Recalcitrant seeds with physiological epicotyl dormancy may limit seedling recruitment of an endangered subtropical oak species

Abstract

Castanopsis kawakamii is an endangered relict oak species inhabiting the southern edge of the subtropical region of China. Its recalcitrant acorns (hereafter seeds) exhibit sequential radicle and epicotyl dormancy, requiring prolonged two-phase release, increasing the risk of viability loss and predation before seedling establishment. Seeds of C. kawakamii were collected from the largest population, Castanopsis kawakamii National Nature Reserve, to assess viability under drying and temperature treatments and to identify environmental cues for radicle and epicotyl emergence. Seeds of C. kawakamii lost viability under 4℃ and at a seed moisture content (MC) < 35%. Radicle dormancy was broken by cold stratification (5/15℃) or by subjecting seeds to winter field conditions, but epicotyl physiological dormancy (PD) persisted and required ~ 30 days of warm stratification (15/25℃) following radicle emergence for release. Seeds dispersed from the parent plant in early autumn exhibited deeper PD than those dispersed in late autumn. In the field, radicle and epicotyl emergence occur mainly in the spring following seed dispersal in autumn but with a 1-month lag between the two events. Over 99% seeds with a non-emerged epicotyl died in April. Seed recalcitrance and the requirement for both cold and warm stratification for seedling establishment may be important in limiting plant regeneration in the natural habitat. To enhance seedling establishment under climate stress, we recommend assisted regeneration via protection of late-autumn seeds, moisture retention through burial, and warm stratification to overcome epicotyl dormancy post-radicle emergence.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-025-07847-2.

Introduction

The transition from seed to seedling is a high-risk period in the life-cycle of most plant species [1, 2], and it is crucial for local adaptation of species and population restoration [3–5]. The endangered species Castanopsis kawakamii Hayata. (Fagaceae) is a Tertiary relict subtropical species confined to southeastern China (including Taiwan) and Vietnam [6]. Population aging and seedling scarcity have been reported across its distributional range [7, 8]. The Castonopsis kawakamii National Forest Park in China is the largest remaining natural population of C. kawakamii (~ 700 ha), yet many mature/aging trees exhibit trunk rot, resulting in hollow trunks [7]. Despite abundant seed production, seedlings and saplings are scarce, raising concerns about the mechanisms restricting regeneration from seeds [9, 10]. Potential factors such as the seed coat (testa), desiccation sensitivity, and canopy shade may limit the recruitment of seedlings of C. kawakamii [7, 10], and we have observed that up to 4 months are required for complete germination, i.e. emergence of both root and shoot.

Based on their response to desiccation, seeds are categorized as orthodox (< 7% a seed moisture content [MC]), intermediate (10–12% MC), or recalcitrant (20–30% MC) [11, 12]. Unlike orthodox seeds, which undergo dehydration tolerance at maturity, recalcitrant seeds are desiccation and low-temperature sensitive [13]. Also, recalcitrant seeds lose viability at a relatively high MC, making them vulnerable to environmental stress [4, 14]. Low temperatures can further accelerate loss of viability of recalcitrant seeds [15, 16], but some recalcitrant seeds require cold (c. 0 to 10 ℃) moist stratification to break epicotyl physiological dormancy (PD) [17].

Desiccation sensitivity has been demonstrated in seeds of various oak species (Fagaceae) [4, 18]. These seeds lack maturation dehydration at the end of development, retaining a high MC at dispersal and exhibiting extreme sensitivity to low temperatures and dehydration [14]. This desiccation sensitivity of seeds can restrict the geographical distribution of species and has important implications for conservation and population restoration efforts [19].

Epicotyl PD has been observed in C. chinensis, C. purpurella, and C. sclerophylla [20, 21], resulting in a delay in timing of epicotyl emergence. Thus, we assume that seeds of C. kawakamii also have epicotyl PD and exhibit a delay between root and shoot emergence. Epicotyl PD occurs in various genera such as Brownea [22], Lecythis [23], Humboldtia, Yunnanopilia [24], Chionanthus [25], and oak species [4, 18, 26, 27].

Early studies on oak species [28, 29] demonstrated that epicotyl PD in Quercus robur can be released by cold stratification, ensuring that shoot emergence aligns with optimal conditions for seedling establishment and early growth in spring [26]. Seeds of species in sections Quercus, Lobatae, and Protoquercus of Fagaceae typically exhibit PD [30]. Radicles of Q. alba, Q. prinus, and Q. robur (in section Quercus) have little or no PD, and they emerge immediately upon dispersal [18, 27]. However, the epicotyl has PD, which is broken by cold stratification during winter [31, 32]. In seeds of some species such as those of Q. cerris [33] and Q. nigra [34] in sections Cerris and Lobatae, respectively, both the radicle and epicotyl have PD, which is broken by cold stratification during winter.

Seeds exhibiting both recalcitrance and epicotyl PD present a unique challenge for conservation and regeneration. Although a few species have been identified with this combination of traits, including Cyclobalanopsis chungii [30, 35], Humboldtia laurifolia, Cynometra cauliflora [22], Paeonia ostia [36] and many oak species [4, 37], comprehensive studies on their dormancy-breaking and germination requirements and maintenance of seed viability are limited [27].

This study assessed seed viability in C. kawakamii under various drying and temperature regimes. We also identified the environmental cues triggering radicle and epicotyl emergence. We hypothesize that the combined effects of seed recalcitrance and epicotyl physiological dormancy significantly constrain seedling establishment in this species by preventing complete germination. Specifically, we aimed to elucidate the physiological mechanisms governing seed viability, dormancy break, and seedling recruitment, providing crucial insights for conservation and restoration strategies for this endangered species.

Materials and methods

Study site and seed collection

Castanopsis kawakamii National Forest Park (“San ming ge shi kao guo jia sen lin gong yuan” in Chinese pronunciation, 26°8′~26°13′ N, 117°25′~117°30′ E) is the world largest population of C. kawakamii Hayata (Fagaceae). Average annual precipitation in this region is 1,819.68 mm (1952–2019), with the majority of it occurring from April to June (Fig. S1). The period from October to January is relatively dry, with only 13.5% of the annual precipitation. Average minimum temperatures in December, January, and February fall below 5℃, with January reaching a low of 2.9℃.

The plant samples were identified by Prof. Zhenying Huang, a botanist at the Institute of Botany, Chinese Academic of Sciences, and voucher specimens (e.g., voucher no. Fag2211-CK01 to Fag 2211-CK06) have been deposited in the National Park Research Center, Sanming University. We declare that the experimental research on plants in this study complied with institutional, national, and international guidelines, including the Convention on the International Trade in Endangered Species of Wild Fauna and Flora (CITES). And we confirm that the Administrative Committee of Castanopsis kawakamii National Forest Park granted permission for the collection of samples and experimental materials for this study and assisted in the material collection. We have acknowledged their contribution.

Freshly matured seeds were collected in Castanopsis kawakamii National Forest Park, specifically within a mountainous experimental area measuring approximately 500 × 500 m, encompassing the foothill, slope, and summit regions. The seeds naturally fall during rainfall upon maturation, and we collected over 100 kg freshly matured seeds from beneath the trees after rain events. And intact, fully developed seeds that showed no signs of predation or insect damage were selected for use in our studies. Seeds were collected three times during the seed dispersal season: early (November 15, 2022), mid (December 15, 2022), and late (January 15, 2023). This design was implemented to test the hypothesis that the duration of natural cold stratification influences dormancy status; the collection points represent seeds with zero, one, and two months of field stratification, respectively.

Species biology and seed/embryo characteristics

According to the report of FOC (Flora of China) [6], C. kawakamii Hayata is a Tertiary relict subtropical species confined to southeastern China and Vietnam. Tree characteristics: Mature individuals are 15–28 m tall, growing in sparse or dense mountainous forests below ca. 1,000 m elevation. This species exhibits a prolonged reproductive cycle. Flowering occurs in March-April, with fruits maturing 18 months later in August-October of the following year. Each spiny cupule (6–8 cm diameter) typically contains a single nut, protected by the cupule’s thick walls and spine-like bracts. At maturity, the cupule splits into segments to release the orangish-brown puberulent nut.

Seed desiccation/low temperature sensitivity (i.e. seed recalcitrance)

Seed sensitivity to desiccation

Freshly matured seeds were randomly sampled in November 15, 2022 and divided into seven treatment groups, with four replicates per group, totaling 28 samples. Each sample contained 25 seeds. After weighing (Sartorius AG, Germany, with a precision of 0.001 g, all subsequent weighings hereafter were performed using this instrument), seeds were embedded in a large excess of dry silica gel (1,500 g) for dehydration. The silica gel was monitored daily and was not replaced as the quantity was sufficient to prevent saturation throughout the experiment. Seed MC (base on the mass of fresh matured seeds) was determined by weighing every 12 h until their weight stabilized, and viability was assessed using 0.4% TTC staining at MCs of 40%, 30%, 25%, 20%, 15%, 10%, and 5%. Additionally, water absorption characteristics of the seeds were investigated. Intact seeds, scarified seeds, and seeds with their coat removed were placed in water separately, and they were weighed at time 0 and after 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 48, and 100 h of water absorption, until seed weight no longer increased.

Seed sensitivity to low temperature

Newly matured seeds collected in November 15, 2022 were randomly sampled and divided into 24 portions (6 temperature treatments × 4 replicates), with each portion containing 25 seeds. The seeds were placed on two layers of filter paper, covered with wet sand (with 12% MC, wet weight basis), and enclosed in airtight metal boxes (20 cm × 10 cm × 10 cm deep). These boxes were placed in incubators (LI180C, Jfu Scientific Equipment Co., Ltd., Shanghai, China) at constant temperatures of 0, 4, and 6℃ and at fluctuating temperatures of 0/10, 5/15, and 10/20℃ (12 h dark/12 h light). Seed viability was assessed using TTC on the 15th and 30th days to evaluate the effects of low temperatures on seed viability. The prepared seeds were treated with a 0.2% (w/v) solution of 2,3,5-triphenyltetrazolium chloride and maintained at 25 ℃ for 4 h in the dark. Viability was assessed following incubation based on the characteristic red coloration of living tissues [38].

Germination/dormancy characteristics

Initial germination experiment

Freshly-collected seeds for each dispersal period were randomly selected and divided into 4 treatment groups×3 periods×4 replicates, 48 samples with 25 seeds per replicate. The germination experiments were conducted at four temperature regimes: 5/15℃ (representing the average monthly minimum temperature of 6℃ and the average monthly maximum temperature of 15℃ in January, February, and December; 12 h light/12 h dark), 10/20℃ (representing the climatic temperatures in March, April, and November), 15/25℃ (representing the climatic temperatures in May and October), and 20/30℃ (representing the climatic temperatures in June, July, August, and September). Water- saturated peat moss was used as a substrate for seed incubation. Germination was defined as emergence of the radicle from the testa to a length of ≥ 5 mm. The incubation period lasted for 30 days, and number of seeds with an emerged radicle was recorded daily.

Kind of seed dormancy

(1) Effect of Cold Stratification on Breaking Radicle Dormancy: In the “Seed Sensitivity to Low Temperature” experiment, seed mortality was 97.02%, 100% and 100% at 4℃, 0/10℃ and 0℃ after 1 month, respectively; therefore, we conducted the cold stratification treatments at 5/15℃ (12 h dark/12 h light) and 6℃. The alternation temperature regime of 5/15℃ simulated the ambient daily minimum and maximum temperature, respectively, from December to February, while 6℃ simulated the average daily field temperature during the same period. Seed germination was tested after 0 (control), 2, 4, 8, and 12 weeks of stratification, and seeds were incubated in light at 5/15℃, 10/20℃, 15/25℃, and 20/30℃ (12 h dark/12 h light). (2) Breaking Epicotyl dormancy: Since epicotyl emergence was not observed in response to cold stratification, we incubated seeds with an emerged radicle at 6℃, 5/15℃, and 15/25℃ (12 h dark/12 h light, simulating average spring temperatures) for an additional 60 days, which represent the temperature regimes during early and late spring, which is the actual seedling recruitment season for plant species. This extended period of incubation was used to determine the effects of cold stratification at 5/15℃ and warm stratification at 15/25℃ on the release of epicotyl dormancy after the radicle had emerged.

Differences in dormancy across different maturation periods

To investigate dormancy characteristics of seeds dispersed at different times, we used seed lots that matured and fell in November 15, 2022, December 15, 2022, January 15, 2023, and February 13, 2023. To evaluate radicle and epicotyl dormancy, we employed three stratification treatments: winter, field cold stratification (starting on November 30), cold stratification at 6℃, and warm stratification at 25℃. Following stratification for 1 month, seeds were incubated at 5/15℃ and 15/25℃ (12 h dark/12 h light) for 30 days, and radicle and epicotyl emergence were checked daily.

Seed germination under field condition

During the seed maturation and dispersal season, freshly matured seeds collected on 21 November 2022 were screened in the laboratory to ensure complete development and absence of predator damage. A total of 56 seed bags were prepared, each containing 400 seeds × 4 replicates × 14 sampling times. The cloth bags, made of lightweight, soft, breathable brown material similar in color to the soil, were spread out in the primary habitat of C. kawakamii, free from human interference, to create an artificial soil seed bank. The bags were placed flat on the ground under canopy at November 20, 2022, with the seeds evenly spread and pressed down to ensure contact with the soil. The buried soil seed bank was enclosed with wire mesh to prevent predation. This setup was established in late October 2022. Emergence of radicles and shoots were monitored monthly November 2022 to March 2023.

Statistical analyses

All data analyses were done with R software [39]. The response variables were expressed as mean + SE (non-transformed data appear in all figures). Linear regression was used to quantify the relationship between seed water loss (independent variable) and seed viability (dependent variable). We fitted a general linear model (GLM) specifying a binomial distribution and a logit link function [40] to evaluate how radicle and epicotyl emergence probabilities were influenced by incubation temperature, storage condition, and stratification time. Model significance was determined at p < 0.05. Following a significant GLM result, we conducted post-hoc multiple comparisons with Tukey’s honest significant difference (HSD) method, utilizing the glht function in the R package multcomp [41].

Result

Seed/embryo characteristics

Seeds of C. kawakamii are irregularly spherical, with an average mass of 2.11 ± 0.04 g (mean ± SE, n = 100) (Fig. 1a). During germination process, the radicle emerges, after which the hypocotyl elongates (Fig. 1b). The radicle then elongates, and lateral roots develop (Fig. 1c-e). After epicotyl dormancy is broken, the cotyledon petioles elongate, pushing the upper hypocotyl out of the seed, and the epicotyl (shoot) emerges at the junction of the two cotyledonary petioles (Fig. 1e, S2g). The seed is composed of a leathery seed coat (densely covered with retrorse hairs on the inner surface, Fig. 1f), internal cotyledons (Fig. 1g, h), and the embryos (Fig. 1h, i). The two cotyledons account for 99.16% of total embryo mass, while the embryo axis comprises only 0.84 ± 0.11% and was clearly differentiated except for the radicle.

Fig. 1.

Germination process and seed structure/morphology of C. kawakamii seeds. a newly matured seed, (b) hypocotyl emergence, (c) hypocotyl elongation and epicotyl emergence, (d) radicle continues to elongate and produces lateral roots, (e) shoot emergence, (f) and (g) are seed coat and embryo, respectively. h and (i) are embryo sections and embryo structures for germination, respectively.

Seed desiccation/low temperature sensitivity (i.e. seed recalcitrance)

Seeds of C. kawakamii are highly sensitive to dehydration and low temperatures. There was a significant negative correlation between seed viability and water loss (R²= 0.77, p < 0.01) (Fig. 2a). When seed water loss was less than 15%, viability was near 100%, but when it exceeded 35% all seeds lost viability. Thus, seeds are highly sensitive to dehydration. Additionally, storing seeds at constant temperatures of 0 and 4 ℃ (freezing point) for 15 or more days resulted in significant seed mortality (Fig. 2b). Storing seeds at 6℃ for 30 days resulted in a 10% reduction in viability compared to the 15-day treatment (Fig. 2b). Exposure to excessively low winter temperatures (e.g., 0/10 ℃) severely impaired seed survival, with only 15.3% of seeds remaining viable. In contrast, prolonged storage at moderately low temperatures (e.g., 5/15 ℃ for 30 days) supported a high survival rate of 82.4% (Fig. 2b). Furthermore, seeds of C. kawakamii maintained nearly 100% viability under 10/20 ℃ conditions, indicating their suitability for long-term storage at this regime.

Fig. 2.

Effect of desiccation (seed moisture content, a) and temperature (b) on seed viability of C. kawakamii. a The relationship between loss of seed moisture content (%) and seed survival (%). The red line in the graph indicates the fitted equation: Y=112.81-3.12x (R²=0.77, p<0.001). The grey line in the figure shows the 95% confidence. b Effect of different storage temperatures and storage times on seed viability. Seed viability was investigated for 15 and 30 days of constant storage at 0, 4, and 6℃ and variable storage at 0/10, 5/15, and 10/20℃, respectively

Germination/dormancy characteristics

Most early-dispersed seeds were dormant, and radicle emergence was ≤ 5% for seeds at all temperatures (Fig. 3a). For mid-dispersed (15 December) seeds, multiple comparisons using Tukey’s HSD tests revealed that emergence at 20/30℃ was slightly above 5%, but there were no significant differences (p = 0.41) in germination across the different temperatures (p < 0.05). In contrast, radicle emergence percentages of seeds dispersed late in the season (15 January) differed significantly (p < 0.05) at 10/20℃, 15/25℃, and 20/30℃, with percentages increasing as temperatures increased.

Fig. 3.

Germination characteristics (mean + SE) of freshly matured seeds dropped from trees at different periods (a) and water absorption characteristics of seeds (b). Different uppercase letters indicate significant differences in seed germination percentage between different periods of dropping from trees under the same temperature conditions and different lowercase letters significant differences in seed germination percentage between different temperatures for the same period of dropping (p<0.05)

At 5/15℃, radicle emergence was nil for seeds from all dispersal periods. At 10/20℃, only a small number of late-dispersed seeds (< 5%) exhibited radicle emergence, with no significant difference compared to seeds from other dispersal periods (p < 0.05). At 15/25℃, a few mid-dispersed seeds also began to exhibit radicle emergence (< 5%), but the percentage was significantly lower than that of the late-dispersal seeds (p < 0.05). At 20/30℃, radicle emergence of early-dispersed seeds was the lowest (< 5%) and did not differ significantly from mid-dispersal seeds, while late-dispersed seeds exhibited the highest emergence (> 30%) and differed significantly from early- and mid-dispersed seeds (p < 0.05). Thus, radicle emergence follows the pattern: late-dispersed seeds > mid-dispersed seeds > early-dispersed seeds, suggesting that radicle dormancy becomes shallower as the dispersal period progresses.

MC of newly-matured seeds was 46.7%, and after 100 h of water absorption, it increased to only 49.0%, which closely aligned with the water absorption curve of scarified seeds (46.4%−49.3%) (Fig. 3b). Initial MC of seeds with the seed coat removed was 49.6%, and after 100 h of water absorption MC increased to 51.7%. Thus, due to the high MC of newly-matured seeds, relatively little additional water is imbibed by the seeds.

Cold stratification (5/15℃) for 2 to 8 weeks effectively broke radicle dormancy of C. kawakamii seeds (Fig. 4). As the duration of the cold stratification period (5/15℃) increased, radicle emergence steadily increased, with higher incubation temperatures leading to higher percentages (Fig. 4a). When the stratification period was less than 2 weeks, radicle emergence was 0 at 5/15℃. However, after 2 weeks of cold stratification, radicle emergence at 15/25℃ was significantly higher than that of non-stratified seeds, with no significant difference from that at 20/30℃ (p < 0.05). After 4 and 8 weeks of cold stratification, additional radicle emergence occurred at all incubation temperatures, with radicle emergence also beginning at 5/15℃. At 20/30℃, radicle emergence for seeds receiving 8 weeks of cold stratification was about 100%.

Fig. 4.

Effect of stratification environment and incubation temperature on emergence of radicle of C. kawakamii. a 5/15℃ stratification; b field storage. Different uppercase letters represent significant differences in seed germination percentage for different stratification/storage durations at the same temperature and different lowercase letters indicate significant differences in seed germination percentage for different temperatures at the same stratification/storage durations (p<0.05)

Similarly, results of generalized linear model show that the longer the field storage period, the higher the percentage of seeds with radicle emergence (p < 0.05, Fig. 4b). After 4 weeks of field storage root emergence at 20/30℃ increased to approximately 50% and was significantly higher than that of seeds incubated at 10/20℃ (p < 0.05). As field storage time increased to 8 and 12 weeks, radicle emergence of seeds incubated at 20/30℃ was nearly 100%, while it was only ca. 60% for control seeds at 10/20℃.

High-temperature stratification (25℃) was ineffective in breaking radicle dormancy (p = 0.59, Fig. 5c, f). The percentage of seeds with radicle emergence following cold stratification at 6℃ (Fig. 5b, e) was higher (p < 0.05) than that of seeds stored in the field (Fig. 5a, d) or at 25℃ (Fig. 5c, f). After 2 months of cold stratification at January 2023, radicle emergence at 15/25℃ reached 79.0%, while epicotyl emergence was < 2% (Fig. 5e), indicating that cold stratification broke radicle but not epicotyl dormancy of C. kawakamii seeds. After warm stratification at 25℃, neither the radicle nor the epicotyl emerged.

Fig. 5.

Effects of stratification and incubation temperature on germination of radicle and epicotyl of C. kawakamii. a Field stratification followed by incubation at 5/15℃, (b) 6℃ cold stratification followed by incubation at 5/15℃, (c) 25℃ warm stratification followed by incubation at 5/15℃, (d) field stratification followed by incubation at 15/25℃, (e) 6℃ cold stratification followed by incubation at 15/25℃, and (f) 25℃ warm stratification followed by incubation at 15/25℃

When seeds with an emerged radicle were incubated at 6℃ for 4 months at January 2023, a few epicotyls emerged (1.1%), beginning after 40 days of incubation (Fig. 6). Additionally, epicotyl emerged 38.8% at 15/25℃, while only 3.4% emerged at 5/15℃ (3.4%). Epicotyl emergence began after 2 months of storage at 15/25℃, which was 1 month earlier than at 5/15℃.

Fig. 6.

Emergence dynamics of radicle and epicotyl dormancy-released seeds of C. kawakamii over time. a emergence percentage of epicotyl with time at 5/15℃ incubation, (b) emergence of epicotyl with time at 15/25℃ incubation, (c) emergence of radicle with time at 5/15℃ incubation, and (d) emergence of radicle with time at 15/25℃ incubation. Seeds used for the experiment were those that had been cold stratification at 6℃ and then incubated for 60 days

Seed germination under field condition

Between January and February 2023, as temperatures gradually decreased in the field, the percentage of seeds with an emerged radicle began to increase (Fig. 7). By 1 February 2023, radicle emergence had stabilized near 60%, indicating that low temperatures play a key role in breaking radicle dormancy. However, on 20 February 2023 only 3.6% of the seeds had an emerged epicotyl (Fig. 7). By 22 April 2023, 56.8% of the seeds has an emerged radicle, but only 4.2% of the seeds had an emerged radicle and epicotyl.

Fig. 7.

Temperature and germination dynamics of shoots and radicles of C. kawakamii field soil seed bank over time (November 2022 to February 2023)

Discussion

We confirmed that freshly matured seeds of C. kawakamii are recalcitrant and that up to 4 months of exposure to natural temperatures in the habitat (from autumn to spring) are required for both radicle and epicotyl emergence. Both the radicle and the epicotyl have PD, and it is broken when conditions in the habitat are suitable for cold and warm stratification, respectively. The delay between radicle and epicotyl emergence is a kind of epicotyl PD. Consistent with dormancy found in seeds of Quercus robur [32], epicotyl PD of C. kawakamii remains insensitive to environmental cues until the root system is established. As is shown in Fig. 8, although freshly matured seeds exhibit both radicle and epicotyl physiological dormancy (PD), the dormancy status at the time of dispersal varies with dispersal timing. Compared to early-dispersed seeds, late-dispersed seeds have already undergone cold stratification on the mother plant, resulting in released radicle dormancy and consequently rapid root establishment upon germination. However, regardless of dispersal timing, the epicotyl remains dormant in all seeds and requires warm stratification to complete seedling establishment. Our results support the hypothesis that environmental conditions required for radicle and shoot emergence impose significant constraints on seedling recruitment in the endangered species C. kawakamii.

Fig. 8.

Putative pattern of germination of radicle and epicotyl in C. kawakamii seeds. The values indicate the percentage of the total seed pool (across all dispersal periods) that successfully completed radicle, or both radicle and epicotyl emergence under the defined laboratory conditions. RPD: radicle physiological dormancy; RND: radicle non-dormancy; EPD: epicotyl physiological dormancy; END: epicotyl non-dormancy

In addition to the long dormancy-breaking period, seeds of C. kawakamii are recalcitrant. Mortality of C. kawakamii seeds increased as MC declined, and seeds were intolerant of freezing temperatures. Radicles of recalcitrant seeds in Fagaceae generally emerge soon after seed dispersal in autumn [14, 21, 42]. Oak species have evolved mechanisms to delay dehydration, balancing rapid germination with habitat dependency [4]. Seed dispersal of C. kawakamii occurs from November to the following February, during which time the habitat is relatively dry from October to January. Extended attachment to the mother plant beyond January, through delayed dispersal, could be a strategy to sustain seed water relations during a critical period. However, a delay of dispersal until January or February means that most of the cold stratification season needed to break PD of the radicle would have passed. Although acorns have a leathery coat and dense internal trichome layer, which aid in water retention, a substantial proportion of C. kawakami acorns still lose viability during overwintering.

Due to PD, radicle emergence in most C. kawakamii seeds is delayed until spring under natural conditions. Roots elongate and thicken rapidly after germination, suggesting that nutrients for the initial development of the root system are derived from the cotyledons. For oak species in subtropical climates or regions with mild winters, rapid radicle emergence allows for the efficient transfer of nutrients from the cotyledons to the developing root system, which is hypothesized to reduce seed susceptibility to rodent predation [43, 44]. This early nutrient mobilization not only decreases the attractiveness of the seed to herbivores but also confers a competitive advantage by facilitating early establishment and growth during the growing season [18, 29, 44, 45].

In red oak species, which typically inhabit temperate climates, radicle emergence is delayed until spring, as in C. kawakamii, reducing the risk of seedling mortality during the dry/cold winter. In C. fargesii that inhabits a dry winter climate, radicle emergence also is delayed until spring, but this increases the chance of rodent predation [46]. Recent studies have suggested that rodents prefer to eat seeds with an emerged radicle immediately, because the stored nutrients in the seeds would be translocated from the cotyledons to the expanding root after radicle emergence [44, 47], suggesting that the radicle-emerged seeds also face a high risk of predation. Our study suggests that the delay of radicle emergence from C. kawakamii seeds until spring would help protect seedlings from frost damage; however, the extent to which viability of hoarded seeds declines due to recalcitrance remains unclear [21]. Thus, seed mortality also should be considered when assessing adaptability, particularly in oak species whose seed are generally sensitive to desiccation.

A higher percentage of epicotyls emerged in the field than in controlled laboratory conditions. Notably, shoot emergence occurred more than 1 month after radicle emergence in both environments, confirming that C. kawakamii exhibits epicotyl dormancy. Visual inspection revealed that from seed maturation to the development of secondary roots following radicle emergence, the shoot apical meristem remained undifferentiated. Epicotyl development only began after seeds with an emerged radicle had been warm stratified for ca. 1 month. The lower shoot emergence percentage in the laboratory than in the field may be attributed to the limited cultivation period of 2 months, which may have been insufficient for complete breaking of PD of the epicotyl.

Although C. kawakamii exhibits a dual strategy of epicotyl dormancy and recalcitrance, its subtropical distribution alters the ecological trade-offs. Unlike temperate red oaks that delayed germination until spring, which prevents seedlings from being subjected to hard freezes during winter in temperate areas [45, 48], the mild climate in the C. kawakamiii habitat reduces frost-related selection pressure. Consequently, the disadvantages of prolonged dormancy (e.g., intensified seed predation and interspecific competition) become increasingly consequential in the C. kawakamii habitat. Increased temperatures and decreased rainfall in recent decades in subtropical southeastern China, especially in winter [49], pose severe future threats to the seedling establishment of C. kawakamii.

Conclusion

Our study demonstrates that the seeds of C. kawakamii are sensitive to desiccation and low temperatures. Furthermore, they require both cold (winter) and warm (spring) stratification to complete germination, which is defined by the emergence of both the radicle and the epicotyl. These combined physiological traits ultimately limit seedling recruitment in the habitat. Thus, to enhance seedling regeneration, we recommend assisted natural regeneration strategies, including: (1) protecting late-autumn dispersed seeds to exploit their shallower dormancy, (2) maintaining seed moisture (> 35% seed MC) by burying seeds to buffer against surface extremes in winter, and (3) undertake large scale ex situ seed propagation for the production of seedlings for future translocation. Additionally, cultivating seedlings under controlled conditions for subsequent out planting offers a way to overcome recruitment barriers in nature and support population restoration. It is important to note that we focused exclusively on physiological constraints (recalcitrance and radicle/epicotyl dormancy). We did not investigate seed predation, which is another major factor limiting seedlings regeneration in C. kawakamii. Future research could integrate seed predation pressure to develop a more comprehensive understanding of the species recruitment limitation.

Authors’ contributions

S.Z. and Z.W. conceive and design the research. Z.W., L.Q., B.W. conduct the experiments. Y.T. and Z. H. contribute new analytical tools. Z.W., L.Q. analyzed the data. Z.W, L.Q., C.C.B, J.M.B and S.Z. write, review and edit the manuscript. All authors read and approved the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (32401309), the National Key Research and Development Program of China (2022YFF1303202), Natural Science Foundation of Fujian Province (2023J011039), and Fujian Provincial Department of Education Grant (JAT220347).

Data availability

All data generated or analyzed during this study are included in this manuscript. Further inquiries can be directed to the corresponding author.

Declarations

Ethics approval and consent to participate

All experimental studies on the plants in this study were conducted in compliance with relevant national, institutional, and international guidelines.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.