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Article

How Do Pollen Grains of Convallaria majalis L. Respond to Different Habitat Conditions?

by
Dorota Wrońska-Pilarek
1,*,
Jan Bocianowski
2,
Kacper Lechowicz
1,
Blanka Maria Wiatrowska
1,
Magdalena Janyszek-Sołtysiak
3 and
Cezary Beker
4
1
Department of Botany and Forest Habitats, Poznań University of Life Sciences, Wojska Polskiego 71d, 60-625 Poznań, Poland
2
Department of Mathematical and Statistical Methods, Poznań University of Life Sciences, Wojska Polskiego 28, 60-637 Poznań, Poland
3
Department of Botany, Poznań University of Life Sciences, Wojska Polskiego 71c, 60-625 Poznań, Poland
4
Department of Forest Management, Poznań University of Life Sciences, Wojska Polskiego 71c, 60-625 Poznań, Poland
*
Author to whom correspondence should be addressed.
Diversity 2023, 15(4), 501; https://doi.org/10.3390/d15040501
Submission received: 30 December 2022 / Revised: 13 March 2023 / Accepted: 24 March 2023 / Published: 1 April 2023
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Abstract

:
To date, the effect of habitat conditions on the characteristics of pollen has not been extensively investigated; however, it needs to be remembered that it may be highly significant for the quality of their generative reproduction success. It was decided to conduct the analyses on Convallaria majalis as a common species, naturally found in many different forest habitats. Moreover, the investigations covered pollen morphology and for the first time also the variability of pollen grains in this species. The plant material came from 98 natural sites located in Poland, in nine differing forest habitats. In total, 2940 pollen grains were analyzed in terms of five quantitative features (i.e., the length of the longest and shortest polar axes–LA and SA, exine thickness–Ex, the LA/SA and Ex/LA ratios) as well as the following qualitative ones: pollen outline and shape, sulcus type and exine ornamentation. Our studies revealed that the most important pollen characteristics in C. majalis included sulcus type, exine ornamentation, distribution and size of perforations, LA and pollen shape. The study showed the response of pollen to different habitat conditions found in the nine investigated habitats. The Ex/LA ratio and Ex were these pollen characteristics, which exhibited the most marked response to the different habitat conditions. Pollen from two habitats, moist mixed coniferous forest and upland mesic broadleaved forest, exhibited the most distinct characteristics.

1. Introduction

Convallaria majalis L. (Lily of the valley) is a plant widespread throughout the northern hemisphere [1]. This species belongs to the family Asparagaceae [2]. The genus Convallaria L. has typically been described as a monotypic genus with the single species C. majalis [3]. Within this genus, three morphologically similar, although geographically isolated species differing in their distribution, are distinguished, i.e., C. majalis L., C. keiskei Miq. and C. montana Raf. However, some authors are of an opinion that, despite their isolated ranges, these species are similar and morphological differences between them are slight [3,4,5,6,7]. Convallaria majalis is found primarily in Europe, as well as certain regions in Asia and along the eastern coast of North America. In turn, C. keiskei is a species growing in northern and eastern Asia, while C. montana is found mainly in central regions of the USA [7,8,9]. Occasionally, C. majalis is treated as a polymorphic species, within which subspecies are distinguished; however, to date in Poland only the typical species, Convallaria majalis L., has been reported [10,11,12].
Lily of the valley in Poland is common in lowlands, highlands and foothills [13]. In the mountains, it is usually found up to the montane forest areas [14]. According to Zarzycki et al. [15], in Poland it is a plant neutral in relation to the continentalism of semi-shaded localities, growing in moderately warm regions and in the warmest microregions. It prefers soils ranging from dry to mesic, trophically moderately poor with a moderately acid reaction, neutral to alkaline, clayey sands with silt deposits as well as heavy clays and silts lying on mineral humus soils. This plant is not very drought tolerant; it requires minimum 130 days with temperatures above 0 °C, although it also tolerates very low temperatures below −20 °C [15].
In the warmer regions of Central Europe, C. majalis invariably occurs in mixed forests in slightly shaded sites, whereas in the colder regions of north-western Europe it is most frequently observed in forest edges and on slopes of gorges, where it frequently overgrows vast clearings [16,17]. Lily of the valley is a differential species for plant communities Peucedano-Pinetum Mat. (1962) 1973 and Potentillo albae-Quercetum petraeae Libb. 1933. Moreover, it is a frequent component of the forest floor cover in acidophilous oak forests and less frequently oak-hornbeam forests and ravine Norway maple-lime forests [18]. In western Poland, its presence distinguishes mesic mixed coniferous forests from mesic coniferous forests, while the species is also characteristic to mesic mixed broadleaved forests and highland mesic broadleaved forests [19].
Pollen morphology of C. majalis has not been frequently described and the existing descriptions were typically very brief. Erdtman [20] found only one polar colpus (sulcus) in this species and for this reason he classified it to monosulcate plants. It needs to be mentioned here that monocotyledons usually have monosulcate or monoporate pollen grains [21,22,23]. Faegri and Iversen [24] listed the most important pollen traits in this species and classified it together with the genera Paris and Polygonatum to Maianthemum, which representatives were classified as monocolpate with a markedly perforate-profossulate tectum. According to Yan-Cheng and Wu-Xiu [25], pollen grains of C. majalis are large (LA length of the longest polar axis-39.95–49.35 μm) medium-sized (SA length of the shortest polar axis-21.15–30.55 μm), with thick granular and finely reticulate or only finely reticulate exine ornamentation. In turn, Li-Ming and De-Yuan [26] described pollen grains in Convallarieae, including C. majalis, as monosulcate, boat-shaped and with a microperforate exine sculpture. Only a slightly more detailed description of pollen in the analyzed species was given by Aktuna and Heigl [27]. According to those authors, pollen grains of C. majalis are heteropolar monad, spheroidal in shape and with mostly an elliptic outline in the polar view. Pollen grains were defined as medium-sized, since the longest (LA) and shortest (SA) polar axes were 26–30 µm. Exine ornamentation was scabrate or reticulate perforate. Pollen grains were monosulcate with Ubisch bodies. The studies cited revealed that the most important pollen features of C. majalis included the aperture type (one sulcus) and structure, scabrate or reticulate perforate exine ornamentation and the length of the longest polar axis (LA). In the most recent, extensive study by Hu et al. [28] presenting a study of pollen morphology in 70 taxa of 11 genera in the family Liliaceae it was not included C. majalis, classified to that family.
The primary aim of this study was to investigate for the first time the response of pollen grains in C. majalis to various habitat conditions found in nine analyzed forest habitats, where this species is present. It was decided to focus on this plant species, since it is common in Poland and in Europe and grows in many different types of forest habitats ranging from poor to fertile. Another reason for this decision was also connected with the fact that its many populations in Europe are reduced or have completely disappeared as a result of disturbed sexual reproduction [29]. Results of our studies are of significant practical importance because determination, to what extent pollen of the analyzed species responds to variable habitat conditions, may influence successful generative reproduction. Another important aim was also to provide a comprehensive description of pollen morphology in C. majalis, so far missing in palynological literature, as well as investigate the previously not analyzed intraspecific variability of pollen grains in this species.

2. Material and Methods

2.1. Palynological Analysis

The plant material in the form of inflorescences was collected during the vegetation seasons in the years 2018–2022 from natural localities of C. majalis situated in nine forest habitats in various regions of Poland (lowlands, foothills and mountains) (Table S1). The plant material was stored at the Department of Forest Botany and Forest Habitats, the Poznań University of Life Sciences.
In each of the habitats selected for the study, flowers were collected from numerous individual plant specimens. Flowers from each sample were placed in a separate paper envelope, which was described in detail (sample number, locality, geographical coordinates, collector). Then the inflorescences were transferred to the described tubes in order to prepare samples for acetolysis. A total of 98 samples of pollen grains were collected. Each sample consisted of 30 randomly selected, mature and correctly formed pollen grains derived from a single individual plant. In total, 2940 pollen grains were studied. The pollen grains were prepared for light (LM) and scanning electron microscopy (SEM) using the method described by Erdtman [20,30]. The acetolysis mixture was made up of nine parts of acetic acid anhydride and one part of concentrated sulphuric acid, while the process of acetolysis lasted 2.5 min. The prepared material was divided into two parts: one part was immersed in an alcohol solution of glycerin (for LM) and the other in 96% ethyl alcohol (for SEM). The LM study of acetolyzed pollen grain was conducted using a Biolar 2308 (Polish Optical Institutions, Warsaw, Poland) microscope at a magnification of 640×. To study exine pattern morphology in SEM, pollen samples were mounted onto aluminum stubs with double-sided adhesive tape. Stubs were sputter-coated with gold-palladium, and pollen was examined and imaged using a Zeiss Evo 40 (Carl Zeiss AG, Oberkochen, Germany).
Pollen grains were analyzed for five quantitative features, i.e., length of the longest polar axis (LA) and length of the shortest polar axis (SA), exine thickness (Ex) measured along the longest polar axis (LA), the SA/LA and Ex/LA ratios, as well as the following qualitative traits: pollen outline and shape, sulcus structure and ornamentation, and exine ornamentation. The pollen shape classes (LA/SA ratio) were adopted according to the classification proposed by Erdtman [20]: suboblate (0.75–0.88), oblate-spheroidal (0.89–0.99), spheroidal (1.00), prolate-spheroidal (1.01–1.14), subprolate (1.15–1.33), prolate (1.34–2.00) and perprolate (>2.01).
The palynological terminology used in the study follows Punt et al. [31] and Halbritter et al. [32].

2.2. Habitat Analysis

The collected pollen samples were presented in terms of the habitat (forest site type) gradient ranging from forests growing in the poorest habitats up to the most fertile habitats. A total of 98 pollen samples were collected from nine forest habitats (Table 1). The largest number of samples were gathered in habitat D-(37), followed by B (18), A and E (with 11 samples each), G (8), H (6), F (3), and C and I (each with 2 samples). The habitats were also characterized in terms of habitat fertility (soils with a moderate to fertile inherent fertility), soil moisture content (mesic or moist) and height above sea level (lowland vs. upland areas) [33].
The list (Table 1) presents plant communities, which correspond to the nine distinguished habitats. Dependencies between the plant communities and habitats are highly complex, and for this reason the communities with a broad ecological scale were classified to several habitats following Danielewicz and Maliński [34], Sikorska and Lasota [35] and Matuszkiewicz [33].

2.3. Statistical Analysis

The normality of distribution for the five traits, i.e., LA, Ex, SA, LA/SA and Ex/LA, was tested using Shapiro–Wilk’s normality test [36] to verify whether the analysis of variance (ANOVA) met the assumption that the ANOVA model residuals followed a normal distribution. Bartlett’s test was applied to test the homogeneity of variance. Box’s M test was used to verify multivariate normality and homogeneity of variance–covariance matrices. All the traits had a normal distribution. Multivariate analysis of variance (MANOVA) was carried out to determine the multivariate effects of habitats. A one-way analysis of variance (ANOVA) was carried out to determine the effects of habitats on the variability of LA, Ex, SA, LA/SA and Ex/LA. The minimal, maximal and mean values as well as standard deviations (SD) of traits were calculated. Moreover, Fisher’s least significant differences (LSDs), at the 0.05 level, were calculated for individual traits and on this basis homogeneous groups were established. The parallel coordinate plot was proposed as an efficient tool for visualization of five pollen traits in relation to nine habitats [37]. The relationships between the nine observed traits were estimated using Pearson’s linear correlation coefficients based on the means for the habitats. Relationships of the five observed traits were presented in a heatmap. The results were also analyzed using multivariate methods. A canonical variance analysis (CVA) was applied to present a multi-trait assessment of the similarity for (1) the tested habitats and (2) samples in a lower number of dimensions with the least possible loss of information. The Mahalanobis distance was used as a measure of “multitrait” similarity of (1) habitats and (2) samples [38]. Mahalanobis distances were calculated for all (1) habitats and (2) samples. The differences between the analyzed habitats were verified by cluster analysis using the nearest neighbor method and Euclidean distances and presented as a dendrogram. The GenStat v. 22 statistical software package (VSN International, Hemel Hempstead, UK) was used for the analyses.

3. Results

3.1. General Morphological Description of Pollen

A description of pollen grains of the C. majalis is given below and illustrated in the SEM photographs (Figure 1). The morphological observations for the other quantitative features of pollen grains are shown in Section 3.2.
Pollen grains of C. majalis were monosulcate, heteropolar monads (Figure 1a). According to the pollen size classification by Erdtman [20], the pollen grains analyzed were mostly medium (25.1–50 µm; 99.9%), very rarely small (10–25 µm; 0.1%). The values for the LA trait ranged from 18.00 to 58.00 μm. In all the analyzed pollen grains, the mean LA length was 35.86 µm. The mean SA length was 26.53 (18.00–40.00) µm.
The pollen outline in the polar distal and proximal views was mostly elliptic (Figure 1b), while the pollen shape was ellipsoid or boat-shaped. Totally, the mean LA/SA ratio (pollen shape) was 1.37 (0.60–2.40). The most frequently observed pollen grains were prolate (48.95%—1439 grains) and subprolate (37.96%—1116). In addition, 10.51% (309) of the grains were prolate-spheroidal in shape. The least numerously represented grains were the most elongated and the most flattened pollen grains representing three shape classes: perprolate (0.24%—7), oblate-spheroidal (0.07%—2) and oblate (0.03%—1).
For all the measured pollen grains, the mean exine thickness (Ex) was 0.67 (0.20–2.00 µm). The exine thickness constituted approx. 0.02 µm (0.004–0.08 µm) of the LA length.
Exine ornamentation was reticulate and microreticulate because a portion of the lumina had diameters of less than 1 µm as well as striate-perforate. The lumina width was decreasing slightly in the sulcus border zone. The proximal polar area was rather microreticulate, whereas the equatorial and distal areas were finely striate-perforate (Figure 1d). The perforations were very numerous, elliptic or circular in outline. Perforations were rather regularly arranged; however, their number decreased in the polar areas and in the sulcus border zone. The number of perforations calculated in 25 µm2 ranged between 147 and 162. They were different in size (from 0.05 to 0.25 µm); usually their diameter was 0.18–0.22 µm.
All the studied pollen grains have a single elongated aperture (sulcus), thus they belong to the monosulcate class. The sulcus is wide (at the widest section ranging from 3.64 to 5.83 µm), elongated, elliptic, narrowing at the rounded apices. It is approximately as long as the half of the circumference of all the pollen grains (Figure 1c). The sulcus borders are irregular, and its membrane is psilate without any ornamentation.
Pollen grains of C. majalis contain Ubisch bodies, i.e., polymorphic sporopollenin elements produced by the tapetum.

3.2. Pollen Response to Various Conditions Occurring in the Habitats Studied

All the traits had a normal distribution (Table 2).
The results of MANOVA indicated that all the studied habitats were significantly different in terms of all the five observed traits jointly (Wilk’s λ = 0.9002; F = 7.79; p < 0.0001). Analysis of variance indicated that the main habitat effects were significant for all the five traits investigated in this study (Table 3, Table 4, Table 5 and Table 6). The LA trait had the greatest range of values for habitats D (18–48 µm) and G (28–58 µm), while in habitat D scarce pollen grains showed the smallest of the LA values recorded in this study, whereas for G pollen grains exhibited the greatest of all reported lengths. At the same time, in habitat D, as well as A and B, most pollen grains showed medium values of LA (Table 3). In habitat H as well, a considerable range of values was found for this trait. The smallest range of LA values was recorded in habitats C (26–40 µm) and I (28–42 µm) (Figure 2 and Figure 3, Table 3). In turn, in habitat C the smallest mean LA value was observed (34.67 µm) (Table 3).
Trait SA showed the greatest range of values in habitats A (20–40 µm), B (20–38 µm) and D (18–36 µm), with the highest SA values recorded in pollen from habitat A, while they were lowest in the case of D and H. Markedly, the smallest range of SA values (22–30 µm) was found in habitat I (Figure 2). The greatest number of pollen grains showed medium values for this trait in habitats B and D. On average, the SA values ranged from 25.5 µm (in H) to 27.12 µm (in G) (Table 4, Figure 3).
In all the habitats, pollen grains had exine thickness (Ex) most typically amounting to 0.7 µm, and much less frequently to approx. 1.0 µm. The highest number of pollen grains with the mean exine thickness was recorded in habitats D, B and A. The greatest range of Ex values was observed for habitat E (0.2–2.0 µm), while it was only slightly smaller for habitats D, A, B and H. In habitat E, single pollen grains with the thickest exine were found, while an opposite situation was observed for habitat D, where the pollen grains with the thinnest exine were reported (Figure 2). On average, Ex ranged from 0.6 µm (in I) to 0.7133 µm (in B) (Table 5, Figure 3).
Pollen shape, i.e., the LA/SA ratio of the analyzed pollen grains in all the habitats, was typically slightly elongated. Only single pollen grains were flattened or strongly elongated, while scarce were spherical. Pollen collected in habitat D was markedly the most diverse (from 0.6 to 2.4) in terms of its shape (from flattened, through spherical up to strongly elongated), which may have been due to the highest number of pollen samples (37) coming from that habitat (Figure 2). The smallest number of pollen shape types was observed in pollen collected in habitats I and F. Mean values of pollen grain shape ranged from 1.342 (in B) to 1.418 (in H) (Table 6, Figure 3).
Relationships were observed between some of the observed features. Significant positive correlations were found between LA and SA (0.738), as well as Ex and Ex/LA (0.839). A negative correlation was observed between SA and LA/SA (−0.749) (Figure 4). The obtained correlations between features may be due to the fact that some observations are not independent. This may have affected the analysis. It should be noted, however, that these correlations are not equal to 1.
Individual traits were of varying importance and had different shares in the joint multivariate variation in the studied habitats. Analysis of the first two canonical variates for nine habitats regarding the five quantitative traits is shown in Figure 5. The first two canonical variates accounted for 84.79% of the total variability between the individual habitats (Figure 5). The most significant positive, linear relationship with the first canonical variate, was found for Ex/LA (0.902). The second canonical variate was significantly positively correlated with Ex (0.753) and SA (0.741).
The greatest variation in terms of all the five traits jointly measured with Mahalanobis distances was found for habitats C and F (distance between them amounted to 1.2447). The greatest similarity was found between habitats A and B (0.2511).
In the dendrogram presented in Figure 6, all the examined habitats were divided into two groups as a result of agglomeration grouping using the Euclidean distance method. The first (I) group comprised two habitats: C and H. The second (II) group comprised the other seven habitats. The second group was divided into two subgroups with three (A, B and D) and four habitats (E, F, G and I), respectively.

3.3. Intraspecific Variability of Pollen Grains Depending on the Analyzed Habitats

In six habitats (A, B, D, E, G and H), significant variation was observed between samples for all the observed traits (Table 7). In habitat C, no significant variation was found between samples for LA/SA only, while in habitat F it was observed for Ex/LA. In habitat I, significant variation between samples was observed for two traits: LA/SA and Ex/LA (Table 7). The habitats with a larger number of samples tend to have greater variability than the habitats with a smaller number of samples.
Analysis of the first two canonical variates for 98 samples from nine habitats regarding the five quantitative traits is shown in Figure 7. The first two canonical variates accounted for 73.88% and 12.01%, respectively, of the total variability between the individual samples (Figure 7). A division into two main groups differentiating all the 98 samples was observed. The most significant positive, linear relationship with the first canonical variate was found for Ex/LA (0.483), while negative for LA (−0.372) and LA/SA (−0.214). The second canonical variate was significantly positively correlated with Ex (0.984), SA (0.234) and Ex/LA (0.858), whereas it was negatively correlated with LA/SA (−0.280).
The greatest variation in terms of all the five traits jointly measured with Mahalanobis distances was found for samples 29D and 82D (distance between them amounted to 5.978). The greatest similarity was found between samples 15B and 38B (0.158) (see: Table S1).

4. Discussion

Monocotyledons, such as e.g., C. majalis, generally have monosulcate or monoporate pollen grains [39,40]. Penet et al. [41] when investigating several species of Asparagales (including C. majalis) stated that they produce such monosulcate pollen, representing most families of this important monocot clade. Hu et al. [28] analyzed pollen morphology of 70 taxa (except for C. majalis) of 11 genera in the family Liliaceae, to which the genus Convallaria had been previously classified. The results showed that the length of the polar axis, colpus (aperture) morphology and exine ornamentation of pollen grains have important systematic significance. The key role of exine ornamentation shown by Hu et al. [28] was also confirmed by earlier studies by Nair and Sharma [42], who considered exine ornamentation a significant morphological character, considerably facilitating the categorization of various genera and species within the family Liliaceae, at that time comprising also the genus Convallaria.
In the scarce palynological studies published to date on C. majalis, similar traits were considered as having the highest diagnostic value: sulcus type and structure, exine ornamentation, length of the longest polar axis (LA) and pollen shape. Our analyses indicate that an important feature is also connected with the distribution and size of perforations. We agree with the other researchers [24,25,27] that the sulcus features are the most important in the diagnosis of C. majalis pollen grains. According to the classification presented by Halbritter and Hesse [43], a simple sulcus occurs in all the samples studied in this research. One sulcus type was identified. It is about as long as the half of the circumference of the pollen grain. A similar sulcus type is found, e.g., in the genus Maianthemum coming from the same family [44]. In other monosulcate monocotyledons, other sulcus types are also found, which differ in the length, width and distribution on pollen grains, e.g., in Allium ampeloprasum L. [45] or Iris reichenbachii Heuff. [32]. In the opinion of Erdtman [46] and Penet et al. [41], apertures as the areas where the external layer (exine) is thinner compared to the rest of the grain are one of the most variable features of pollen grains. Apertures play a crucial role during pollen germination because they are the areas through which the pollen tube emerges. In our study, we recorded a considerable variability in exine thickness (Ex) (from 0.2 to 2.0 µm), which proved to be a feature responding to the conditions observed in the analyzed habitats. Its response was particularly important, because the successful pollen tube penetration depends on exine thickness in the sulcus membrane. The greatest range of Ex values was observed for the upland habitat E (upland mesic mixed broadleaved forest), with relatively harsh climatic conditions, and there pollen grains with the thickest exine were collected. Slightly smaller, comparable Ex values were recorded for similar, less fertile coniferous habitats A and B, with similar Ex values also observed in pollen from habitats D and H, differing slightly in terms of their habitat conditions. Exine ornamentation and the distribution and size of perforations are important traits for species identification; however, they do not differentiate pollen coming from the studied nine habitats. In C. majalis in different pollen areas, two types of exine ornamentation are observed (reticulate or microreticulate and finely striate with perforations). In earlier studies on the tribe Convallarieae, Yan-Cheng and Wu-Xiu [25] reported that in C. majalis only the microreticulate exine ornamentation is found, whereas Aktuna and Heigl [27] distinguished two types of exine ornamentation, which is consistent with our observations. The feature LA exhibited a large range of values (from 18.00 to 58.00 μm), with the mean value of 35.86 µm. In contrast, Yan-Cheng and Wu-Xiu [25] reported a much smaller range of this trait and its mean values (from 39.95 to 49.35 μm) were higher than those recorded in our analyses. This may have been caused by the fact that it was given not only for C. majalis, but also for three other species from the tribe Convallarieae. The LA trait showed the greatest range for two fertile habitats D and G, while in the less fertile habitat D single pollen grains had the smallest lengths, whereas in the fertile habitat G they reached the highest values. Additionally, in the fertile, upland habitat H, a considerable range of values was observed for this trait. The smallest range of LA values was recorded in two moist habitats, of which C is found on poor soils and I on fertile soils. Pollen collected in the poor, moist habitat C also had the smallest mean LA value (34.67 µm). These results indicate the lesser or greater effect of the habitat conditions on the length of the analyzed pollen grains. On fertile soils, C. majalis forms greater (longer) pollen than in less fertile, moist habitats. The most diverse shape (LA/SA ratio), ranging from flattened, through spherical up to strongly elongated, was recorded for pollen collected in a relatively fertile habitat D. This may have been caused by the highest number of samples (37) coming from this habitat, in which the investigated species grows most frequently in Poland. It may also be connected with the conditions prevalent in this habitat, which are optimal for C. majalis. The smallest number of pollen shape types was observed in pollen collected in moist forest habitats I and F. Mean values of pollen shape ranged from 1.342 (in the relatively poor habitat B) to 1.418 (in the fertile, upland habitat H) (Table 6, Figure 3). In the least fertile habitat, the response of pollen was connected with reduced length (LA) (Figure 2).
Interesting results were provided by the analysis of the impact of different habitat conditions in the nine studied habitats on pollen morphology in C. majalis. The recorded results are rather inconclusive. On the one hand, it turned out that the most similar features were observed in pollen collected in the similar habitats A and B (Figure 5), being similar in terms of all characteristics. They are two coniferous forest habitats, with the most similar species composition among the investigated habitats, and which both are found on poor soils typically with an acid reaction. On the other hand, pollen from habitats A and D also turned out to be very similar (in this case, also similar with respect to all characteristics), and in this case the habitat conditions and the species compositions differ considerably, since habitat D is found on relatively fertile soils, and it is a broadleaved forest habitat. In turn, the most different traits among the investigated habitats were observed for pollen from habitats C and H (especially in terms of length of the longest polar axis and the ratio of the length of the longest polar axis and length of the shortest polar axis), which differ greatly in terms of their habitat conditions (Figure 5 and Figure 6). The former is found on relatively infertile soils in lowland moist mixed coniferous forest, whereas the other is an upland mesic broadleaved forest, growing on fertile soils. The other habitats had such similar pollen that, in the dendrogram, they constituted a large joint group, which nevertheless was divided into two subgroups with three (A, B and D) and four habitats (E, F, G and I), respectively. Some of the habitats in these two subgroups have similar conditions (e.g., A and B, G and H), whereas other differ considerably, mainly in terms of their fertility and soil moisture content (e.g., A and D, or F and H). Among the investigated habitats, the greatest variation in terms of all the five analyzed pollen features was found for habitats C and F (especially for the length of the longest polar axis, length of the shortest polar axis and exine thickness). These are two moist forest habitats, with the former found on less fertile, while the latter in more fertile soils. Moist habitats are not optimal for C. majalis, thus possibly pollen responded with greater variability as an adaptation to conditions being less advantageous for this species. All the 98 studied pollen samples of C. majalis were divided into two groups, differentiated based on two pollen features, Ex/LA and LA/SA, with no impact of the habitat types (Figure 7). The habitats with a larger number of samples tend to have greater variability than those with a smaller number of samples.
Summing up, diverse habitat conditions in the nine investigated natural forest habitats were found to influence the pollen features of C. majalis. This impact varied and was not always marked. Similar conclusions were drawn by Chwedorzewska et al. [47] and Kosiński et al. [11], when investigating the morphological characteristics of inflorescences and leaves in C. majalis coming both from natural localities and from various habitats, as well as cultivated plants. Those authors also showed an impact of the habitat conditions on the characteristics of the analyzed plant organs, with the impact of the habitat being much more marked in cultivated plants rather than in wild growing populations. This conclusion may explain the occasionally rather weak response of pollen recorded in our study. Such a result of our study may have been influenced by the limited number of samples collected from individual habitats. The importance of the investigations cited above needs to be stressed also in view of the decreasing number of C. majalis populations in some regions of Europe. Studies on the genetic structure of 20 populations of this species in central Belgium showed that a majority of populations consisted of a single genotype [29]. A population consisting of multiple genotypes mainly occurred in locations with a thin litter layer and high soil phosphorus levels, suggesting environment-mediated sporadic recruitment from seed. Lack of sexual recruitment in spatially isolated C. majalis populations has resulted in almost monoclonal populations with reduced or absent sexual reproduction, potentially constraining their long-term persistence [29].
In view of the complicated habitat-plant relationships, the authors of this paper were aware that it would be difficult to precisely determine how habitat conditions affect individual characteristics of pollen grains, and thus the generative reproduction of the investigated species. We assumed that, possibly in fertile habitats, C. majalis would form larger pollen grains with a thinner exine than is the case in these least fertile habitats; however, this hypothesis was not always confirmed. For this reason, our results are inconclusive, which is not surprising in view of the very large number of variable environmental conditions influencing natural, forest populations of C. majalis.

5. Conclusions

The most important pollen grain features in C. majalis comprise sulcus type, exine ornamentation, distribution and size of perforations, LA and pollen shape (LA/SA ratio).
The most marked response to the different habitat conditions prevalent in the nine investigated habitats was observed for Ex/LA and Ex. This is an important finding, since exine thickness (Ex) in the area of apertures (e.g., the sulcus membrane) is of key importance for effective pollen tube penetration and thus for the entire generative reproduction process. The habitat had a lesser impact on the LA/SA ratio, SA and LA. Nevertheless, when analyzing LA and the LA/SA ratio it was found that, in the more fertile habitats, C. majalis frequently formed slightly longer and more elongated pollen grains than it does in poor habitats.
Dependencies between the pollen features and the investigated habitats were complex. Similar features were identified both in pollen coming from relatively similar and from markedly different habitats. In turn, the greatest variation in terms of all the five analyzed pollen features for habitats C and F may be a response to habitat conditions found in these two moist forest habitats, being less advantageous for C. majalis.
All the 98 studied pollen samples of C. majalis were divided into two groups (Figure 7), comprising pollen with more or less similar characteristics based on two pollen features: Ex/LA and LA/SA. Habitat types had no impact on this division.
Conditions in the nine studied forest habitats were found to have an impact on the pollen features of C. majalis; however, for some habitats it was more, while for others it was less marked. However, the obtained results may have been influenced also by the limited number of samples collected from some habitats and the fact that pollen grains are the most conservative plant organs, which is why their characteristics did not always respond to the diverse habitat conditions found in different forest habitats. Nevertheless, the obtained results confirmed the need to conduct further studies on the impact of habitat conditions on pollen in other forest plant species.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/d15040501/s1, Table S1: List of localities of the Convallaria majalis studied.

Author Contributions

Conceptualization, D.W.-P. and J.B.; methodology, D.W.-P., J.B., K.L., B.M.W. and C.B., software, J.B.; validation, D.W.-P. and J.B.; formal analysis, J.B. and D.W.-P.; investigation, D.W.-P., K.L. and B.M.W.; data curation, D.W.-P. and J.B.; writing—original draft preparation, D.W.-P., J.B., M.J.-S. and B.M.W.; writing—review and editing, D.W.-P. and J.B.; visualization, J.B. and K.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Acknowledgments

We kindly thank Anna Binczarowska (English language proofreader) for linguistic support. We would like to thank the reviewers for their detailed and valuable comments on the manuscript.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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Figure 1. Pollen grains of Convallaria majalis; (a) group of pollen grains in the distal and proximal polar view with the sulcus seen in the center of one pollen grain, (b) proximal polar view, (c) equatorial view (long axis) and sulcus with membrane, (d) finely striate exine ornamentation with perforations.
Figure 1. Pollen grains of Convallaria majalis; (a) group of pollen grains in the distal and proximal polar view with the sulcus seen in the center of one pollen grain, (b) proximal polar view, (c) equatorial view (long axis) and sulcus with membrane, (d) finely striate exine ornamentation with perforations.
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Figure 2. Density plots of the length of the longest polar axis (LA), exine thickness (Ex), length of the shortest polar axis (SA) and the LA/SA ratio, determined by habitats.
Figure 2. Density plots of the length of the longest polar axis (LA), exine thickness (Ex), length of the shortest polar axis (SA) and the LA/SA ratio, determined by habitats.
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Figure 3. Parallel coordinate plots (PCPs) for nine habitats and five traits: length of the longest polar axis (LA), exine thickness (Ex), length of the shortest polar axis (SA) as well as the SA/LA and Ex/LA ratios. Habitats A–I—see Table 1.
Figure 3. Parallel coordinate plots (PCPs) for nine habitats and five traits: length of the longest polar axis (LA), exine thickness (Ex), length of the shortest polar axis (SA) as well as the SA/LA and Ex/LA ratios. Habitats A–I—see Table 1.
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Figure 4. Heatmap for Pearson’s correlation coefficients between observed traits (rcr = 0.666) for habitats. The heatmap shows a graphical representation of the correlation matrix between pairs of observed traits. LA: length of longest axis, Ex: thickness of exine, SA: length of shortest axis. * p < 0.05; ** p < 0.01.
Figure 4. Heatmap for Pearson’s correlation coefficients between observed traits (rcr = 0.666) for habitats. The heatmap shows a graphical representation of the correlation matrix between pairs of observed traits. LA: length of longest axis, Ex: thickness of exine, SA: length of shortest axis. * p < 0.05; ** p < 0.01.
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Figure 5. Distribution of nine habitats in the space of the first two canonical variables (V1 and V2), A–I abbreviated habitat names (see: Table 1).
Figure 5. Distribution of nine habitats in the space of the first two canonical variables (V1 and V2), A–I abbreviated habitat names (see: Table 1).
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Figure 6. Clustering (an average is taken over all the units in the two merged clusters) of habitats based on five morphological traits. The length of the lines indicates the similarity/distance between two habitats or among two groups of habitats and stated hierarchical clusters, A–I abbreviated habitat names (see: Table 1).
Figure 6. Clustering (an average is taken over all the units in the two merged clusters) of habitats based on five morphological traits. The length of the lines indicates the similarity/distance between two habitats or among two groups of habitats and stated hierarchical clusters, A–I abbreviated habitat names (see: Table 1).
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Figure 7. Distribution of 98 samples in the space of the first two canonical variables (V1 and V2), A–I abbreviated habitat names (see: Table 1).
Figure 7. Distribution of 98 samples in the space of the first two canonical variables (V1 and V2), A–I abbreviated habitat names (see: Table 1).
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Table
Table 1. Habitats from which the plant material originated, listed ranging from the poorest to the most fertile along with corresponding plant communities.
Table 1. Habitats from which the plant material originated, listed ranging from the poorest to the most fertile along with corresponding plant communities.
No.HabitatsAbbreviationPlant Communities Corresponding to Forest Habitats
1Mesic coniferous forestALeucobryo-Pinetum, Peucedano-Pinetum, Empetro-Pinetum
2Mesic mixed coniferous forestBQuerco robosris-Pinetum typicum, Serratulo-Pinetum, Fago-Quercetum typicum, Betulo-Quercetum typicum, Calamagrostio arundinaceae-Quercetum
3Moist mixed coniferous forestCQuerco roboris-Pinetum molinietosum, Fago-Quercetum molinietosum, Betulo-Quercetum molinietosum,, Querco-Piceetum typicum, Calamagrostio villosae-Pinetum, Molinio caeruleae-Quercetum roboris
4Mesic mixed broadleaved forestDQuerco roboris-Pinetum coryletosum, Luzulo pilosae-Fagetum typicum, cladonietosum, dryopteridetosum, Stellario holosteae-Carpinetum deschampsietosum, Galio sylvatici-Carpinetum politrichetosum, lathyretosumTilio-Carpinetum typicum, calamagrostietosum, caricetosum brizoides, Potentillo albae-Quercetum petraeae
5Upland mesic mixed broadleaved forestEQuerco roboris-Pinetum typicum, Luzulo luzuloidis-Quercetum petraeae, Luzulo luzuloidis-Fagetum, Abietetum polonicum typicum, Luzulo pilosae-Fagetum, Tilio-Carpinetum luzuletosum, Galio-Carpinetum luzuletosum
6Moist mixed broadleaved forestFFago-Quercetum molinietosum, Stellario holosteae-Carpinetum typicum, Galio sylvatici-Carpinetum polytrichetosum, Tilio cordatae-Carpinetum calamagrostietosum, caricetosum brizoides, Querco roboris-Pinetum coryletosum, molinietosum, Querco-Piceetum typicum, dryopteridetosum, Molinio caeruleae-Quercetum roboris
7Mesic broadleaved forestGGalio odorati-Fagetum (typicum, deschampsietosum, festucetosum sylvaticae, corydaletosum), Stellario-Carpinetum typicum, Galio sylvatici-Carpinetum typicum, Tilio cordatae-Carpinetum typicum
8Upland mesic broadleaved forestHTilio-Carpinetum typicum, Carici-Fagetum convallarietosum, Dentario glandulosae-Fagetum typicum, Dentario enneaphyllidis-Fagetum typicum, Galio odorati-Fagetum, Aceri-Tilietum, Phyllitido-Aceretum, Luzulo luzuloidis-Quercetum petraeae, Tilio-Carpinetum luzuletosum, Galio-Carpinetum luzuletosum
9Moist broadleaved forestIStellario holosteae-Carpinetum betuli ficarietosum, Galio-Carpinetum corydaletosum, Tilio-Carpinetum stachyetosum, corydaletosum
Table
Table 2. Results of Shapiro–Wilk’s normality test and Bartlett’s test for particular traits as well as Box’s M test for equality of the covariance matrices.
Table 2. Results of Shapiro–Wilk’s normality test and Bartlett’s test for particular traits as well as Box’s M test for equality of the covariance matrices.
MethodParametersTrait
LAExSALA/SAEx/LA
Shapiro–Wilk test for normalityTest statistic W0.96610.74440.95780.96940.9198
p-value<0.001<0.001<0.001<0.001<0.001
Bartlett’s test for homogeneity of variancesChi-square20.1784.2322.2752.4372.56
degrees of freedom88888
p-value0.010<0.0010.004<0.001<0.001
Box’s test for equality of the covariance matricesM: 1247.32; degrees of freedom: 120; p-value: <0.001
Table
Table 3. Mean values, minimal, maximal and standard deviations (SD) as well as homogenous groups for length of the longest polar axis (LA), A–I abbreviated habitat names (see: Table 1).
Table 3. Mean values, minimal, maximal and standard deviations (SD) as well as homogenous groups for length of the longest polar axis (LA), A–I abbreviated habitat names (see: Table 1).
HabitatMeanMinMaxSD
A35.95 a28443.081
B35.97 a26463.348
C34.67 b26403.545
D35.87 a18483.548
E35.73 a24443.386
F36.2 a30463.745
G36.13 a28583.881
H35.63 a22463.603
I35.43 ab28423.212
LSD0.050.903
F-ANOVA1.52 *
In column, means followed by the same superscript letters are not significantly different. * p < 0.05.
Table
Table 4. Mean values, minimal, maximal and standard deviations (SD) as well as homogenous groups for length of the shortest polar axis (SA), A–I abbreviated habitat names (see: Table 1).
Table 4. Mean values, minimal, maximal and standard deviations (SD) as well as homogenous groups for length of the shortest polar axis (SA), A–I abbreviated habitat names (see: Table 1).
HabitatMeanMinMaxSD
A26.55 abc20403.019
B27.03 ab20382.766
C25.63 de20322.98
D26.48 abc18362.913
E26.3 bcd20322.609
F26.42 abc20322.804
G27.12 a20342.838
H25.5 e18323.055
I25.83 cde22302.027
LSD0.050.741
F-ANOVA7.8 ***
In column, means followed by the same superscript letters are not significantly different. *** p < 0.001.
Table
Table 5. Mean values, minimal, maximal and standard deviations (SD) as well as homogenous groups of exine thickness (Ex), A–I abbreviated habitat names (see: Table 1).
Table 5. Mean values, minimal, maximal and standard deviations (SD) as well as homogenous groups of exine thickness (Ex), A–I abbreviated habitat names (see: Table 1).
HabitatMeanMinMaxSD
A0.7103 ab0.21.60.2958
B0.7133 a0.21.60.2866
C0.6933 ab0.210.213
D0.6856 abc0.21.60.2528
E0.6218 cd0.220.2324
F0.6178 d0.210.2236
G0.6063 d0.210.2041
H0.645 bcd0.21.60.2317
I0.6 d0.210.1647
LSD0.050.066
F-ANOVA8.11 ***
In column, means followed by the same superscript letters are not significantly different. *** p < 0.001.
Table
Table 6. Mean values, minimal, maximal and standard deviations (SD) as well as homogenous groups of the ratio of the length of the longest polar axis and length of the shortest polar axis (LA/SA), A–I abbreviated habitat names (see: Table 1).
Table 6. Mean values, minimal, maximal and standard deviations (SD) as well as homogenous groups of the ratio of the length of the longest polar axis and length of the shortest polar axis (LA/SA), A–I abbreviated habitat names (see: Table 1).
HabitatMeanMinMaxSD
A1.37 ab120.186
B1.342 b120.172
C1.366 b11.90.188
D1.371 ab0.6432.4440.206
E1.371 ab11.9090.179
F1.388 ab11.9090.223
G1.348 b12.20.212
H1.418 a12.10.230
I1.378 ab1.0671.750.154
LSD0.050.051
F-ANOVA3.1 **
In column, means followed by the same superscript letters are not significantly different. ** p < 0.01.
Table
Table 7. F-statistic value from one-way analysis of variance for a comparison of samples in particular habitats for five quantitative traits, A–I abbreviated habitat names (see: Table 1).
Table 7. F-statistic value from one-way analysis of variance for a comparison of samples in particular habitats for five quantitative traits, A–I abbreviated habitat names (see: Table 1).
F-ANOVATrait
HabitatLAExSALA/SAEx/LA
A4.09 ***28.25 ***9.23 ***3.59 ***23.00 ***
B6.86 ***11.84 ***7.14 ***4.90 ***12.09 ***
C20.62 ***0.238.08 **0.5324.40 ***
D9.69 ***9.45 ***4.03 ***5.00 ***12.93 ***
E2.10 *10.90 ***2.75 **3.09 ***11.61 ***
F16.78 ***5.17 **4.83 *12.93 ***1.21
G8.18 ***7.33 ***9.09 ***15.37 ***13.39 ***
H6.98 ***7.47 ***5.85 ***4.55 ***14.95 ***
I1.93.72.835.66 *4.91 *
* p < 0.05; ** p < 0.01; *** p < 0.001; LA: length of the longest polar axis, Ex: exine thickness, SA: length of the shortest polar axis.
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Wrońska-Pilarek, D.; Bocianowski, J.; Lechowicz, K.; Wiatrowska, B.M.; Janyszek-Sołtysiak, M.; Beker, C. How Do Pollen Grains of Convallaria majalis L. Respond to Different Habitat Conditions? Diversity 2023, 15, 501. https://doi.org/10.3390/d15040501

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Wrońska-Pilarek D, Bocianowski J, Lechowicz K, Wiatrowska BM, Janyszek-Sołtysiak M, Beker C. How Do Pollen Grains of Convallaria majalis L. Respond to Different Habitat Conditions? Diversity. 2023; 15(4):501. https://doi.org/10.3390/d15040501

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Wrońska-Pilarek, Dorota, Jan Bocianowski, Kacper Lechowicz, Blanka Maria Wiatrowska, Magdalena Janyszek-Sołtysiak, and Cezary Beker. 2023. "How Do Pollen Grains of Convallaria majalis L. Respond to Different Habitat Conditions?" Diversity 15, no. 4: 501. https://doi.org/10.3390/d15040501

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Wrońska-Pilarek, D., Bocianowski, J., Lechowicz, K., Wiatrowska, B. M., Janyszek-Sołtysiak, M., & Beker, C. (2023). How Do Pollen Grains of Convallaria majalis L. Respond to Different Habitat Conditions? Diversity, 15(4), 501. https://doi.org/10.3390/d15040501

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