Introduction

Global climate change contributes to increased soil salinity due to rising sea levels and changing rainfall patterns. This fact negatively affects plant development, especially germination and initial seedling growth1. The osmotic stress caused by salinity decrease germination, plant nutrient uptake, elevate ion toxicity, as well as confer morphological (e.g., reducing root and shoot length, changing root architecture), biochemical (e.g., decreased chlorophyll and carotenoid content, excessive Na+ accumulation, lower K+/Na+ ratio), and physiological changes (e.g., decreasing stomatal conductance, changes in chloroplast structure, lower photosystem II efficiency)2,3,4,5.

During evolution, plants have developed different stress tolerance mechanisms to survive in different environments, including Antarctic and desertic ones. Among them can be mentioned the accumulation of protective osmolytes, scavenging of reactive oxygen species, selective ion uptake, changing root architecture, protection of the photosynthetic system, and reduction of their metabolism and growth6. Also, researchers have identified seed heteromorphism as a survival strategy that helps plants grow and endure in harsh conditions7. In this regard, heteromorphism is evident when a species produces seeds with different morphology, size, weight, and/or color. These characteristics influence seed germination success, dormancy, and vigor7. Studies on the impact of seed heteromorphism on germination often use scanning electron microscopy (SEM) to analyze testa microstructure, as color and morphological alterations are sometimes linked8,9. Moreover, heteromorphic seeds in the presence of abiotic stress conditions such as salinity, temperature, and illumination changes show different germination rates10,11. The ratio of one type of heteromorphic seed to another varies with populations, varieties, climatic, and environmental conditions11,12. Then, a greater germination percentage of heteromorphic seeds on certain ecosystem conditions suggest that these seeds have higher adaptability to this environment than other ones. Hence, color heteromorphism may serve as an indicator of the specific stress condition prevailing in each habitat and its impact on the ecological dynamics of plant species13.

Colobanthus quitensis (Kunth) Bartl. is considered a model plant for biotechnological studies and for understanding adaptive mechanisms to extreme environments, as well as an indicator of the effects of climate change in Antarctica14,15. It is an extremophile species distributed from southern Mexico to the north of the Antarctic Peninsula16. Thus, to perform successful scientific research, deep understanding of the physiology and genetics of model plant species17,18, like C. quitensis is crucial. Certain populations of this species are located along the coast19, which exposes them to marine aerosols directly or enables them to grow in soils drenched with saltwater20. Recent studies have categorized C. quitensis as moderately tolerant to salinity, capable of withstanding from 150 mM NaCl in common garden conditions and up to 400 mM NaCl in vitro, showing morphological, physiological, and biochemical adaptations to salinity stress21,22,23. Therefore, throughout the germination process, seeds do not exhibit the same responses or adaptive strategies against salinity as observed in mature plants7. For instance, C. quitensis seeds show a reduction in their germination and seedling survival rates when exposed to salt concentrations exceeding 100 mM NaCl21,22. Therefore, further research is required to distinguish the salinity tolerance of C. quitensis seeds among its populations. Seed stress can affect plant reproduction and productivity24. Then, germination tests are a rapid and effective alternative to detect genotypes resistant to environmental stresses25.

Seed heteromorphism has been previously described in different families such as Fabaceae8, Amaranthaceae11,13, and Caryophyllaceae26, to which C. quitensis belongs. There are several reports that studied the germination process in different populations of C. quitensis27,28,29,30,31, but none have mentioned the presence of heteromorphism or its influence on germination. These studies documented variations in germination rates among C. quitensis populations. These variations may relate to dormancy30, but seed traits likely influenced the results, as dark brown seeds showed higher germination rates.

Whe hypothesized that the color heteromorphism observed in C. quitensis confers plasticity in the response to salinity, which could be influenced by local adaptation acquired by the mother plants. This work aims to answer three research questions: (I) Are there differences in germination among different populations of C. quitensis? (II) Does color heteromorphism influence the germination rate of C. quitensis populations? (III) Does the presence of seeds color heteromorphism in C. quitensis constitute an adaptation mechanism of salinity tolerance? Therefore, the objective of this work was to analyze whether seeds from different populations of C. quitensis, grown under controlled conditions, vary in their tolerance to salinity depending on their color heteromorphism. For this purpose, the morphology of light and dark brown seeds was analyzed for structural differences, as well as in vitro germination analyses of heteromorphic seeds in the presence and absence of salinity.

Results

Electron microscopy analysis

SEM analysis was conducted to ascertain any morphological disparities among C. quitensis seeds of varying colors. According to SEM micrographs, both the light brown and dark brown seeds of C. quitensis populations are triangular and kidney shaped. Furthermore, morphological examination of the seeds reveals a uniform, mostly smooth tegument surface, with the presence of small puzzle-like striations near the micropyle area (Fig. 1). Overall, there were no discernible differences in the seed coat with different colors.

Fig. 1
figure 1

SEM micrographs of heteromorphic Colobanthus quitensis seeds side view. Lateral view of light brown seeds from (a) Arctowski, (b) La Marisma, (c) Laredo and (d) Conguillío populations; and dark brown seeds from (e) Arctowski, (f) La Marisma, (g) Laredo and (h) Conguillío populations. Close-up of the light brown seed micropyle area from (i) Arctowski, (j) La Marisma, (k) Laredo and (l) Conguillío populations; and dark brown seeds from (m) Arctowski, (n) La Marisma, (o) Laredo and (p) Conguillío populations. Yellow arrows indicate the position of the micropile. Red arrow indicates the presence of surface striations.

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Effect of population on seed germination

In each population, the germination percentage of C. quitensis seeds was over 40% (Fig. 2a). The pPA and pC populations showed the highest germination percentage, 78% and 66%, respectively, with no statistical difference (p < 0.05). Conversely, the germination percentage of pL (41%) and pA (46%) populations were statistically similar (p < 0.05), but 1.9-fold and 1.6-fold lower (p < 0.05) than pPA population.

Fig. 2
figure 2

Germination indicators of different populations of Colobanthus quitensis. (a) Germination and (b) time to reach 50% germination (T50). Bars represent mean ± SD (n = 10). Different letters indicate statistically significant differences (p < 0.05) according to one-way ANOVA followed by a Tukey HSD test.

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On the other hand, the time at which 50% germination is reached (T50) was higher than 10 days for all populations (Fig. 2b). A significantly lower T50 (p < 0.05) was observed in pPA (10.8 days) compared to pL (14.48 days) and pC (18.4 days). While the T50 of pA (12.39 days) does not differ statistically (p > 0.05) from the T50 of pPA and pL.

Effect of color heteromorphism on seed germination

Seed color had a significant effect on germination rates (Supplementary Table 1), which was evidenced by the fact that dark brown seeds in all populations had a significantly (p < 0.05) higher germination percentage than light brown seeds (Fig. 3a). The dark seeds of pPA (91%) and pC (84%) achieved the highest germination percentages. It should be noted that light brown seeds of pA and pL barely exceeded 20% germination.

Fig. 3
figure 3

Germination indicators of different populations of Colobanthus quitensis. (a) Germination and (b) time to reach 50% germination (T50) of heteromorphic seeds from Arctowski (pA), La Marisma (pPA), Laredo (pL) and Conguillío populations (pC). Bars represent mean ± SD (n = 5). Different letters indicate statistically significant differences (p < 0.05) according to one-way ANOVA followed by a Tukey HSD test.

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Also, the dark brown seeds required significantly (p < 0.05) less time to reach 50% germination than the light ones. From all populations, the light (11.7 days) and dark (9.9 days) pPA seeds reached their T50 faster (Fig. 3b).

Interplay between color heteromorphism and salinity on seed germination

The presence of NaCl salt in the medium affected the germination of all C. quitensis populations, while seed color only showed influence on the germination of pA and pC populations. The interaction of both factors had significant effect (p < 0.05) in germination percentage of all populations except for pC (Supplementary Table 1).

Both light and dark seeds show a general trend of germination percentage decreasing with increasing salinity. When the results were analyzed, we could observe that dark seeds from the pA and pC populations have a higher germination percentage than light seeds. Conversely, for the pPA and PL populations, light seeds tend to have a higher germination percentage than dark brown seeds (Fig. 4).

Fig. 4
figure 4

Germination percentage of heteromorphic seeds of Colobanthus quitensis treated with different concentrations of sodium chloride (50–200 mM). Light and dark brown seeds from (a) Arctowski (pA), (b) La Marisma (pPA), (c) Laredo (pL) and (d) Conguillío populations (pC). Bars represent mean ± SD (n = 5). Different letters indicate statistically significant differences (p < 0.05) according to factorial analysis (2 × 5), followed by a Tukey HSD test.

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The germination percentage of dark brown seeds from pA populations in NaCl 50 mM was the highest (73%), then it decreased to 52% at 100 mM (Fig. 4a). The presence of 50 and 100 mM NaCl did not generate differences in germination between light and dark brown seeds in the pPA population (Fig. 4b). On the other hand, the pL and pC populations showed a higher susceptibility to salt concentrations higher than 150 mM NaCl (Fig. 4c, d).

Salinity in the medium affected the T50 of all populations (Fig. 5), while seed color only influenced the T50 of pL and pC (p < 0.05). The interaction between these factors significantly affected the T50 of pL and pC (p < 0.05) (Supplementary Table 2).

Fig. 5
figure 5

Time to reach 50% germination (T50) of heteromorphic seeds of Colobanthus quitensis subjected to different concentrations of sodium chloride. Light and dark brown seeds from (a) Arctowski (pA), (b) La Marisma (pPA), (c) Laredo (pL) and (d) Conguillío populations (pC). The bars represent the mean ± SD (n = 75). Different letters indicate statistically significant differences (p < 0.05) according to factorial analysis (2 × 5), followed by a Tukey HSD test.

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The pPA population had the lowest T50 in the presence of 200 mM NaCl for both seed types (14 days), compared to the other populations (Fig. 5b). In contrast, the pC population maintained a high T50 regardless of NaCl concentration. For example, dark seeds took 17 days to reach their T50 under control conditions (0 mM NaCl) and 23 days in the presence of 200 mM NaCl (Fig. 5d).

The cumulative germination analysis indicates that the dark brown seeds of pA (Fig. 6b) and pPA (Fig. 6d) began to germinate faster than their light brown counterparts (Fig. 6a, c). Increasing the salt concentration in the in vitro medium resulted in a decreased germination percentage and an increased germination time. Salinity has the most severe negative effect on the light brown seeds of pA (Fig. 6a), as well as on both seed colors of the pL (Fig. 6e, f) and pC (Fig. 6g, h) populations. The dark brown pA seeds (Fig. 6b) and both seed colors of pPA (Fig. 6c, d) showed better tolerance in the presence of 50 mM and 100 mM NaCl in the in vitro germination medium.

Fig. 6
figure 6

Cumulative germination of Colobanthus quitensis under different salinity concentration. Light brown (C) and dark brown (O) seeds of Arctowski population (pA) (a, b), La Marisma (pPA) (c, d), Laredo (pL) (e, f) and Conguillío (pC) (g, h) (n = 75).

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Discussion

This study, for the first time, provides scientific evidence and describes the presence of color heteromorphism in the seeds of four C. quitensis populations grown under controlled conditions, originating from habitats with varying marine influence: pA, a coastal Antarctic population; pPA, a sub-Antarctic coastal population that develops in soils flooded by seawater; pL, a sub-Antarctic coastal population; and pC, an Andean population not exposed to marine influence (Supplementary Fig. 1). This phenotypic characteristic in the seeds contributes to the salinity tolerance of the different C. quitensis populations.

The C. quitensis triangular and kidney-shaped seed morphology (Fig. 1) agrees with previous descriptions, where it is mentioned that seeds tend to be flattened and wider towards the cotyledon area32. Conversely, variations in seed testa microstructure, dormancy, and germination are associated with seed coloration33. In particular, the effect of seed color on germination varies among species8,9.

SEM analysis performed in lateral view reflected little variation in testa structure, with striations evident only in the dark pPA seeds (Fig. 1d). However, a close-up of the seed micropyle area revealed that heteromorphic seeds from all populations show striations that form small pieces of a puzzle (Fig. 1i-p). Similar shallow marks were previously detected on the surface of the periclinal walls of the pA testa32. It is possible that light and dark brown seeds differ in their biochemical and structural composition at the deeper layers of the seed coat, so more detailed analyses, including the use of Fourier-transform infrared spectroscopy, may be required34. Genes regulating the synthesis of flavonols and proanthocyanidin pigments, along with other chemicals, are known to play a crucial role in determining seed coat color and their arrangement is found in the different layers of the seed coat35,36. However, this study did not cover these aspects, so further research is necessary to understand the factors that lead to heteromorphism in C. quitensis. Seed coat characteristics play an ecological role in species dispersal, longevity, water uptake capacity, and germination in ecosystems dominated by changes in temperature, salinity, and water availability37.

The wide distribution of C. quitensis has generated genetic and morphological differentiation among its populations27,38, which influences their germination30. Plants growing in cold climates are usually small in size and slower growing than their counterparts. Therefore, they produce small seeds with dormancy, showing low germination speed and high temperature requirements for germination39. Antarctic populations of C. quitensis have been reported to have lower germination relative to sub-Antarctic or Andean populations27, respectively. However, in this study, the Antarctic population pA showed equal germination percentage and T50 to the sub-Antarctic population pL (Fig. 2a, b).

Seed heteromorphism is a critical adaptive mechanism that enables plants to maintain high germination rates under stress conditions like high salinity and intense UV radiation40, significantly influencing the ecological resilience of plants to changes in the environment41. C. quitensis frequently encounters these stressors within its natural habitats, making this mechanism particularly vital. Factors such as seed maturity, flavonoid and pigment concentrations, environmental variations, and genetic influences all play a role in testa pigmentation42. Consequently, under increasing salt concentrations, seed heteromorphism is expected to differentially affect the germination of C. quitensis populations (Figs. 4, 5 and 6). However, this character only contributed to the higher salinity tolerance of dark brown pA seeds, inferring, therefore, that seed color heteromorphism might be fundamental for this population to tolerate increased salinity conditions to a greater extent in Antarctic.

The testa thickness of heteromorphic seeds is determined by the amount of suberin, cutin, and lignin in the cell walls, as well as the presence of fatty acids in the intercellular spaces. This thickness influences seed permeability, impacting the rate of water uptake by the embryo, dormancy, and germination43,44. Additionally, the levels of phytohormones such as abscisic acid, indole-3-acetic acid, and zeatin riboside vary with seed color, potentially affecting the germination process45. Understanding these complex interactions provides valuable insights into the resilience mechanisms of C. quitensis, offering potential strategies for enhancing crop tolerance to abiotic stressors.

Varieties or genotypes among species exhibit different levels of tolerance to the same stress46, a phenomenon observable from the early stages of plant development47. For instance, dark brown pA seeds demonstrated a higher germination percentage than light brown seeds up to 150 mM NaCl (Fig. 4a). However, in the presence of 150 and 200 mM NaCl, light brown pPA seeds showed a higher germination percentage than dark brown seeds (Fig. 5b). This suggests that within a population, the higher germination percentage of one seed type over another indicates the possible presence of biochemical compounds that enable tolerance to NaCl. Both pA and pPA populations thrive in areas exposed to saline conditions (Supplementary Fig. 1a, b)19,20, suggesting they possess genetically determined mechanisms that confer salinity tolerance. Previous research has shown that the addition of 50 mM NaCl to in vitro culture media did not affect the germination percentage of pPA compared to seeds germinated under non-saline (control) conditions21,22.

Nevertheless, in this investigation, pPA seeds with a dark brown color exhibited a greater germination percentage when the in vitro germination media did not contain NaCl (Figs. 3a and 4b). The pPA has a higher tolerance to salt than pL, even though both species originate in similar locations and are associated with coastal ecosystems (Supplementary Fig. 1b, c). A possible reason could be that the pPA ecosystem undergoes frequent flooding of the soil with seawater, exposing the roots to continuous and direct contact with saltwater20. This may have led to the development of stronger genetic mechanisms in this population that can tolerate salinity. Because the habitats and microclimates that comprise a natural landscape are ever-changing. Therefore, the populations that grow there tend to show differences in response to environmental pressures48. However, pL is subject to strong anthropic pressure that has influenced its habitat and therefore the ecology of this population (M. Cuba-Díaz, pers. comm.). This could also explain why, although they grow in Punta Arenas associated with coastal environments, pL and pPA have different levels of salt tolerance.

Both pL and pC showed low tolerance to salinity (Fig. 4c, d). However, at concentrations up to 100 mM NaCl, pPA seeds and only the dark brown seeds of pA had a germination percentage higher than 50% (Fig. 4a, b). The observed difference in salinity tolerance between pPA and pA could be attributed to the different ways these populations are exposed to salinity: pPA is found in areas that are flooded with seawater, while pA receives marine spray. This distinction should be analyzed in depth. For this experiment, seeds were obtained from plants grown under controlled conditions and temperatures, without exposure to salinity for at least 8 to 10 months49. Nonetheless, seeds from pPA and the dark brown seeds from pA exhibited greater tolerance to salinity, suggesting the presence of genetically inherited mechanisms that confer tolerance, even in the absence of direct exposure to saline conditions during their production.

For each population, it is evident that when the concentration of NaCl increases, seeds of all colors are impacted. In fact, there was a delay in seed germination, starting with an increase in salt concentration (Fig. 4). In numerous species, studies have documented the negative influence of salinity46,50,51 because the increase of Na+ ions in the medium decreases the water uptake rate of seeds, generating drought stress, thereby delaying and decreasing germination percentage52. Furthermore, it generates toxicity that changes and disrupts the activity of enzymes involved in nutrient mobilization, such as α-amylase, and decreases the content of gibberellins required for germination50,53.

Researchers have observed that C. quitensis does not germinate quickly under laboratory conditions. The development of long germination periods is considered an adaptive trait in species that occur in ecosystems with a high degree of stress or disturbance54. However, in this study, pA and pPA required less time to achieve 50% germination (Fig. 5a, b) than pL and pC (Fig. 5c, d). This is advantageous because faster-germinating plants are less susceptible to disease and experience less competition for environmental resources, both of which are critical for healthy growth and subsequent seed production55.

This research shows that there is a close relationship between seed color and the germination of C. quitensis in response to salinity. Although this phenomenon was not observed in the other three populations, it is possible, according to the literature, that seed heteromorphism may be a useful mechanism for tolerating other types of environmental stress, in the remaining populations of C. quitensis. Considering that the thickness of the testa, the composition and abundance of chemical compounds in the seed coat, and the presence of secondary metabolites in seeds may provide relevant information about the ecology of species in extreme environments7. Finally, there is a need for further investigation focusing on seed heteromorphism to clarify the causes of its origin and its implications for the species’ ecology (Fig. 7).

Fig. 7
figure 7

Diagram of the main research results and proposal for future research focused on the heteromorphism of Colobanthus quitensis seeds.

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While heteromorphism has been documented in the Caryophyllaceae family26, this is the first report of such a phenomenon in C. quitensis, highlighting an additional adaptive strategy this species uses to endure the harsh conditions of its native habitats. Heteromorphism in seeds has been reported across numerous plant families, including Amaranthaceae41, Poaceae56, Fabaceae57, and Asteraceae58, many of which contain crops of economic importance. The discovery of heteromorphism in C. quitensis opens the possibility of leveraging this trait to understand seed survival mechanisms under variable environmental conditions. This knowledge could be instrumental in selecting populations, varieties, or genotypes of commercially significant species that exhibit enhanced tolerance to abiotic stresses, such as increased soil salinity.

Conclusion

This work represents the first effort to identify the existence of color heteromorphism in C. quitensis seeds from different habitats. The studied populations vary in their germination capacity, with dark brown seeds showing greater success than light brown ones. The most salt-tolerant population is pPA, where the heteromorphic seeds respond equally to the presence of NaCl. This opens the possibility of using pPA as a biotechnological model to investigate the mechanisms of salinity tolerance in extremophilic species. In pA, dark brown seeds showed a higher germination percentage than light brown seeds in the presence of NaCl, showing that heteromorphism is one of the mechanisms used by this population to cope with saline conditions. Heteromorphism in pL and pC did not influence their ability to withstand salt in the medium, making them the most susceptible populations. Finally, seed heteromorphism related color of seed coat in C. quitensis may be one of the strategies used by this species to tolerate the harsh conditions within its natural habitat, although the advantages of this phenomenon for the species need further study.

Materials and methods

Plant material

For this study, seeds from four populations of C. quitensis plants were analyzed 8 to 10 months after their collection in the field. These plants were kept in a common garden belonging to the Antarctic plant collection of the Laboratorio de Biotecnología y Estudios Ambientales of Universidad de Concepción, Chile. Seeds were coded with the name of the location where the plants were collected: Arctowski (pA) (King George Island, South Shetland Islands, Antarctica; 62°09’S; 58°28’W), La Marisma (pPA) (Santa María Point, South of Punta Arenas, Chile; 53°22’S; 70°58’W), Laredo (pL) (Laredo sector, North of Punta Arenas, Chile; 52°58’S; 70°49’W) and Conguillío (pC) (Conguillío National Park, Araucanía Region, Chile; 38°36’S; 71°36’W) (Supplementary Fig. 1). The populations pA, pPA, and pL are coastal populations, so they are constantly exposed to marine spray. Notably, pPA inhabits areas that are continuously flooded with seawater, while pC is located in the mountain range and does not receive marine influence.

C. quitensis plants were grown in growth chambers at 13 ± 1 °C, with a 16/8 h light/dark photoperiod, a light intensity of 120 ± 20 µmol m2 s− 1 and 85–90% relative humidity. Seeds from each population were collected when the flower capsules were fully opened, and the seeds were matured (Fig. 8a). Mature seeds were considered as those able to tolerate desiccation59 and whose color varied from immature seeds. The capsules were dried at room temperature for 2–3 days. Subsequently, seeds were manually extracted, sorted by color into light and dark brown seeds (Fig. 8b, c), and stored in hermetically sealed Eppendorf tubes at 4 °C until use.

Fig. 8
figure 8

Open floral capsule of Colobanthus quitensis showing mature seeds ready to be collected (a), light brown seeds (b) and dark brown seeds (c).

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Electron microscopy analysis

The light and dark brown seed testa of the different populations was analyzed by scanning electron microscopy (SEM) (model JSM-6380) to detect differences in their morphology. Seeds from different colors were fixed to a holding plate and sputtered with an Au layer using a Leica EM ACE600 high vacuum coater. SEM images were taken using an acceleration voltage of 30 kV modified from Kellman-Sopyla et al.32.

Seed selection and conditioning

Seeds were submerged for 24 h in distilled water, and seeds that floated were considered non-viable and those that did not were considered viable60. All viable seeds were scarified with 1% (v/v) H2SO4 during 30s30 and subsequently disinfected with 70% (v/v) ethanol for 30 s in vortex and 5% (v/v) NaClO for 7 min in vortex, followed by three times washes with sterile distilled water21.

Effect of population, seed color heteromorphism and salinity on seed germination

For the experiments, 10 cm diameter Petri dishes were used containing 20 mL of MS culture medium61, 3% sucrose, 0.7% agar were prepared. Seeds were placed on dishes for in vitro germination at 20 ± 2 °C, with a 16 h light/ 8 h dark photoperiod and a light intensity of 45 ± 2 µmol m-2 s-1.

To evaluate the effect of populations (n = 4) on germination without making distinctions in seed color, an experimental design was carried out with 10 replicates per treatment (n = 40). Seeds were randomly selected, maintaining the color proportions for each population of C. quitensis and 15 seeds per replicate were placed to germinate in the mentioned medium.

In addition, to evaluate the effect of seed color2 within the population, five replicates of 15 seeds of the same color were used for each replicate and placed to germinate on the same medium.

Finally, to explore the combined effect of seed color and salt concentration, five concentrations of NaCl (0, 50, 100, 150 and 200 mM) were added to the medium. Five replicates of 15 seeds per color and NaCl concentration, respectively, were used.

For the three tests, the number of germinated seeds on each plate was recorded every 48 h for 32 days. Germinated seeds were considered to be those whose radicle was at least twice the size of the seed28. Data was calculated for germination percentage (GP) according to the Eq. 1.

$$\:\:GP=\frac{n}{N}*100$$
(1)

Where n is the number of germinated seeds at the end of the experiment and N is the total number of seeds. In addition, it was determined the time at which 50% germination is reached (T50) according to the Eq. 262.

$$\:T50=ti+\frac{(\frac{n}{2}-ni)(tj-ti)}{(nj-ni)}$$
(2)

Where n is the number of germinated seeds at the end of the experiment; ni and nj are the cumulative number of germinated seeds per adjacent count at tj and ti times, respectively, where \(\:ni<\frac{N}{2}<nj\).

Based on these count data, it was obtained cumulative germination rate as the fraction of the number of germinating seeds per Petri dish every two days.

Statistical analysis

To analyze the effect of populations and seed heteromorphism on germination percentage and T50 of each population, a one-way ANOVA was performed. Germination percentage and T50 were considered as dependent variables, and population and seed color were used as independent variables. To evaluate whether color heteromorphism affects germination response to salt stress, a factorial analysis was performed. Germination percentage and T50 were again the dependent variables, while seed color and different salinity treatments acted as independent variables. In both analyses, a post hoc Tukey’s Honestly Significant Difference (HSD) analysis was employed with a 95% confidence interval. These analyses were executed using the “aov” function within the R Studio program (R Core Team, 2023). Subsequently, the graphs were generated utilizing the “ggplot2” package63.