table 1) and exine sculpture of the third type. Three investigated populations in the large lake Moldino in Tver region (108-110) have various exine sculpture (Table 2) and very similar macromorphology (Fig. 3). We found the third type of exine sculpture in all herbarium specimens of N. tetragona and in all populations of N. tetragona collected by us near lake Bajkal (201, 203 and 204). This type of exine sculpture was also found in one lake in Tver region (population 106: Table 2). Plants in this population differed from N. candida morphotype by linear filaments of inner stamens, main veins which are bent along the full length of the leaf and strongly invaginated stigma (Table 1). All other populations have pollen of the first, second and forth types of exine sculpture without any dependence on plant macromorphology or region of growing (Table 2).

Discussion

Dependence of Nymphaea size characters on the organic content in the water

Contrary to widespread opinion (Komarov 1937, Heslop-Harisson 1955, Kupriyanova 1976, Dubyna 1982, Papchnekov 2003), there was no significant dependence of Nymphaea size characters on content of organic in the water. From the one hand, we can suppose that range of organic content in the investigated reservoirs was not large enough for the detection; from the other hand, special quantitative investigations of organic content were not carried out. Small northern lakes with floating mats are traditionally classified as oligotrophic contrary to mesotrophic lakes of the middle Russia (Kupriyanova 1976). Our quantitative investigations do not demonstrate any considerable differences in organic content for investigated lakes. Consequently, the size characters of Nymphaea are not so clearly caused by organic content in the water as it was thought before.

Some notes on Nymphaea taxonomy in the European Russia

Our data let us distinguish three Nymphaea morphotypes in the European Russia, which correspond to the literature descriptions of N. alba, N. candida and N. tetragona. These species can be separated by several macromorphological characters of live plants, as it was shown in the abovementioned diagnostic key. These species differ also by the shape of fresh leaves, but these differences are not clear so we do not recommend to use leaf shape as a diagnostic character. Our investigations did not reveal any definite dependence of leaf shape on leaf position on the rhizome. Therefore, one can analyze leaf shape without taking into account the leaf position. Pollen morphology, in our opinion, has much less diagnostic value that it was thought before (Komarov 1937, Kupriyanova 1976, Dubyna 1982, Uotila 2000). Sizes of pollen grains for N. alba, N. candida and N. tetragona overlap considerably; moreover, this character has large variation within population. Only N. tetragona can be distinguished on the base of exine sculpture while N. alba and N. candida do not differ on this character, contrary to researchers worked with small samples (Kupriyanova, 1976, Muntendam et al., 1996).

We found plants with typical for N. alba morphology only in the most southern part of European Russia (delta of the Volga river, Astrakhan region), plants with typical for N. tetragona macromorphology were found only in the Western Siberia. Our investigations do not support data about N. alba and N. tetragona in the middle Russia (Lisitsyna et al. 1993; Papchenkov 2003). According to our data, variety of Nymphaea morphotypes in the middle Russia can not be dissected into more or less discrete groups.

Separate marginal morphotypes of the morphological continuum, existing in middle Russia, can be interpreted as N. alba and N. tetragona. Specimens that have intermediate morphology between imaginary center of continuum (typical N. candida) and its marginal morphotypes can be also interpreted as hybrids N. alba × N. candida = N. × borealis and N. tetragona × N. candida = N. × sundvickii. This approach is driven to its logical end by V.G. Papchenkov (2003). However, this interpretation does not correspond with generally accepted species determination (Grant 1981). Distinguishing separate species on the base on small differences in morphology is not appropriate especially for plants with prevalence of vegetative reproduction over sexual (Elven et al. 2004), such us water-lilies (Heslop-Harrison 1955, Dubyna 1982).

We suppose that in the middle Russia there is only N. candida, a very polymorphic species. Our data about high morphological variability of N. candida agree with data of K.I. Aleksandrova (1996) for Lipetsk region of European Russia and contradict to data of D.V. Dubyna (1982) for the Ukraine.

Our point of view is supported by the information about existence of “hybrids” in the absence of parental species (Uotila 2000, Papchenkov 2003). As far as we know, these hybrids were distinguished only on the morphological basis so plants with unusual combinations of morphological characters lacking the hybrid original can be easily taken as “hybrids”. Our studies of pollen fertility show that plants with unusual combinations of morphological characters almost always have highly fertile pollen and therefore can be hardly consider as hybrids (Komarov 1937, Heslop-Harrison 1955, Neuhausl & Tomsovic 1957 as cited in Dubyna 1982).

Division of the investigated populations of N. candida on basis of the pollen size corresponds to their division on basis of macromorphological character set which probably means that two chromosomal races of N. candida exist in the middle Russia. This assumption agrees with data about the correlation between pollen size and ploidy level (Possubnaya-Arnol'di 1976) and about various chromosomal numbers known for the European N. candida (Heslop-Harisson 1955, Dubyna 1982, Wiersema 1988, Krupkina 2001). To test this hypothesis in future, it is essential to estimate ploidy level. However, these estimations will be troubled by big chromosomal numbers (up to more then 100) and small sizes of the chromosomes (Heslop-Harrison 1955). It is also essential to carry out DNA analysis for decision a question about species diversity in Nymphaea genus in the European Russia (Muntendam et al. 1996), what, as far as we know, was not done for the European Nymphaea species.

Acknowledgements

We are grateful to everybody, who helped us in field collection of the material in various ways: all members of summer practices of Moscow South-West High School (1543), led by Dr. S.M. Glagolev; E. Chibelyov (natural-historical reserve “Arkaim”), Dr. K.I. Aleksandrova (Lipetsk University), Dr. V.I. Sutula (Bajkal Zapovednik) and Dr. A.K. Gorbunov (Astrakhan Zapovednik). We are also grateful to Dr. S.V. Polevova (Moscow State University) for consultations on palynology, Dr. O.V. Anisimova (Moscow State University) for estimation of the organic content in the water, Dr. I.Ya. Pavlinov (Moscow Zoological Museum) for consultations on geometric morphometry and Dr. S.R. Mayorov (Moscow State University) for all his support.

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