Overview of Allelopathic Potential of Lemna minor L. Obtained from a Shallow Eutrophic Lake

Allelopathy is an interaction that releases allelochemicals (chemicals that act allelopathically) from plants into the environment that can limit or stimulate the development, reproduction, and survival of target organisms and alter the environment. Lemna minor L. contains chemicals that are allelopathic, such as phenolic acids. Chemical compounds contained in L. minor may have a significant impact on the development and the rate of multiplication and lead to stronger competition, which may enhance the allelopathic potential. Allelopathic potential may exist between L. minor and C. glomerata (L) Kütz. because they occupy a similar space in the aquatic ecosystem, have a similar preference for the amount of light, and compete for similar habitat resources. L. minor and C. glomerata can form dense populations on the water surface. Allelopathy can be seen as a wish to dominate one of the plants in the aquatic ecosystem. By creating a place for the development of extensive mats, an interspecific interaction is created and one of the species achieves competitive success. It is most effective as a result of the release of chemicals by macrophytes into the aquatic environment. Therefore, allelopathy plays a significant role in the formation, stabilization, and dynamics of the structure of plant communities.


Introduction
The plant community is shaped by specific plant species that coexist in a particular place at a particular time [1]. Their presence in a community is determined by many ecological aspects, including biotic and abiotic factors [2]. Interspecies relationships are an important biotic factor, as a result of which individuals compete with each other. These interactions take place directly or indirectly in the individual-individual and individualenvironment relationships [1]. As a consequence, it is possible to inhibit the development of one of the co-occurring species, which ensures the competitive success of another species. One example of such an interaction is allelopathy. Allelopathy is a series of interactions that occur in the environment between various organisms [3]. As a result of this process, mixtures of allelopathic chemicals are released from plants into the environment [3]. Allelochemicals can directly or indirectly limit or stimulate the development, reproduction, and survival of target organisms, also affecting the environment [4].
Large amounts of nutrients are present in eutrophic water bodies. The consequence of this is the excessive development of biological life, including the massive appearance of phytoplankton organisms and other aquatic plants that create blooms on the water surface and reduce the transparency of the water. An excessive development of green algae, mainly Cladophora (Chlorophyta) species, is observed in eutrophic water reservoirs. Lemna minor, belonging to the Lemnaceae family, is also a common aquatic plant.
Lemna minor is a small plant (pleustophyte) with a simple morphological structure. It consists of a single thallus and has no root [17,18]. The plant reproduces mainly vegetatively [19]. In summer, L. minor forms single-species, dense, and compact clusters that float freely on the water surface [9,20]. These patches inhibit the development of other macrophytes, limiting their access to light by up to 99% [21]. The increase in plant biomass over a short time contributes to the eutrophication of the aquatic ecosystem. To survive the colder months, the pleustophyte forms small turions that are filled with starch and sink to the bottom of the water reservoir [10,22]. Most often, L. minor occurs in waters with a temperature of 6 to 33 • C. It also has a wide range of pH tolerance, from 5 to 9 [23]. L. minor prefers shallow places with low water turbulence that are rich in electrolytes [21,24]. The growth, survival rate, and growth rate are influenced by various ecological factors, e.g., pH, water temperature, concentration of nutrients, presence and concentration of toxins in water, as well as competition with other plants for light and nutrients [22]. However, temperature and light availability are the factors that best stimulate the proper growth and development of L. minor [22]. It has the ability to absorb minerals as well as phosphorus and potassium that are present in nutrient-rich water [22]. A threat to the development of L. minor is the intense growth of algae, including the green algae of the genus Cladophora, which limit the plant's access to light and nutrients [22]. As a result, it has limited space and resources to create dense colonies on the surface of a body of water.
Cladophora glomerata (L) Kütz. is a green alga that dominates in eutrophic waters [25,26]. Due to the diversified morphology of the plant, the thallus may be long and branched in flowing waters and short and bushy in stagnant waters [27]. The lifting force of water enables the movement of the thallus, which can change its shape and place of occurrence. It creates filamentous forms, called grippers, by means of which it attaches to the ground or floats in the water column [27]. C. glomerata forms dense and large populations ("mats") on the surface of a water body [21,28]. Intensive development occurs in spring and autumn, as a result of which it creates massive blooms [27]. The density and size of the biomass often depend on the growing season and the environmental conditions in which it grows [29]. Green alga mats may consist of individuals of one species (Cladophora glomerata) or of several co-occurring species [25]. Although C. glomerata occurs in most types of aquatic ecosystems (from stagnant waters to flowing waters), it is most numerous in eutrophic waters with a high concentration of nitrogen and phosphorus [21,25]. It has preference for a wide range of temperatures and light conditions; therefore, it easily adapts to the ecosystem it currently inhabits [21,25]. Owing to its uncomplicated morphological structure, rapid multiplication rate, and high ecological tolerance, C. glomerata easily forms large populations and occupies considerable space [26,29]. This alga can limit access to light by up to 70%, create anaerobic conditions, and inhibit the supply of nutrients and thus inhibit the development of macrophytes [26,29,30]. C. glomerata, similarly to L. minor, prefers waters with a high content of nutrients as well as nitrogen and phosphorus [29]. An important element for the proper growth of green algae is the optimal chemical composition of water, in which there is a high concentration of nitrates and orthophosphates [25].
As shown in the above literature data, L. minor and C. glomerata are species with similar ecological preferences. Both plants occupy a similar space in the aquatic ecosystem, have a similar preference for the amount of light, and compete for similar habitat resources. Both L. minor and C. glomerata can form dense populations that float on the water surface. As a result, they inhibit the growth of other hydro macrophytes as they restrict their access to light and nutrients. Their chemical composition indicates the presence of substances with an allelopathic effect. The presence of phenols such as phenyl ester, methoxylphenol, coumaric acid, and benzoic acid has been found in the thalli of filamentous green algae [31]. Unfortunately, there is little research explaining the mechanism of interaction between these plant species. However, it can be concluded that there is an allelopathy phenomenon between them, which may be visible when one plant wants to dominate the aquatic ecosystem. Creating a place for the development of extensive mats allows an interspecific interaction (allelopathy) to occur, thanks to which one of the species achieves competitive success. This is an important point that we want to highlight in this article. Therefore, this paper provides an overview of the allelopathic potential of duckweed and reviews the studies on models of Lemna minor and Cladophora glomerata population formation with a focus on their chemical composition. Moreover, with reference to the studies, the aim of the research presented in the paper is to answer the following questions:

1.
Do Lemna minor and Cladophora glomerata secrete allelopathic substances in coexistence?

2.
Does the allelopathic potential occur in competing interactions?

Chemical Composition Analysis of L. minor and C. glomerata
The analysis of the chemical composition of L. minor and C. glomerata showed the content of chemical elements (Tables 6 and 7). In the case of L. minor, the following elements were identified: potassium, calcium, phosphorus, magnesium, nitrogen, iron, boron, copper, zinc, and manganese (Table 6). Among all analyzed elements, potassium (2194 mg·kg −1 ) and calcium (1,3020 mg·kg −1 ) were characterized by the highest content in L. minor compared to other elements. Manganese (68.4 mg·kg −1 ) had the lowest amount in L. minor. In the case of C. glomerata, the following elements were identified: calcium, potassium, magnesium, lead, arsenic, sodium, iron, nickel, manganese, cadmium, copper, zinc, cobalt, and chrome ( Table 7). The highest content was that of calcium (148.2 ± 3.1) in dry mass. Chrome had the lowest content (0.01 ± 0.001) in dry mass. Table 6. Elemental composition observed in the freshwater L. minor filaments.

Element
The Element Content (mg·kg −1 )   (Figures 3 and 4). The test sample contained 30 C. glomerata cells and 30 L. minor individuals. Cell length values in C. glomerata were not statistically significant. The C. glomerata cell width was statistically significant in April (a) and June (a) in relation to the cell width in July (b). Statistically significant differences were also observed in the diameters and thicknesses of L. minor cells. The diameters of L. minor cells showed statistically significant differences among themselves (a, b, c) in April, June, and July. Cell thicknesses also showed significant differences between themselves (a, b, c) in April, June, and July. The highest mean cell length (310 µm) of C. glomerata was in July and the lowest in June (276.5 µm). The greatest difference was between the average widths in June (63 µm) and July (46.1 µm). In the case of L. minor, the highest average diameter (2048.6 µm) and thickness (918 µm) of individuals was in July. The smallest average diameter (1434.6 µm) and thickness (684.5 µm) of individuals was in June.

The Size of the Patches of L. minor and C. glomerata in the Littoral Zone
The mean value, the minimum value, the maximum value, and the standard deviation were analyzed in terms of the size of the patches of C. glomerata and L. minor in the littoral zone (Table 8)

The Content of Phenolic Acids in L. minor and C. glomerata
Phenolic acids were found in all the tested C. glomerata individuals from Oporzyńskie Lake, L. minor individuals from Oporzyńskie Lake, and L. minor from breeding ( Table 9). The phenol content in C. glomerata was significantly higher than the phenol content in L. minor. Table 4 shows that the lowest minimum concentration of phenols (7.418 mg GAE g −1 ) in C. glomerata occurred on 30 April 2021 and the highest minimum concentration of phenols (17.934 mg GAE g −1 ) was on 4 June 2021. The lowest maximum value of phenol concentration was 7.601 mg GAE g −1 (30 April 2021), and the highest maximum was 18.459 mg GAE g −1 (4 June 2021). The lowest average concentration of phenols was 7.515 mg GAE g −1 , which occurred on 30 April 2021. The highest average concentration of phenols was 18.209 mg GAE g −1 , which occurred on 4 June 2021. The concentration of phenols in L. minor collected from Oporzyńskie Lake significantly differed from the concentration of phenols in individuals from the farm and from the concentration of phenols in C. glomerata. The lowest minimum concentration of phenols in the species L. minor from Oporzyńskie Lake was 2.111 mg GAE g −1 (30 April 2021), and the highest minimum concentration of phenols was 9.423 mg GAE g −1 (17 July 2021). The lowest minimum concentration of phenols was 2.892 mg GAE g −1 (30 April 2021) and the highest 9.816 mg GAE g −1 (17 July 2021). The lowest average concentration of phenols was also 2.111 mg GAE g −1 (30 April 2021), and the highest was 9.582 mg GAE g −1 (17 July 2021). The lowest minimal (1.794 mg GAE g −1 ), the lowest maximum (2.006 mg GAE g −1 ), and average (1.902 mg GAE g −1 ) concentrations of phenols in individuals from the cultivation of L. minor occurred on 30 April 2021. The minimum concentration of phenols (2.029 mg GAE g −1 ) occurred on 17 July 2021 and the highest maximum (2.052 mg GAE g −1 ) and the highest mean were recorded on 4 June 2021.  Table 9. The mean value, the minimum value, the maximum value, and the standard deviation of phenol concentration of C. glomerata from Oporzyńskie Lake, L. minor from Oporzyńskie Lake, and L. minor from breeding (mg GAE g −1 ).

Relationships between the Parameters of Dry Mass of L. minor and C. glomerata and the Effect of Polyphenol Concentration and Time
The results of the dry mass content in C. glomerata ( Figure 5) and L. minor ( Figure 6) showed that C. glomerata had a higher dry mass content in June and July than L. minor. In April, C. glomerata had a slightly lower content of dry mass than L. minor. Considering the results separately, the dry mass of C. glomerata had the highest mean value in July and the lowest in April. It was similar for L. minor-the highest average dry mass value was in July and the lowest in April. Both in C. glomerata and L. minor, the mean value of dry matter was statistically significant between the tested trials from April, June, and July (a, b, c). the effect of the observation time ( Figure 8) were analyzed using the GAM model. The first model showed that in both C. glomerata and L. minor, the concentration of polyphenols increased in cells over time (Figure 7). However, in the case of C. glomerata, the biomass increased faster with increasing polyphenol concentration (Figure 7). The biomass of L. minor increased significantly slower with the increase in the concentration of polyphenols as compared to C. glomerata (Figure 7). The second model showed that C. glomerata is characterized by a faster increase in biomass during the observation than L. minor (Figure 8).    The response of C. glomerata and L. minor to the polyphenol content ( Figure 7) and the effect of the observation time ( Figure 8) were analyzed using the GAM model. The first model showed that in both C. glomerata and L. minor, the concentration of polyphenols increased in cells over time (Figure 7). However, in the case of C. glomerata, the biomass increased faster with increasing polyphenol concentration (Figure 7). The biomass of L. minor increased significantly slower with the increase in the concentration of polyphenols as compared to C. glomerata (Figure 7). The second model showed that C. glomerata is characterized by a faster increase in biomass during the observation than L. minor (Figure 8).

Discussion
Taxa co-occurring in the same place may use allelopathy as a strategy in competition for space, nutrients, light, and other ecological factors [26,40]. Competition can result in the replacement or exclusion of a species. It is most effective as a result of the release of chemicals by hydro macrophytes into the aquatic environment. Therefore, allelopathy plays a significant role in the formation, stabilization, and dynamics of the structure of plant communities [6,26,41]. Both Lemna minor and Cladophora glomerata inhabit common habitats, show similar preferences for the availability of light and nutrients, and create dense mats on the water surface. Plants need space for proper development, so they compete with each other for the surface layer of water.
The conducted analyses showed that: (1) Lemna minor and Cladophora glomerata secrete allelopathic substances, (2) allelochemicals have a significant impact on the structure of pleustophyte and alga communities, and (3) allelopathic potential occurs as a result of competition between co-occurring species.
The results of our research show that the average length and width of cells in C. glom-

Discussion
Taxa co-occurring in the same place may use allelopathy as a strategy in competition for space, nutrients, light, and other ecological factors [26,40]. Competition can result in the replacement or exclusion of a species. It is most effective as a result of the release of chemicals by hydro macrophytes into the aquatic environment. Therefore, allelopathy plays a significant role in the formation, stabilization, and dynamics of the structure of plant communities [6,26,41]. Both Lemna minor and Cladophora glomerata inhabit common habitats, show similar preferences for the availability of light and nutrients, and create dense mats on the water surface. Plants need space for proper development, so they compete with each other for the surface layer of water.
The conducted analyses showed that: (1) Lemna minor and Cladophora glomerata secrete allelopathic substances, (2) allelochemicals have a significant impact on the structure of pleustophyte and alga communities, and (3) allelopathic potential occurs as a result of competition between co-occurring species.
The results of our research show that the average length and width of cells in C. glomerata as well as the diameter and thickness of L. minor individuals showed significant differences in the analyzed time (April-June-July). The highest mean length of C. glomerata cells was in July and the lowest in June. In the case of L. minor, the highest average diameter and thickness of individuals was in July (in the fullest of the growing season). The smallest average diameter and thickness of individuals was in June. Considering the size of the patches of C. glomerata in the littoral zone, the largest occurred in July and the smallest in April. L. minor, however, had the largest patches in April and the smallest in July. Additionally, these results show that C. glomerata created more extensive patches on the water surface of Oporzyńskie Lake as compared to L. minor.
The length and width of C. glomerata cells and L. minor individuals as well as the size of their patches ("mats") also showed a relationship with the total phenolic content in these species. It is worth noting that the phenol content in C. glomerata was higher than the phenol content in L. minor. The highest concentration of phenols in green algae had the highest and most similar values in June and July and the lowest in April. L. minor that was collected from Oporzyńskie Lake had the highest concentration of phenols in July and the lowest in April. L. minor from the cultivation had a lower content of phenols compared to L. minor and C. glomerata that were collected from the Oporzyńskie Lake. In this case, phenols reached their highest values in June and lowest in April.
These results show that both C. glomerata and L. minor have allelopathic potential. However, C. glomerata shows greater potential and therefore is a stronger taxon in interspecies competition. C. glomerata has a specific feature. This is the rapid development of the thallus and dynamic multiplication. At the same time, it produces a large quantity of phenolic compounds. Our research confirms these features. Both in C. glomerata and in L. minor, the concentration of polyphenols in the cells increases with time. However, the biomass of C. glomerata grows much faster compared to that of L. minor. As a result, C. glomerata can form broad and dense mats on the water surface and successfully compete for space with duckweed. The macroalga C. glomerata characteristically dominates in eutrophic waters [25] and has a rapid biomass growth over time, and our findings have confirmed this dependence. As a result, the alga excludes the co-occurring taxon and achieves competitive success over other aquatic plants [24,25]. This mechanism provides green algae with a large space for proper development and access to light and nutrients. Phenolic compounds secreted by C. glomerata may influence the formation of communities of small pleustophytes, including L. minor. C. glomerata showed a higher total phenolic content than L. minor. The greater the concentration of green algae in the surface water layers, the more phenols are released into the environment. This inhibits the development of L. minor and limits the size of its patches on the water surface. The strategy of freshwater algae in the process of competition is: intensive development in early spring, taking nutrients from the water, chemical composition of the thallus (presence of proline in the period of high thallus density), changes in the morphological structure, secretion of allelopathic substances, and the formation of spores [26] (from Ozimek 1990).
Lemna minor may also inhibit the development of other hydromacrophytes. This pleustophyte creates dense mats on the water surface. As a result, it reduces the amount of light available for the deeper layers of water, which is necessary for other plants to grow adequately [20]. Both the literature data and our results show that this pleustophyte contains allelopathic chemicals. During coexistence with C. glomerata, a higher content of phenolic compounds was demonstrated in L. minor individuals than in bred individuals. This shows that L. minor also has a competitive strategy. Pleustophyte competes with C. glomerata for space, light, and nutrients, so it uses its allelopathic potential to achieve competitive success. When bred, L. minor does not have to compete with other plants for space and access to nutrients. Therefore, the total phenolic content is lower as it is not necessary for survival. It is likely that these allelochemicals may also inhibit the development of other plants. As a result, L. minor can ensure a competitive advantage in the aquatic ecosystem [20]. However, the mechanism of the allelopathic strategy has not yet been elucidated.

Conclusions
Finally, we would emphasize that our efforts have been directed in two ways: (i) to review the studies on models of Lemna minor and Cladophora glomerata population formation with a focus on their chemical composition and (ii) to mark a particular model of seasonal growth of two species occupying one ecological niche at the same time. In our study, the concentration of polyphenols in the cells of C. glomerata and in L. minor increases with time. The role of rapid biomass growth of one of the competing species seems to be crucial. To assess the effectiveness of Lemna minor allelopathic potential, many factors need to be determined. The research carried out in this article highlights the poorly understood relationships between species (duckweed and macroalga) that coexist in the same place at the same time in the aquatic environment. The mechanisms that would accurately explain the phenomenon of allelopathy between competing taxa are also unknown. These issues require further research, which we will certainly undertake in well-defined aquatic systems during laboratory experiments.

Raw Material Collection and Identification
Freshwater Cladophora glomerata thalli and Lemna minor specimens were collected from the shallow Lake Oporzyńskie (N 52 • 55 70", E 17 • 09 60"), situated in the northern part of the Wielkopolska region (western Poland), in the April-July period of 2021, when the algal biomass was at its annual minimum and maximum. Characterization of physical and chemical parameters during the intensive development of C. glomerata in the lake has been performed earlier [24,25,42]. Morphometric measurements of the length and width of cells and individuals were taken using a light microscope (LM).

Element Analysis of Lemna minor and Cladophora glomerata Raw Material
The content of selected elements, such as Ca, Mg, Na, K, Fe, Zn, Cu, As, Cd, Ni, Pb, Cr, Mn, and Co, was determined in the Lemna and Cladophora biomass. The elemental analysis was carried out in an inductively coupled plasma-optical emission spectrometer (Varian ICP-OES VISTA-MPX) by the ICP-OES method. The concentration of elements was expressed as µg·g −1 of dry matter or g·g −1 of dry matter or mg·kg −1 (in the case of Lemna minor). Then the analysis error was calculated.

Ultrasound-Assisted Extraction (UAE)
Ultrasound-assisted extracts were made out of raw, powdered material of C. glomerata and L. minor. In each case, for the preparation of extracts for spectrometric analysis, 10 g of dry weight of material was extracted in an ultrasonic bath with two portions of methanol as a solvent (2 × 100 mL), over a total time of 1 h. After 30 min, the first portion of the solvent was removed and a new portion was added to continue the extraction for another 30 min. The temperature of the ultrasonic bath did not exceed 35 • C. The extracts were filtrated, and the filtrates were combined. The solvent was removed in the rotary evaporator. To prepare samples for spectrometric analysis, the methanolic solutions of extracts were made up to a concentration of 10.00 ± 0.06 mg mL −1 .

Determination of Total Phenolic Compounds in Extracts
A calibration curve was prepared by dissolving gallic acid in 70% methanol to obtain a stock solution with a concentration of 1 mg/mL, after which subsequent dilutions were made in a range of concentrations from 0.1 mg/mL to 1 mg/mL. Then 20 µL of gallic acid solution with a particular concentration was added, along with 1.58 mL of distilled water, 0.1 mL of Folin-Ciocalteu reagent, and 0.3 mL of saturated solution of sodium bicarbonate (Na2CO3). The final reaction volume was 2 mL, and the final concentration of gallic acid ranged between 0.001 and 0.01 mg/mL [43]. A reaction mixture with real samples was prepared in the same way as the samples for the calibration curve, and the extract solution was added instead of gallic acid solution. The result was expressed as gallic acid equivalent (GAE) using the equation C [mg QE/gextract] = C [mg/mL] × (V1 [mL]/V2 [mL]) × (V3 [mL]/m [g]), where C (mg/mL) is the concentration from the calibration curve, V1 is the total volume of the reaction vessel, V2 is the volume of the extract/standard added to the reaction, V3 is the volume in which the extract was dissolved, and m is the mass of the extract dissolved in V3 to prepare a real sample of extract. After keeping the reaction vessels in darkness for 2 h, the samples were measured with a UV/VIS spectrometer at 760 nm. Each sample was prepared and measured in triplicate. Data are the mean ± the SD values.

Data Analysis
Statistical analyses were performed with STATISTICA software version 12.0. To confirm the significance of differences between the analyzed Lemna minor and Cladophora glomerata features in time, a one-way ANOVA followed by Tukey's RIR post hoc test was used. Differences were considered to be significant at p < 0.05. These statistical analyses were performed using the R 3.0.1 statistical package (R Development Core Team 2013, using the vegan package; [44]).
Changes of Lemna and Cladophora biomass occurrence in response to time were modeled using the GAMs [45]. We used Poisson distribution, while smooth term complexity was selected using the Akaike information criterion [46].