Effects of cadmium on gametophyte growth and archegonia development in the endangered floating fern Ceratopteris pteridoides in China

Abstract

Ceratopteris pteridoides is an endangered annual floating fern in China. This study tested the effect of cadmium on gametophyte growth and archegonia development of the aquatic fern C. pteridoides in culture. The results show that C. pteridoides spores germinated after being treated with different cadmium, but it was not significant compared with the reference group (45.63%). With the increase of cadmium, the rates of hermaphrodite gametophyte, the number of archegonia of C. pteridoides decreased, and archegonium differentiation was delayed gradually, indicating that cadmium caused adverse effects on sexual organ differentiation of C. pteridoides, further exacerbating the endangered status of this species in China. We suggest that it is necessary to pay more attention to protecting the primary habitats of C. pteridoides in China and reduce the impact of human activities from excessive aquaculture and industrial wastewater discharge on the water body in its living water body.

Key Policy Highlights

Ceratopteris pteridoides is an endangered aquatic fern, and listed in the second category of protected wild plants in China.

With the increase of the concentration of Cd²⁺, the proportion of hermaphroditic gametophyte and the number of archegonium decreased gradually, indicating that cadmium caused adverse effects on sexual organ differentiation of C. pteridoides.

We suggest that it is necessary to protect the primary habitat of the species, especially the protection of its living water body.

Keywords:

• Ceratopteris pteridoides
• endanger
• cadmium
• gametophyte growth
• archegonia development

1. Introduction

Ceratopteris pteridoides (Hook.) Hieron. (Parkeriaceae), is an endangered, annual, floating, fern (Yu 1999), growing mainly in ponds, lakes, rivers and ditches in Central and South America, Southeastern Asia, Eastern India and China (Hickok et al. 1995). In China, the species is endangered, and was listed in the second category of protected wild plants in 1999 (Yu 1999) and 2021 (National Forestry and Grassland Administration, PRC 2021). Sexual reproduction by spores and subsequent gametophytes, and clonal reproduction are the main methods of reproduction of this species (Yu 1999; Hickok et al. 1995). Previous research showed that aquatic ferns could represent ideal species for studying the effects of water pollutants on the entire life cycle under laboratory conditions (Gupta et al. 1992). The growth and differentiation of fern gametophytes can be affected by several physical and chemical factors (Hickok and Kiriluk 1984). Dong et al. (2012, 2022) reported that water chemistry with pH values and dissolved oxygen might have significant influences on the sexual reproduction of C. pteridoides, and could be closely correlated with the decline and extirpation of the species. Several earlier studies reported that the effect of auxins (such as IBA, IAA, NAA, a-naphthaleneacetic acid, 2,4,5-trichlorophenoxy acetic acid, 2,4-D, ABA, GA), antheridiogen, cadmium on sexual differentiation and gametophyte growth in the fern Ceratopteris richardii, Ceratopteris thalictroides (Banks et al. 1993; Hickok et al. 1995; Gregorich and Fisher 2006; Dong et al. 2012; Ganger and Sturey 2012). Herbicides Bensulfuron-methyl and quinclorac, and pH values affected sexual reproduction and gametophyte growth in aquatic fern C. pteridoides (Tao et al. 2008; Dong et al. 2022). Bora and Sarma (2021) reported anatomical and ultrastructural alterations in C. pteridoides under cadmium stress. Monashree et al. (2020) estimated the tolerance mechanism of translocation and subcellular distribution for cadmium in C. pteridoides. Wang et al. (2021) and Ji (2019) reported that some pteridophytes such as Phyllitis japonica, Sphaeropteris lepifera, Alsophila costularis and Alsophila gigantea suffered from chlorosis of gametophyte chloroplasts and damage to gametophyte development under cadmium (Cd²⁺) stress, thus affecting reproductive development. Cadmium is a widely present non-essential element in organisms in aquatic environments and is recognized as one of the main pollutants in the water environment (Adam et al. 2019; Long et al. 2022). Its concentration in polluted water bodies ranges from 6 to 360 μmol L⁻¹, the highest concentration in wastewater can reach 2224 μmol L⁻¹ (Deng et al. 2014). Cadmium in the water environment mainly comes from the development and utilization of metal mining, urban domestic sewage, agricultural production and cadmium-containing exhaust gases in the atmosphere (Hua et al. 2019). With the rapid development of industrialization, a large amount of cadmium-containing wastewater is discharged, and human production and household waste disposed of improperly, the problem of cadmium pollution in the water environment is becoming increasingly serious (Qu et al., 2016). Cadmium has a long half-life in living organisms and is not easily degraded. Even at lower concentrations, it has strong biological toxicity and hence poses a serious threat to plants, animals and microorganisms, and can subsequently harm human health through the food chain. However, studies on the effect of heavy metals on sexual reproduction and gametophyte growth of C. pteridoides have not been reported. The principal aims of this study were to investigate the effect of heavy metals cadmium (Cd) on gametophyte growth and archegonia development in the endangered floating fern C. pteridoides, and to provide information for the conservation of the species.

2. Materials and methods

2.1. Plant material

In this study, the mature spores of C. pteridoides come from Houguan Lake (N 30°30′, E 114°08′) in Wuhan, China. The spores are kept in airtight, dry bags at room temperature.

2.2. Growth condition

In this experiment, the concentration gradient of Cd²⁺ was set according to the maximum allowable discharge concentration of 0.1 mg L⁻¹ Cd in China’s water pollutant discharge limit standard and surface water environmental quality standard (GB 3838-2002). Cd²⁺ concentrations of 0, 0.01, 0.1, 1, 5, 10 and 15 mg L⁻¹ were made in Knop’s nutrient solution containing 1.5% agar (Gupta et al. 1992; Dong et al. 2022). A reference group (Cd²⁺ concentrations was 0 mg L⁻¹) using Knop’s nutrient solution containing 1.5% agar was performed simultaneously with the experimental run. The main process is as follows: first, disinfect the spores, about 500–600 sterilized spores are inoculated in each Petri dish, and then culture the spores in SPRX-600B intelligent artificial climate box. A total of 8 days after spore sowing, the spore germination rate was recorded under a Jiangnan BM2000 digital microscope (Dong et al. 2022). Three horizons for observation in each culture plate of three replicate plates were selected randomly. A total of 14 days after spore sowing, gametophyte size and occupancy were recorded for the three culture plates representing each Cd²⁺ concentration. Two gametophytes were selected in each horizon and photographed under the digital microscope. Each gametophyte area was measured as pixels through digital photographs. A total of 16 days after spore sowing, the number of hermaphroditic gametophytes in each culture plate was recorded, and recorded one time per 2-day interval, for a total of three times (Dong et al. 2022).

2.2.1. Data collection and statistical analysis

All parameters were analyzed using SPSS 17.0 software. Level of significance was set at p < .05. Gametophyte areas of C. pteridoides were fitted using Log-Logistic Concentration Model (Seefeldt et al. 1995): $$U=C+\hspace{0.17em}\frac{D-C}{1+\mathit{\text{exp}}\left[\text{B}\hspace{0.17em}\times \left(\mathit{\text{log}}\hspace{0.17em}\left(x\right)-\mathit{\text{log}}\hspace{0.17em}\left({\text{EC}}_{50}\right)\right)\right]}$$

Here, U represents response of the gametophyte area to different concentrations of Cd²⁺, namely the size of the gametophyte area after fitting, C and D represent response of the upper and lower limits of the gametophyte area to different concentrations of Cd²⁺ respectively, EC₅₀ represents the concentration of Cd²⁺ where the gametophyte area was inhibited and reached half effect. B represents the slope of the curve at EC₅₀. In the present study, the responses of upper and lower limits of the gametophyte area to different concentrations of Cd²⁺ were set as the control value and 0, respectively.

3. Results

3.1. Effect of Cd²⁺ on spore germination

Spores of C. pteridoides germinated after being treated with different heavy metal Cd²⁺. With the increase of Cd²⁺concentration, spore germination rates decreased (Figure 1). The spore germination rate of reference group was 45.63 ± 5.48%. In the maximum allowable discharge concentration of 0.1 mg L⁻¹ Cd²⁺ in GB 3838-2002, spore germination rate was 37.27 ± 5.17%. At 15 mg L⁻¹ Cd²⁺, the spore germination rate was 25.77 ± 3.30%, which was the lowest. However, an ANOVA analysis revealed that spore germination rates of different Cd²⁺ were no significant difference than that of the reference group (45.63%, Figure 1).

Figure 1. Effect of Cd²⁺ on spore germination of C. pteridoides. Data are means ± SD (n = 9).

3.2. Effect of Cd²⁺ on gametophyte growth

Gametophyte area (mm²) was greatest in the reference group, when Cd²⁺ concentration is 15 mg L⁻¹, the gametophyte area is the lowest (Figure 2). However, an ANOVA showed gametophyte areas of different Cd²⁺ treatments were no significant difference than that of the reference group. EC50 is equal to 0.5 mg L⁻¹.

Figure 2. Effect of Cd²⁺ on gametophyte growth of C. pteridoides. Data are means ± SD (n = 18).

3.3. Effect of Cd²⁺ on hermaphrodite gametophytes

After 14 days of C. pteridoide spore culture, the control group gametophytes formed large, cordate hermaphroditic gametophytes and male gametophytes, indicating that sex differentiation had occurred (Figure 3). Hermaphrodite gametophyte rate was 52.8 ± 2.45% in the reference group. The proportion of hermaphrodite gametophyte tended to decrease with the increase of Cd²⁺ concentration. At Cd²⁺concentration of 15 mg L⁻¹, the hermaphrodite gametophyte rate was the lowest, which was 33.43 ± 7.19%. An ANOVA analysis exhibited that hermaphrodite gametophyte rates in 5, 10, and 15 mg L⁻¹ Cd²⁺ were significantly lower (p ﹤.05) than that of the reference group (Figure 4).

Figure 3. Cordate hermaphrodites of C. pteridoides in digital microscope.

Figure 4. Effect of Cd²⁺ on hermaphrodite gametophytes of C. pteridoides. Data are means ± SD (n = 3). *Level of significance was set at p < .05.

3.4. Effect of Cd²⁺ on time of archegonia emergence

Sixteen days after sowing C. pteridoides spores, archegonia were observed in the reference group. With the increase of Cd²⁺concentration, the number of archegonia decreased gradually. In the range of 0.1–15 mg L⁻¹, the formation time of archegonia on the gametophyte was delayed with the increase of Cd²⁺ concentration. Compared to the reference group, time of archegonia emergence was delayed four days when Cd²⁺ concentration was 15 mg L⁻¹, and the means number of archegonia was also reduced (Table 1).

Table 1. Effect of Cd²⁺ on time and number of archegonia emergence in C. pteridoides.

Cd^2+ (mg L^−1)	Time of archegonia emergence (days)	The means number of archegonia
0	16	11
0.01	16	9.5
0.1	17	9
1.0	19	8
5.0	19	9
10	20	7.5
15	20	6.5

4. Discussion

Earlier studies revealed that dose-dependent effects of cadmium on the spore germination and sporophyte development in some ferns differed in degree and magnitude (Gupta and Devi 1991). Gupta et al. (1992) reported that the germination rate of the second generation of C. thalictroides decreased gradually with the increase of cadmium concentration from 0.1 to 2.5 mg L⁻¹, but did not affect the germination rate of the first-generation spores, indicating that the damage of cadmium concentration to spore germination showed a cumulative systemic effect. In this study, although spore germination rates of floating C. pteridoides (these spores are equivalent to the first-generation spores of C. thalictroides) decreased with the increase of Cd²⁺concentration from 0.01 to 15 mg L⁻¹, it was not significant compared with the reference group (45.63%; Figure 1), indicating that different Cd treatments did not affect the germination rate of the first-generation spores of C. pteridoides. The results were similar to those reported by Gupta et al. (1992) on the germination rate of the first-generation C. thalictroides spores treated with cadmium. However, in this study, the effect of cadmium on the germination of the second-generation spores of C. pteridoides was not investigated.

This study showed that the rates of hermaphrodite gametophyte of C. pteridoides reduced with the increase of cadmium concentration, and showed that those in 5, 10, and 15 mg L⁻¹ Cd were significantly lower (p < .05) than that of the reference group (Figure 4). Meanwhile, with the increase of Cd²⁺concentration, the number of archegonia reduced and archegonium differentiation was delayed gradually. These results indicated that cadmium concentration higher than 5 mg L⁻¹ caused adverse effects on the sexual organ differentiation and reproduction of C. pteridoides. The results of affecting sexual reproduction under Cd-treated C. pteridoides in this study were similar to those reported in previous studies of Ceratopteris and some other ferns. For example, Gupta et al. (1992) exhibited that times of antheridia and archegonia formation in C. thalictroides was delayed in concentrations up to 0. l mg L⁻¹ Cd. Wang et al. (2021) also reported that times of antheridia and archegonia formation in the endangered fern P. japonica were delayed, and the number of archegonia reduced with the increase of Cd²⁺. Cadmium absorption can cause cell damage, interfere with the normal physiological metabolic process of cells, and affect plant growth and development (Li et al. 2022). It is well known that cadmium particularly interferes with the function of cations such as calcium and zinc, thus affecting growth and differentiation in plants (Ernst 1980). The visible toxic effects on the aerial parts of sporophytes were chlorosis, reduction in the size of vascular bundles and damage to the radial walls of the endodermal cells (Gupta et al. 1992). C. pteridoides treated with cadmium showed that the stomata of leaves were closed, the diameter of xylem tracheids of stem and root was reduced, the chloroplast and mitochondrial membrane system were damaged, and the components of chloroplast were disordered (Ji 2019). Gupta et al. (1992) showed that the chlorophyll and protein contents of C. thalictroides gametophytes treated with cadmium decreased gradually. In 1.0 mg L⁻¹ cadmium, the total chlorophyll decreased by approximately 50%. Wang et al. (2021) observed that chloroplast of gametophyte of P. japonica turned yellow and dissolution under 0.4 mg/kg Cd stress, and the gametophyte growth and sexual differentiation were affected by the increase of Cd concentration. Under the treatment of 0.8 mg/kg Cd, most of the antheridia could not be released sperm freely, accompanied by macular, and there was obvious necrosis of the archegonia, which might lead to the failure of fertilization, to affect sexual reproduction (Wang et al. 2021). These studies give insights into the metabolic changes in ferns, which lead to various phytotoxic symptoms caused by cadmium. The effect of cadmium concentration is more common during the formation of gametophytes which is possibly attributed to more cadmium concentration accumulated and increases toxicity, and consequently affecting the sexual reproduction.

Early studies have shown that the reduction and extinction of C. pteridoides in China are related to the destruction and loss of primary habitats, especially the increase in water pollution and the reduction of wetland areas (Dong et al. 2012, 2022). Based on the analysis of eight heavy metals contamination in surface sediments of 31 major lakes in China, Ding et al. (2017) reported that the average pollution degree of cadmium was the highest, the average content was 0. 497 mg/kg, and the pollution class of Cd and Hg were moderately and heavily polluted respectively in many lakes, indicating that the potential ecological risks in the sediments of lakes in China were mainly caused by Cd and Hg. Heavy metal pollution in sediments of eastern and central lakes is more serious than that of western lakes in China (Ding et al. 2017). In China, C. pteridoides occur mainly in eastern and central lakes, ponds and rivers. C. pteridoides used in this study were collected from lakes in central China.

Thus, in the study, water environmental factors such as the changes in cadmium concentration might have significant influences on the sexual reproduction of C. pteridoides, further exacerbating the endangered degree of this species in China. Therefore, it is necessary to pay more attention to protecting more habitats of C. pteridoides in China and reduce the impact of human activities such as excessive aquaculture and industrial wastewater discharge on the water body in which C. pteridoides live.

Previous studies have shown that Ceratopteris species are sensitive to water pollutants (Gupta et al. 1992; Deng et al. 2014; Dong et al. 2012, 2022). Gupta et al. (1992) reported that dose-dependent toxicity of Cd manifestations, including ultrastructural pathomorphological changes were detected in all stages of the entire life cycle (gametophyte, juvenile sporophyte, mature sporophytes, and spores) of the aquatic fern C. thalictroides, with gametophytes being the most sensitive. Deng et al. (2014) showed that cadmium can lead to a decrease in the growth and photosynthesis of endangered aquatic fern C. pteridoides, thus enhancing its mortality risk. An efficient strategy to restore environmental quality for this endangered fern implies the reduction of Cd pollution in the environment. In this study, our results showed that cadmium had an adverse effect on the differentiation of sexual organs of C. pteridoides. Field investigations and water quality analysis also indicate that the survival of C. pteridoides is closely related to water quality changes (Dong et al. 2012, 2022). The study therefore recommends C. pteridoides to be used as an indicator species for water pollution.

In conclusion, with the increase of the cadmium concentration, the proportion of hermaphroditic gametophyte and the number of archegonium decreased gradually. Since the reproductive organ is a crucial part of the conservation of species, to conserve this endangered C. pteridoides species, it is necessary to protect the primary habitat of C. pteridoides, especially the protection of its living water body.

Acknowledgment

The authors would like to thank Jifei Zhang for his assistance.

Disclosure statement

The authors have no conflicts of interest to declare.

Data availability statement

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

Additional information

Funding

This work were financed by the National Natural Science Foundation of China (32070379), Department of Education of Hubei Province: Support Action Plan Project of science and Technology of 100 schools and 100 counties-universities serve the rural revitalization (2021–2025) (BXLBX0358), Department of Science and Technology of Hubei Province: Key Research and Development Plan Project of Hubei Province (grant number: 2022BBA0064), and the Open Foundation of Hubei Engineering Research Center for Protection and Utilization of Special Biological Resources in the Hanjiang River Basin (2020-07, 2020-11, 2021-05).