Abstract We investigated how growth, gametophore morphology and sporophyte production in Physcomitrium sphaericum (C.F. Ludw.) Brid., Funariaceae, which is influenced by temperatures under in vitro conditions.  Physcomitrium sphaericum was cultured with a 1/4 strength standard Gamborg B5 (GB) supplemented with 1% sucrose and 0.45% w/v agar at pH 5.8 and grown under 18 ± 2/16 ± 2°C, 25 ± 2/23 ± 2°C, and 35 ± 2/33 ± 2°C day/night temperature, respectively. Morphological development of P. sphaericum was best at 18 ± 2/16 ± 2°C with large plant sizes and sporophyte production (about 27 sporophytes per colony), but no sporophytes formed at temperatures between 25°C and 35°C. By adapting to minuet plant sizes (about 1 mm in height) with rudimentary tissues and organs as shown by smaller leaves, shorter laminal cells, and numerous vegetative diaspore production, P. sphaericum may tolerate temperatures as high as 35°C. These adaptations under in vitro stresses may predict wild plant characteristics when encountering climate change.

Keywords: Dwarf bladder-moss, In vitro sporophyte production, Physcomitrium sphaericum, Temperatures variations   

Funding: The authors are grateful for the research funding provided by the Chiang Mai University, Chiang Mai, Thailand.

Citation: Printarakul, N., Chantanaorrapint, S., Chotikadachanarong, K., Srisukkham, W. and Jampeetong, A. 2025. Effects of temperature on growth, gametophore morphology, and sporophyte production of Physcomitrium sphaericum under in vitro conditions. Natural and Life Sciences Communications. 24(3): e2025051.

INTRODUCTION

One contemporary problem related to environmental conservation is climate change. The richness and composition of species may change as a result of long-term temperature shifts and changes in weather patterns. Many plant and animal species have changed their global distribution ranges. Also, an increase in surface temperature causes endangered species to become extinct. Bryophytes are one of the plant groups considered vulnerable to global climate change. Most of bryophytes required low temperature for growth and have a low capacity for adaptation to high temperatures (He et al., 2016; Marschall, 2017). Hence, losses in bryophyte diversity due to the sensitivity to raised temperature has been documented. Long-term warming has been linked to a decline in species richness and bryophyte cover in northern Sweden's alpine sub-arctic plant communities (Alatalo et al., 2020). Hespanhol et al. (2022) found that bryophyte species with smaller niche breadths and more marginalities are expected to be more sensitive to climate change compared to generalist species (Thuiller et al., 2005; Broennimann et al., 2006). Because of their unique characteristics and physiology, bryophytes are extremely dependent on their external surroundings for survival and reproduction. However, little is known about effects of swift and ongoing climatic change on growth and morphology in bryophytes, including their ability to survive as populations or individual species. 

Physcomitrium sphaericum (C.F. Ludw.) Brid., Funariaceae is a rare temperate Eurasian element with a sporadic occurrence, typically reliant on seasonally exposed muddy soils (Smith, 2004; Atherton et al., 2010; Ellis et al., 2020). In the IUCN threatened categories of the European red list of bryophytes, this taxon has been categorized as a vulnerable species (Hodgetts et al., 2019). Physcomitrium sphaericum has mostly been found in the northern hemisphere, including China, Japan, Russia, Europe, and North America (Li et al., 2003; Ellis et al., 2020). However, it is also found as a native species in tropical regions (reported as P. sphaericum (C.F. Ludw.) Fürnr.), such as Malaysia (Eddy, 1996; Yong et al., 2013) and Thailand (Printarakul et al., 2014). The status of P. sphaericum in many countries, including Thailand, is unclear (Printarakul et al., 2014). More research is needed to better understand its distribution pattern and other biological and ecological aspects. In Chiang Mai Province, Northern Thailand, this species is currently traded in the aquarium and terrarium markets, and the compound annual growth rate (CAGR) for the global level is not projected and estimated (unpublished data). Hohe et al. (2002) found that sporophyte induction in Physcomitrium (Physcomitriella) patens (Hedw.) Mitt., Funariaceae was triggered when the plants grew under low temperature (˂18°C). Nevertheless, nothing is known about how temperature affects the rare P. sphaericum, and no research has been published on how temperature affects the morphology of gametophores and sporophyte production in this moss.   

Long-term sustainability for protecting natural ecosystems and preventing the extinction of species is required especially as ex situ conservation, which seeks to ensure the long-term survival of habitats and species in their natural environments (Ros et al., 2013; Sabovljević et al., 2014). Several recent research efforts have assisted in the proliferation of bryophytes using a biotechnological in vitro technique, which is essential for obtaining critical biomass and investigating physiological strategies to avoid extinction of several endangered species. Recent in vitro experiments found that effects of copper accumulation promoted protonema transition from chloronema to caulonema and increased bud and gametophore production in a rare Cu moss, Scopelophila cataractae (Mitt.) Broth., Pottiaceae (Nomura et al. 2015; Printarakul et al., 2022). Prior evaluating the impacts of global warming and climate change on sensitive and endangered mosses, it is essential to study these mosses under ex situ conservation conditions, particularly using in vitro techniques. Hence, this study aims to assess growth, gametophore morphology, and sporophyte induction in P. sphaericum in vitro culture, with a focus on temperature effects. The study provides new insight into plant adaptation to various temperatures, which is useful for predicting climate change effects in this moss species, and it will also provide a valuable guideline for the ex situ conservation of P. sphaericum in the future. 

MATERIAL AND METHODS

Plant collection and plant stock preparation  

Fresh materials of P. sphaericum were collected from Doi Suthep-Pui National Park (Printarakul 05092021) and Chiang Dao Wildlife Sanctuary (Printarakul 7513), Chiang Mai Province, Northern Thailand, after receiving permission and authorization from the Department of National Parks, Wildlife, and Plant Conservation of Thailand. Voucher specimens were prepared and deposited in the Forest Herbarium (BKF), Chiang Mai University Herbarium (CMUB) and Prince of Songkla University Herbarium (PSU). This study followed IUCN’s policy statement on research involving species at risk of extinction and convention on the trade in endangered species of wild fauna and flora. Living plants were acclimated in the growth chamber at a temperature of 22°C and a relative humidity of 80–85%. A photon flux density was approximately 110 μmol m^-2 s^-1, supplied by LED E27 daylight bulbs (6500K, AC 220 V, 9 W, 800 lumens). The photoperiod was 16/8 h day/night cycle controlled using an AL-06 digital electronic timer (24 Hours timer, AC 220-240 V 50Hz, 10 A). 

At approximately 8 weeks, twenty new shoots (3 mm long pieces) were selected and effectively sterilized for 3 min using a 3% bleach solution (Haiter^TM, 6% NaOCl, prepared v/v with deionized water), and then rinsed 3 times using sterilized water (by adding 0.1% bleach), 1 min each. Individual sterilized shoots were then cultured in a sterilized-transparent plastic container (60 mm diameter×40 mm height, sterilized with 5% bleach v/v prepared with deionized water) containing 30 mL of standard tissue culture medium. The tissue culture medium was a 1/4 strength standard Gamborg B5 (GB) (Gamborg et al., 1968) to which 1% sucrose and 0.45% w/v agar were added. The pH was adjusted to 5.8 using 1 M HNO₃ and 1 M NaOH. This modified tissue culture medium was selected and used for the experiments because it produced the highest dry weight with the best yield of plant cultures, and morphology of gametophores resembled that of wild type gametophores. All plants were grown in a growth chamber under the same conditions as plant acclimation stated above.  

Experimental set-up

Approximately 16 weeks after the new colony formed, 10 gametophores from plant stocks were chosen and were then placed in 30 mL of the sterilized-transparent plastic cup with a lid containing the 1/4 strength standard Gamborg B5 (GB) (Gamborg et al., 1968) to which 1% sucrose and 0.45% w/v agar were added and pH was adjusted to 5.8 using 1 M HNO₃ and 1 M NaOH. Then, each container (n = 10) was placed in the growth chamber and the plants were grown for 8 weeks with the following temperature regimes: 18 ± 2/16 ± 2°C, 25 ± 2/23 ± 2°C, and 35 ± 2/33 ± 2°C day/night temperature at 80–85 % relative air humidity. The photoperiod was 12/12 h day/night cycle controlled using an AL-06 digital electronic timer (24 Hours timer, AC 220-240 V 50Hz, 10 A). The photon flux density was approximately 110 μmol m^-2 s^-1 supplied by LED E27 daylight bulbs (6500K, AC 220 V, 9 W, 800 lumens). 

Growth and morphology of gametophore study

Colony diameter and total dry biomass of P. sphaericum were recorded. Stem height, leaf size, the size of median and basal laminal cells, number and size of chloroplast (n = 25), and gametophore morphology were examined under a 400x light-transmitted microscope (Nikon, Eclipse E200) and a stereo-microscope (Olympus, SZ-30).  

Sporophyte production

The number of sporophytes per colony was counted under the stereo-microscope (Olympus, SZ-30).  

Statistical analysis

Statistical analysis was performed using the SPSS 17 software (SPSS Inc., USA). Significantly different means of each character were determined among treatments using one-way ANOVA with a 95% confidence level and the least significant difference (LSD) statistical test to represent differences among the treatments.

RESULTS

Growth and morphology of gametophore

Temperature significantly affected growth of P. sphaericum. The highest colony diameter and total dry mass was found in the plants grown at 18 ± 2/16 ± 2°C (Table 1, Figure 1). At 18 ± 2/16 ± 2°C gametophores were predominantly large, with an average stem height of about 7.4 cm (Figure  1) and large leaves of around 2.5×1.1 mm (Table 1, Figure 1). At higher temperatures, 25 ± 2/23 ± 2°C and 35 ± 2/33 ± 2°C, smaller leaves were observed (Figure 1). The average values of median and basal laminal cells were 57.2×30.1 μm (Figure 1), and 135.7×39.5 μm (Figure 1), respectively. However, smaller laminal cells producing vegetative diaspores were found at higher temperatures (Table 1, Figure 1). The plants grown at 18 ± 2/16 ± 2°C produced high number and large size of chloroplast compared with the higher temperature (Table 1, Figure 1). Shorter and deformed rhizoids were also noted for the gametophores grown under high temperature, particularly at 35/33°C (Figure 1). High temperatures over 25°C also had a negative effect on the sexual organ production and resulted in smaller gametangia than usual with organ dysfunction to infertility. 

Table 1. Results of one-way ANOVA of morphological characteristics and sporophyte production of P. sphaericum grown with different temperatures.

Different letters superscripts indicate significant differences between treatment, *** P <0.001

Sporophyte production 

In approximately 8 weeks, the gametophores grown at 18 ± 2/16 ± 2°C developed sporophytes. However, temperatures above 25°C inhibited the development of sporophyte structures in vitro.

Figure 1. Physcomitrium sphaericum grown on the 1/4 strength standard Gamborg B5 (GB) (Gamborg et al., 1968) to which 1% sucrose was added for 8 weeks at different temperatures  (18 ± 2/16 ± 2°C, 25 ± 2/23 ± 2°C, and 35 ± 2/33 ± 2°C day/night temperature): colony (a-c); mature plants (d); normal leaves (e-g); median laminal cells (h-j); basal laminal cells (k-m); caducous leaf producing vegetative diaspores from 35 ± 2/33 ± 2°C (n); and compact growth habit aberration from 35 ± 2/33 ± 2°C (o).

DISCUSSION

Growth and gametophore morphology of P. sphaericum were affected by temperature. Generally, P. sphaericum grew well at low temperature (18 ± 2/16 ± 2°C day/night temperature). They produced a large colony with big leaf size compared with the plants grown at higher temperatures. This moss species may favor a low temperature as a result of their ancestors’ origin and adaptation to extremely cool climates during the Pleistocene glaciations (He et al., 2016). In Thailand, P. sphaericum has currently been found in Chiang Mai Province at the altitude range of 350–1,400 m elevation, and they produced spore only in cool climate periods (Printarakul et al., 2014). Since P. sphaericum is found in temperate regions, its presence in Northern Thailand is most likely due to its tiny, resilient spores, which, like some other mosses, can disperse over large distances when carried by wind (He et al., 2016; Marschall, 2017). It appears that the traits of their ancestor are still present, allowing them to survive in a variety of settings, with temperature serving as a major constraint on their range. Furthermore, it is a general pattern that most bryophytes show better photosynthesis at lower temperatures. The temperature optima for photosynthesis of temperate species ranges from 15–25°C, whereas tropical bryophytes have an upper limit of 25°C (He et al., 2016). Although, we did not determine photosynthesis of P. sphaericum, the amount of chloroplast as well as its size was consistent with a greater growth under low temperature (18°C). Whereas growth and colonial size were reduced at temperature ≥ 25°C, there was also less chloroplast production. This is especially true for chlorophyllose cells, such that chloroplasts few in number, become packed, and reduce efficiency, thus potentially decreasing photosynthesis. This resulted in low dry weight and small colony diameters with smaller gametophores and shorter cells were observed in high temperature. Thus, it was expected that the optimum temperature for photosynthesis of this moss might be around 18°C. 

Most bryophytes prefer a temperature range of 15–25°C for optimal development (Furness and Grime, 1982). Hohe et al. (2002) found that sporophyte growth in the moss model P. patens in the field was strongly influenced by short-day length (8 h light/16 h dark) and low temperature (˂18°C).  Similarly, sporophytes of P. patens grew in vitro in response to a short-day length (8 h light/16 h dark photoperiod) and a low temperature (18°C) (Hohe and Reski, 2005). It has been confirmed that the response of P. patens to low temperature and short day length was likely correlated with the expression level of a MADS-Box gene (Hohe et al., 2002; Hohe and Reski, 2005). Low temperature and short-day length substantially regulated sporophyte induction. We found that P. sphaericum produced sporophytes in approximately 8 weeks of culture after acclimating to low temperature (18 ± 2/16 ± 2°C). At high temperature we found some sex organs in smaller gametangia. However, no sporophyte was developed at 25 ± 2/23 ± 2°C and 35 ± 2/32 ± 2°C. This could indicate that despite existence of the sex organs, they may have been affected by heat and dysfunction. Likewise, increasing temperatures, according to Bopp and Bhatla (1990), could inhibit the induction of sex organs in several moss species. When compared to cultures at low temperature (15°C) for P. patens on modified Knop medium, elevated temperatures also inhibited sporophyte development (Hohe et al., 2002).  Hence, the optimal temperature for growth of gametophore and development of sporophyte (after fertilization) in this rare moss was lower than 25°C. 

Temperatures exceeding 25°C in vitro cultivation frequently damage bryophyte tissues (Duckett et al., 2004). In this study, P. sphaericum survived at high temperatures up to 35°C in vitro cultures. However, the plants reduced size into minute gametophores and produced numerous vegetative diaspores, which was also the case found in many mosses grown under dryness and high temperatures (Stark et al., 2007; He et al., 2016). Production of vegetative diaspores may be the result of promoted ABA hormone production as a physiological response to high-temperature stress (Arif et al., 2019). Ability of P. sphaerium to survive in high temperatures by reducing its size to minute gametophores could be a key factor for why P. sphaericum occurrence in tropical regions where temperature is higher, such as Thailand (Printarakul et al., 2014), although it is found mostly in the northern hemisphere of the globe (Li et al., 2003; Ellis et al., 2020) where temperature is cooler. Contrastingly, it has been documented that warming temperature can increase sexual reproduction leading to increase sporophyte production and size in some Antarctic mosses, especially Bartramia patens Brid., and Hennediella antarctica (Ångström) Ochyra and Matteri (Casanova-Katny et al., 2016; Prather et al., 2019). These different responses suggest that global warming significantly influences moss population and dispersal patterns. Therefore, the morphological resilience of
P. sphaericum under different in vitro temperature conditions aids in understanding of plant stress physiology and its impact on regeneration response in this species or other moss species under future climate change scenarios.

CONCLUSION

Growth and gametophore morphology of P. sphaericum was influenced by temperature. The plants grew well at 18 ± 2/16 ± 2°C day/night temperature with large colony and numerous sporophyte production. At higher temperatures (above 25°C), the plants developed a tiny or densely bud-like habit with small leaves, and they lacked sporophyte production. However, the plants produced numerous vegetative diaspores. It shows that P. sphaericum has a physiological defense mechanism against thermal stress, which enables this plant species to adapt and tolerate high temperature.

ACKNOWLEDGEMENTS

We would like to thank the Department of National Parks, Wildlife and Plant Conservation of Thailand for permission to collect moss specimens (The sample collection permit number is TS 0907.4/2044). We thank Prof. Henrik Balslev and Assoc. Prof. Weeradej Meeinkuirt for their kind help to comment and improve the English text. The first author would like to thank Ms. Kanonrat Adulkittichai for her kind help and support in moss tissue culture laboratory. 

AUTHOR CONTRIBUTIONS 

Narin Printarakul conducted the experiment, performed the statistical analysis and data visualization, and wrote the manuscript. Sahut Chantanaorrapint identified moss specimens. Kittisak Chotikadachanarong assisted in moss tissue culture. Worawut Srisukkham designed and constructed plant growth chamber. Arunothai Jampeetong conceptualization, designed and investigated the experiment, wrote and edited the manuscript. All authors have read and approved of the final manuscript.

CONFLICT OF INTEREST

The authors declare that they hold no competing interests.

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