Germination of different gyrogonite types of Chara intermedia A. Braun 1836

SUMMARY The paper presents the germination of different types of gyrogonites of Chara intermedia A. Braun 1836. The study material was collected from the surface layer of sediments (sediment gyrogonites) and from dead C. intermedia specimens inhabiting a post-excavation pit. As a result of a low level of water and seasonal drying, two morphological types of gyrogonites taken from the thallus were distinguished: fully ripe gyrogonites and gyrogonites in oosporangium remains. The highest germination rate was recorded for the sediment gyrogonites. At the end of the experiment, about 28% of germinating gyrogonites originating from sediments were observed. The value of this parameter was over 3-fold higher than that of fully ripe gyrogonites produced by the thallus and more than 5-fold higher in relation to gyrogonites in the oosporangium remains. The results of this experiment indicate that the germination of the two morphological types of gyrogonites taken from plants depends on the degree of their maturity and can take place under limited light conditions. Drying of charophyte thallus in shallow water bodies may have a signiicant impact on the degree of maturity of gyrogonites, their morphological differentiation, and sediment seed bank characteristics.


INTRODUCTION
Characeae (charophytes or stoneworts) are macroscopic green algae that occur in different types of surface waters, especially in freshwater bodies. These organisms widely appear in lowing waters, and speciic species also inhabit brackish and salty water (10,13,33). Investigation of this group of macroalgae is extremely important because they play a signiicant role in the functioning of aquatic ecosystems (23). They bind biogens, take part in stabilization of sediments, and play a basic role in the maintenance of clear water state (9,23,24,46,48). As one of the irst colonizers, charophytes play a key role in restoring vegetation in water bodies (1,46). Re-establishment of these submerged macrophytes is a clear signal of improvement of the water ecological status, as well as the desired effect of renaturation (31). however, charophytes are sensitive to changes in water transparency, especially in shallow lakes, which very often leads to the disappearance of these macrophytes and encroachment of other competing species (2,27,42,45,47).
Stoneworts reproduce by vegetative (for example through bulbils, vegetative fragments) and sexual methods. As a result of fertilization, a zygote (oospore) is formed. hence, oospores are mature oogonia (15,18). Most species enter the ultimate ripening stage (calciication) of the oosporangium, resulting in the formation of gyrogonites (including Chara intermedia) (38). In the ield study, it turned out that the gyrogonites from the dried dead thalli remained with an outer integument cover and the outer wall of spiral cells. Probably, this morphotype is not at full morphological maturity because disadvantageous habitat conditions contribute to impeding the growth and development of gyrogonites.
Research on ecological water systems is an attempt to clarify the role of submerged seed banks in the colonization, vegetation dynamics, and biodiversity (26,29,39). In some species of Characeae, the dispersion, colonization and maintenance of the population depends only on the oospore OLhA BUDNYK, PIOTR SUGIER, zBIGNIEW CIERECh Pobrane z czasopisma Annales C -Biologia http://biologia.annales.umcs.pl Data: 03/06/2020 05:46:07 U M C S 51 bank (3). In the case of many studies, attention is focused on the resources and structure of the oospore bank, which is characterized by longevity (31,42). It turns out that oospores of charophytes may re-establish from oospore banks after several decades (40,41,42). however, less attention is focused on the germination of diaspores forming the so-called "seed rain". In available literature, the lack of results concerning the morphological diversity of oospores, which relected the degree of maturity, in turn determines properties of the underwater seed bank.
In this study, we focused on this problem, given the disadvantageous habitat conditions prevailing in shallow water bodies predominant in the summer of 2015, which were not recorded in the last few decades, including a low level of water and seasonal drying of plants. Therefore, the aim of this study was to determine the germination of C. intermedia gyrogonites (calciied oospores), a species commonly occurring in the water bodies in eastern Poland.

MATERIALS AND METhODS
The ield study was performed in a post-excavation water body (peat pit) (N: 51 o 21'42"; E: 23 o 14'51") formed several decades ago on a calcareous fen. The water level of the studied peat pit was monitored during the last several years (unpublished data). The summer of 2015 was very dry, which was conirmed in the meteorological data from the Łęczna-Włodawa Lakeland (www. en.tutiempo.net (49), Włodawa). Such a drastic decrease in the water level was not reported in the earlier vegetation seasons. It caused the emergence of desiccated reproductive plants with immature gyrogonites and sediment desiccation. Probably, this caused gyrogonites to remain in the oosporangium and inhibited their growth and germination.
The materials used in the studies were gyrogonites of C. intermedia collected from a dried dead thallus and from sediments. The method of selection of the gyrogonites for germination is described as follows: ten randomly chosen collection sites (each 0.1 m 2 ) were selected in the peat pit. At each point, all the individuals of C. intermedia were taken and ten sediment cores from the top layer (0-10 cm thick) were sampled with the use of a plastic tube (5.6 cm in diameter). Sedimentary gyrogonites (type 1) were washed with water on a coarse sieve to remove some debris and then through a ine 0.2 mm sieve. From reproductive but dried dead plants, gyrogonites (type 2) and gyrogonites in oosporangium remains (type 3) were obtained. The latter morphotype has not been described to date. Sediment samples and gyrogonites were stored in water at 4°C within 4 weeks of collection until the experiment.
The morphometric analysis of gyrogonites was carried out according to horn af Rantzien (21) and haas (19) by means of a stereoscopic microscope Olympus SzX 16, using a Stream Motion program. The features of the diaspores were observed and imaged under a scanning electron microscope TESCAN VEGA3-LMU at the Laboratory of the Department of Botany and Mycology and the Department of zoology of Maria Curie-Skłodowska University. In the laboratory conditions, it was found that C. intermedia was the dominant species and Chara globularis had a smaller share.
The germination experiment was conducted in a controlled vegetation chamber in the dark at a temperature of 10°C in water in four repeats. The pooled material was homogenized, and the three selected gyrogonite types in ive series (each 100 gyrogonites) were chosen. The irst portion of the material was analysed after 25 days, and the next series were examined regularly at 10-day intervals, performing microscopic observations. Based on the data obtained, the germination of all distinguished gyrogonite types was determined. Apparently viable gyrogonites were identiied as those having turgid, healthy starch reserves when squeezed with forceps (6). The germination percentage was calculated as the number of germinated gyrogonites divided by the number of apparently viable gyrogonites.

GERMINATION OF DIFFERENT GYROGONITE TYPES OF ChaRa inteRmedia A. BRAUN 1836
Pobrane z czasopisma Annales C -Biologia http://biologia.annales.umcs.pl Data: 03/06/2020 05:46:07 The Kruskal-Wallis test was used to compare two or more independent samples. The Mann-Whitney test was used to verify the differences between gyrogonites types. The level of signiicance was chosen as p<0.05. The Statistica 11.0 software program was used to analyse all data.

rESuLtS
The germination dynamics of the gyrogonite morphotypes varied (Figs.  1-3). After twenty-ive days of the experiment, the share of germinating sediment oospores was about 21%, whereas ten days later it was about 24%, and the increase was statistically signiicant (Fig. 1). Germination was markedly higher after forty-ive days, and the rate of oospore germination was approximately 30%. During the two following terms of observations, a similar percent of germinating oospores was recorded, and there were no statistically signiicant differences.
In the case of gyrogonites taken from the thallus, after twenty-ive days of the experiment, more than 5% of germinating gyrogonites were found (Fig. 2). In the following days of observation, the percentage of germinating gyrogonites was in the range of 6-9%; because of the large variability of the data, the differences between the values of this parameter were not statistically signiicant (Fig. 2).  Germination of gyrogonites in oosporangium remains was initiated only thirty-ive days after the beginning of the experiment, and their percentage share was over 1% (Fig. 3). Twenty days later, the rate of oospore germination was about 3%. The highest germination percentage of these gyrogonites was noted at the end of the experiment (about 5%), and statistically signiicant differences were found.
Based on the experimental observations, it was found that gyrogonites in the oosporangium remains were characterized by the lowest ability of germination (an average of 1%), and the highest germination ability was exhibited by gyrogonites recovered from the underwater seed bank (Fig. 4). At the end of the experiment, about 28% of germinating gyrogonites from the sediments were observed, and their ability of germination was 3-fold higher than that of gyrogonites produced by the thalli and more than 5-fold higher in relation to gyrogonites produced by the thalli in oosporangium remains (Fig. 4).   (37,41,44). Germination can proceed without a requirement for light (23). hence, there are relatively few experimental studies concerning germination of charophytes in the dark (37,41,44).
De Winton et al. (14) suggest that germination is possible in the absence of light exposure, which is in accordance with the results of the present studies (Figs. 1-4). Also, the data for the other species show that germination in the dark of oospores of Nitella furcata ssp. megacarpa is successful (7.9%) (36). In our experiment conducted in the dark conditions, the rate of germination was several times higher and amounted to 30% (Fig. 1). Thus, a short impulse of light is suficient to trigger germination of charophytes in the dark.
Nevertheless, light is required to activate germination following the phase of dormancy (36). Dormant oospores are not capable of germination. This is important in the case of prevailing disadvantageous conditions (desiccation or winter) (7,30). Generally, fresh oospores, i.e. those produced during the growing season, show deeper secondary dormancy than those originating from sediments (44). Our study results indicate that the gyrogonites produced by the thalli are probably in secondary dormancy. This fact is evidenced by the low germination during the experiment. The average germinability of gyrogonites produced by plants was over 3-fold lower than the average germinability of the theoretically older gyrogonites found in the sediments (Fig. 4).
The disadvantageous habitat conditions prevailing in shallow water bodies can have a signiicant impact on the degree of oospore maturity and morphological diversity (Fig. 5). This results in diverse germinability of diaspores (Figs. 2-3). According to the researchers of this subject, except the morphological aspects of gyrogonites, crucial is the knowledge relating the ecological conditions leading to the formation of oospores, calciication of the fertilized oogonium, and inally the formation of gyrogonites (38). For most species of charophytes, after the oospore has been formed, the process of maturation and formation of gyrogonite is continued (17,38). The unpredictable habitat conditions in the post-excavation pit in summer 2015 led to appearance of charophytes above the water surface. The low water level in the peat pit caused drying of C. intermedia thalli; consequently, different morphological types of body plants developed, which characterized different degree of maturity and germinability. Probably, gyrogonites in oosporangium remains need additional time to mature. This may be the major cause of their delayed germination (Figs. 2-3).
Summing up, disadvantageous habitat conditions prevailing in shallow water bodies can have a signiicant effect on the degree of oospore maturity and morphological variability. The two distinguished morphological types of gyrogonites collected from the thalli are characterized by different degrees of maturity and germination ability, while those deposited in sediments may affect the structure and properties of the underwater seed bank.