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

Recalcitrant seeds with physiological epicotyl dormancy may limit seedling recruitment of an endangered subtropical oak species | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Recalcitrant seeds with physiological epicotyl dormancy may limit seedling recruitment of an endangered subtropical oak species Zhaoren Wang, Lanyu Qin, Jerry M Baskin, Carol C Baskin, Bomeng Wu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7259877/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Dec, 2025 Read the published version in BMC Plant Biology → Version 1 posted 14 You are reading this latest preprint version 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 determine environmental cues for radicle and epicotyl emergence. Seeds of C. kawakamii rapidly lost viability under low temperature and at a seed moisture content (MC) < 35%. Cold stratification (5/15°C) or field winter temperatures broke radicle dormancy, but epicotyl physiological dormancy (PD) persisted and required ~ 30 days of warm stratification (15/25°C) 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. Almost all 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. Castanopsis kawakamii cold/warm stratification endangered plant epicotyl dormancy germination phenology seed recalcitrance soil seedbank Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 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 Hay. (Fagaceae) is a Tertiary relict subtropical species confined to southeastern China 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 , 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 ]. One potential factor limiting recruitment of seedlings is that seeds of C. kawakamii are desiccation sensitive (recalcitrant) [ 7 ], 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% 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 C) moist stratification to break 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 ]. Occurring epicotyl PD has been observed in Castanopsis 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 ]. In this study, we hypothesize that the combined effects of recalcitrance and epicotyl PD in acorns (hereafter seeds) of C. kawakamii in the Castonopsis kawakamii National Forest Park could significantly reduce seedling establishment due to lack of 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. It is important to gain a good understanding of the requirements for plant regeneration from seeds, in view of the changes in temperature and precipitation patterns that may occur in the natural habitat of this species due to global warming. Materials and Methods Study site and seed collection Castanopsis kawakamii National Forest Park (26°8′~26°13′N, 117°25′~117°30′E) is the world largest population of C. kawakamii Hay. (Fagaceae). Average annual precipitation in this region is 1,819.68 mm (1952–2019), with the majority of it occurring from April to June (Figure 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°C, with January reaching a low of 2.9°C. The plant samples were identified by Prof. Zhenying Huang, a botanist at the Institute of Botany, Chinese Academy of Sciences. 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. The voucher specimens of the plant materials have been deposited at the National Park Research Center, Sanming University. The seed dispersal and dormancy-breaking periods of C. kawakamii overlap with the dry, cold season, which is not favorable for the long-term field storage of recalcitrant seeds. Seed yield of C. kawakamii in the study area is high. Freshly matured seeds were collected in Castanopsis kawakamii National Forest Park, specifically within a mountainous experimental area measuring approximately 500 × 500 meters, encompassing the foothill, slope, and summit regions. The seeds naturally fall during rainfall upon maturation, and we collected the newly 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 (seeds that fell on November 15), mid (seeds that fell on December 15), and late (seeds that fell on January 15 of the following year). The size and weight of 100 randomly selected seeds were measured for each collection date. Some seeds were used for anatomical observations, while others were used in germination experiments. Effect of Dehydration and Low Temperature on Seed Viability Seed Sensitivity to desiccation. Newly matured seeds were randomly sampled and divided into seven treatment groups, with four replicates per group, totaling 28 samples. Each sample contained 25 seeds. After weighing, seeds were embedded in dry silica gel for dehydration, and their MC was determined by weighing every 12 h until seed weight no longer decreased. Seed viability was assessed using 0.4% TTC (triphenyltetrazolium chloride) 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 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 at constant temperatures of 0, 4, and 6°C and at fluctuating temperatures of 10/0, 5/15, and 10/20°C. Seed viability was assessed using TTC on the 15th and 30th days to evaluate the effects of low temperatures on seed viability. Seed Dormancy Release Experiment 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°C (representing the average monthly minimum temperature of 6°C and the average monthly maximum temperature of 15°C in January, February, and December; 12h light/12h dark), 10/20°C (representing the climatic temperatures in March, April, and November), 15/25°C (representing the climatic temperatures in May and October), and 20/30°C (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 high (97.02% after 1 month) at 4°C; therefore, we conducted the cold stratification treatments at 5/15°C and 6°C. The alternation temperature regime of 5/15°C simulated the ambient daily minimum and maximum temperature, respectively, from December to February, while 6°C 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°C, 10/20°C, 15/25°C, and 20/30°C. (2) Breaking Epicotyl dormancy : Since epicotyl emergence was not observed in response to cold stratification, we incubated seeds with an emerged radicle at 6°C, 5/15°C, and 15/25°C (12h dark/12h light, simulating average spring temperatures) for an additional 60 days. This extended period of incubation was used to determine the effects of cold stratification at 5/15°C and warm stratification at 15/25°C 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 2022, December 2022, January 2023, and February 2023. To evaluate radicle and epicotyl dormancy, we employed three stratification treatments: winter, field cold stratification (starting on November 30), cold stratification at 6°C, and warm stratification at 25°C. Following stratification for 1 month, seeds were incubated at 5/15°C and 15/25°C (12h dark/12h light) for 30 days, and radicle and epicotyl emergence were checked daily. Field Soil Seed Bank Germination Experiment. 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 with the seeds evenly spread and pressed down to ensure contact with the soil. The artificial soil seed bank was enclosed with wire mesh to prevent predation. This setup was established in late October 2022, and the temperature of the test site was continuously monitored using a temperature recorder. Emergence of radicles and shoots were monitored monthly November 2022 to March 2023. Statistical analyses All data analyses were done with R software [ 38 ]. The response variables were expressed as mean ± SE or mean + SE (non-transformed data appear in all figures). A general linear model (GLM) with family = binomial (‘logit’) model [ 39 ] was used to test whether different experimental treatments, including incubation temperature, storage condition, and length of stratification time as variables, radicle/epicotyl emergence as response variables, significances on emergence followed by multiple contrasts with Tukey’s HSD tests using the ‘glht’ function of the ‘multcomp’ package, level of significance was set at P < 0.05 [ 40 ]. Result Morphology of Seed Germination Seeds of C. kawakamii are irregularly spherical, with an average mass of 2.11 ± 0.04 g (mean ± SE, n = 100) (Fig. 1 a). The pericarp is leathery and densely covered with retrorse hairs on the inner surface (Fig. 1 f). 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 i). Under suitable conditions, the radicle emerges, after which the hypocotyl elongates (Fig. 1 b). The radicle then elongates, and lateral roots develop (Figs. 1 d, e). After epicotyl dormancy is broken, the cotyledon petioles elongate, pushing the upper hypocotyl out of the seed. The epicotyl(shoot) emerges at the junction of the two cotyledonary petioles (Fig. 1 e, S2). 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. 2 a). 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 ≤ 4°C (freezing point) for 15 or more days resulted in significant seed mortality. Storing seeds at 6°C for 30 days increased mortality by 10% compared to storing them for 15 days (Fig. 2 b). Excessively low winter temperatures (e.g., 0/10°C) caused high (84.7%) seed mortality, while prolonged exposure to moderately low temperatures (e.g., 5/15°C for 30 days, Fig. 2 b) lead to 17.6% mortality. However, at 10/20°C C. kawakamii seeds can be stored for an extended period with nearly 100% of them remaining viabile. Initial Germination Characteristics and Seed Coat Permeability Most early-dispersed seeds were dormant, and radicle emergence was ≤ 5% for seeds at all temperatures (Fig. 3 a). For mid-dispersed (15 December) seeds, emergence at 20/30°C was slightly above 5%, but there were no significant differences 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°C, 15/25°C, and 20/30°C, with percentages increasing as temperatures increased. At 5/15°C, radicle emergence was nil for seeds from all dispersal periods. At 10/20°C, 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°C, 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°C, radicle emergence of early-dispersed seeds was the lowest ( 30%) and differed significantly from early- and mid-dispersed seeds ( P 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 hours 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. 3 b). Initial MC of seeds with the seed coat removed was 49.6%, and after 100 hours 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. Seed Dormancy Release Cold stratification (5/15°C) for 2 to 8 weeks effectively broke radicle dormancy of C. kawakamii seeds (Fig. 4 ). As duration of the cold stratification period (5/15°C) increased, radicle emergence steadily increased, with higher incubation temperatures leading to higher percentages (Fig. 4 a). When the stratification period was less than 2 weeks, radicle emergence was minimal at 5/15°C. However, after 2 weeks of cold stratification, radicle emergence at 15/25°C was significantly higher than that of non-stratified seeds, with no significant difference from that at 20/30°C ( 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°C. At 20/30°C, radicle emergence for seeds receiving 8 weeks of cold stratification was about 100%. Similarly, the longer the field storage period, the higher the percentage of seeds with radicle emergence (Fig. 4 b). After 2 weeks of field storage, the percentage of seeds with radicle emergence at 10/20°C was slightly higher than that of seeds incubated at 20/30°C, although the difference was non-significant ( P < 0.05). After 4 weeks of field storage root emergence at 20/30°C increased to approximately 50% and was significantly higher than that of seeds incubated at 10/20°C. As field storage time increased to 8 and 12 weeks, radicle emergence of seeds incubated at 20/30°C was nearly 100%, while it was only ca . 60% for control seeds at 10/20°C. High-temperature stratification (25℃) was ineffective in breaking radicle dormancy (Figs. 5 c, f). The percentage of seeds with radicle emergence following cold stratification at 6℃ (Figs. 5 b, e) was higher than that of seeds stored in the field or at 25℃ (Figs. 5 a, d and Figs. 5 c, f). After 2 months of cold stratification, radicle emergence at 15/25℃ reached 79.0%, while epicotyl emergence was < 2% (Fig. 5 e), 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 (< 2%) (Fig. 5 e). When seeds with an emerged radicle were incubated at 6°C for 4 months, a few epicotyls emerged (1.1%), beginning after 40 days of incubation (Fig. 6 ). Additionally, epicotyl emergence was higher at 15/25℃ (38.8%) than that 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℃. 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. 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. 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 . In addition to the long dormancy-breaking period, seeds of C. lawakamii 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 , 41 ]. 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. It seems that delayed seed dispersal to after January would help ensure a continued water supply from the mother plant for the seeds. 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 reduces seed susceptibility to rodent predation [ 42 , 43 ]. 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 , 43 , 44 ]. 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 [ 45 ]. Recent studies have suggested that rodents prefer to eat seeds with an emerged radicle immediately, because the stored nutrients in the acorns would be translocated from the cotyledons to the expanding root after radicle emergence [ 43 , 46 ], 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. Anatomical observations revealed that from seed maturation to the development of secondary roots following radicle emergence, the shoot apical meristem remained undifferenced. 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 [ 44 , 47 ], 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 [ 48 ], pose severe future threats to the seedling establishment of C. kawakamii . Conclusion Desiccation and low temperature sensitivity of C. kawakamii seeds and their requirement for cold (winter) and warm (spring) stratification for complete germination, i.e. emergence of both the radicle and epicotyl. 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 soil moisture (> 35% seed MC) by burying seeds to buffer against surface extremes in winter, and (3) applying post-radicle artificial warm stratification (15/25°C, ~ 30 days) to break epicotyl dormancy, to achieve effective population recovery. 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 that they do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted. Data statement All data generated or analyzed during this study are included in this manuscript. Further inquiries can be directed to the corresponding author. 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). Author Contribution 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. Acknowledgement The authors thank equipment provided by Administrative Committee of Castanopsis kawakamii National Forest Park and Sanming University (Sanming city, Fujian province, China) experimental platforms for the assistance for this study. Special thanks are extended to undergraduate students Huijing Jin, Shunan, Yang and Jiaxing Jing from the School of Economics and Management, Sanming University, for their contributions to sample collection and processing. Data Availability All data generated or analyzed during this study are included in this manuscript. Further inquiries can be directed to the corresponding author. References Harper JL. Population biology of plants. London, UK: Academic; 1977. Wang Z, Baskin JM, Baskin CC, Liu G, Ye X, Yang X, Huang Z. Soil salinity regulates spatial-temporal heterogeneity of seed germination and seedbank persistence of an annual diaspore-trimorphic halophyte in northern China. BMC Plant Bio. 2024;24:604. https://doi.org/10.1186/s12870-024-05307-x . Donohue K, Casas RRD, Burghardt L. Germination, post germination adaptation, and species ecological ranges. Ann Rev Ecol Syst. 2010;41:293–319. https://doi.org/10.1146/annurev-ecolsys-102209-144715 . Xia K, Daws MI, Peng LL. Climate drives patterns of seed traits in Quercus species across China. New Phytol. 2022;234:1629–38. https://doi.org/10.1111/nph.18103 . Wang Z, Baskin JM, Baskin CC, Yang X, Liu G, Ye X, Huang Z, Cornelissen JHC. Great granny still ruling from the grave: Phenotypical response of plant performance and seed functional traits to salt stress affects multiple generations of a halophyte. J Ecol. 2022;110:117–28. https://doi.org/10.1111/1365-2745.13789 . Huang C, Chang Y, Bruce B. Fagaceae. In: Chun Woonyong & Huang Chengchiu, eds. Fl. Reipubl. Popularis Sin. 1998;22:1-332. https://www.iplant.cn/info/Fagaceae?t=foc Liu JF, He ZS, Hong W, Zheng S, Wang Z. Conservation ecology of endangered plant Castanopsis kawakamii . J Beijing Forestry Univ. 2011;33:136–43. (in Chinese with English abstract). http://j.bjfu.edu.cn/en/article/id/9662 . Hong WJ, Zeng SJ, Ma DW, Wang P, Lai Z, Zhong W, Liao Y, Yang L, Zhuang X. Community characteristics with the population of Castanopsis kawakamii in Lianhuashan-Baipenzhu Nature Reserve, Guangdong. Forestry Environ Sci. 2016;32:10–6. (in Chinese with English abstract). Liu JF. Studies on structure and dynamics characteristics of Castanopsis kawakamii Hayata population[M]. Beijing Forestry Univ. 2004. (in Chinese with English abstract). He ZS, Liu FJ, Zheng SQ, Su S, Hong W, Wu Z, Xu D, Wu C. Studies on the seeds dispersal and seedlings regeneration in gaps and understory of Castanopsis kawakamii natural forest. J Trop Subtrop Bot. 2012;20:506–12. https://api.semanticscholar.org/CorpusID:87572748 . Roberts EH. Predicting the storage life of seeds. Seed Sci Technol. 1973;1:499–514. Pritchard HW. Classification of seed storage ‘types’ for ex-situ conservation in relation to temperature and moisture. In: Havens EO, Maunder K, editors. Ex-Situ Plant Conservation. Supporting Species Survival in the Wild;Guerrant. Washington, DC, USA: M., Eds.;Island; 2004. pp. 139–61. Masaki T, Wijenayake PR. How long do tree seeds survive in soil seedbanks? A multi-species comparison in an old-growth deciduous temperate forest. J Res. 2024;29:374–82. https://doi.org/10.1080/13416979.2024.2327979 . Berjak P, Pammenter NW. Implications of the lack of desiccation tolerance in recalcitrant seeds. Front Plant Sci. 2014;5:123. https://doi.org/10.3389/fpls.2013.00478 . Hong TD, Ellis RH. Contrasting seed storage behaviour among different species of Meliaceae. Seed Sci Technol. 1998;26:77–95. Liu X, Xiao Y, Wang Y, Wang R, Huang R, Liang H, Jiang Y, Jiang Y. Seed germination ecology of endangered plant Horsfieldia hainanensis Merr. In China. BMC Plant Biol. 2024;24: 486. https://doi.org/10.1186/s12870-024-05208-z Pritchard HW, Steadman KJ, Nash JV, Jones C. Kinetics of dormancy release and the high temperature gemination response in Aesculus hippocastanum seeds. J Exp Bot. 1999;50:1507–14. https://doi.org/10.1093/jxb/50.338.1507 . Rodríguez-Calcerrada J, Sancho-Knapik D, Martin-StPaul NK, Limousin JM, McDowell NG, Gil-Pelegrín E. Drought-induced oak decline—factors involved, physiological dysfunctions, and potential attenuation by forestry practices. In: Gil-Pelegrín E, Peguero-Pina JJ, Sancho-Knapik D, editors. Oaks physiological ecology exploring the functional diversity of genus Quercus L. Cham: Springer International Publishing; 2017. pp. 419–51. https://doi.org/10.1007/978-3-319-69099-5_13 . Walters C, Berjak P, Pammenter N, Kennedy K, Raven P. Preservation of recalcitrant seeds. Science. 2013;339:915–6. https://doi.org/10.1126/science.123093 . Li J, Jaganathan GK, Kang H, Liu B. The co-occurrence of physiological and epicotyl physiological dormancy in three desiccation-sensitive Castanopsis (Fagaceae) acorns from China with specific reference to the embryonic axis position. Forests. 2023;14:2330. https://doi.org/10.3390/f14122330 . Jaganathan GK, Phartyal SS. A classification system for germination in desiccation-sensitive Fagaceae acorns: with particular focus on physiological and epicotyl dormancy. Bot J Linn Soc. 2025;207:197–207. https://doi.org/10.1093/botlinnean/boae049 . Jayasuriya KMGG, Wijetunga ASTB, Baskin JM, Baskin CC. Physiological epicotyl dormancy and recalcitrant storage behaviour in seeds of two tropical Fabaceae (subfamily Caesalpinioideae) species. AoB Plants. 2012;2012:pls044. https://doi.org/10.1093/aobpla/pls044 . Flores EM. Seed biology, pp. 13–124 in Vozzo JA, editor Tropical tree seed manual. USDA Forest Service Agriculture Handbook 721, 2002. https://doi.org/10.1093/aob/mch046 Yang G, Yang L, Wang Y, Shen S. Physiological epicotyl dormancy and its alleviation in seeds of Yunnanopilia longistaminea : the first report of physiological epicotyl dormancy in China. PeerJ. 2017;5:e3435. https://doi.org/10.7717/peerj.3435 . Rong Z, Yi Y, Shen X, Deng S, Xu L, Li J, Mou J, Deng Z. Breaking deep epicotyl physiological dormancy in Chionanthus hionanthus retusus (Oleaceae) seeds. Seed Sci Technol. 2024;52:67–77. https://doi.org/10.15258/sst.2024.52.1.07 . McCartan SA, Jinks RL, Barsoum N. Using thermal time models to predict the impact of assisted migration on the synchronization of germination and shoot emergence of oak ( Quercus robur L). Ann For Sci. 2015;72:479–87. https://doi.org/10.1007/s13595-014-0454-5 . Baskin JM, Baskin CC. The great diversity in kinds of seed dormancy: a revision of the Nikolaeva-Baskin classification system for primary seed dormancy. Seed Sci Res. 2021;31:249–77. https://doi.org/10.1017/S096025852100026X . Hopper GM, Smith DW, Parrish DJ. Germination and seedling growth of northern red oak: effects of stratification and pericarp removal. For Sci. 1985;31:31–9. https://doi.org/10.1093/forestscience/31.1.31 . Wigston DL. Epicotyl dormancy in Quercus robur L. Quart J For. 1987;81:110–2. Sun X, Song Y, Ge B, Dai X, Kozlowski G. Intermediate epicotyl physiological dormancy in the recalcitrant seed of Quercus chungii F.P.Metcalf with the elongated cotyledonary petiole. Forests. 2021;12:263. https://doi.org/10.3390/f12030263 . Farmer R. Epicotyl dormancy in white and chestnut Oaks. For Sci. 1977;23:329–32. https://doi.org/10.1093/forestscience/23.3.329 . Jastrzębowski S, Ukalska J, Walck JL. Does the lag time between radicle and epicotyl emergences in acorns of pedunculate oak ( Quercus robur L.) depend on the duration of cold stratification and poststratification temperatures? Modelling with the sigmoidal growth curves approach. Seed Sci Res. 2021;31:105–115. 1 https://doi.org/0.1017/S096025852100009X. Macchia F, Cavallaro V, Vita F, Sburlino G. Acorn dormancy and aridity as factors of Quercus Cerris L. distribution. In: Teller A, Mathy P, Jeffers JNR, editors. Responses of forest ecosystems to environmental changes. Netherlands, Dordrecht: Springer; 1992. pp. 633–4. https://doi.org/10.1007/978-94-011-2866-7_91 . Hawkins TS. The influence of dormancy break requirements on germination and viability responses to winter submergence in acorns of three bottomland red oak (Sect. Lobatae) species. For Sci. 2019;65:556–61. https://doi.org/10.1093/forsci/fxz028 . Zhang K, Ji Y, Yao L, Liu H, Zhang Y, Baskin JM, Baskin CC, Zhang L, Xu C, Tao J, Prinzing A. Paternal intergenerational plasticity in the plant species Paeonia ostii : Implications for parental fitness and offspring performance. Funct Ecol. 2024;38:832–47. https://doi.org/10.1111/1365-2435.14506 . Zhang K, Pan H, Baskin CC, Baskin JM, Xiong Z, Cao W, Yao L, Tang B, Zhang C, Tao J. Epicotyl morphophysiological dormancy in seeds of Paeonia ostii (Paeoniaceae): seasonal temperature regulation of germination phenology. Environ Exp Bot. 2022;194:104742. https://doi.org/10.1016/j.envexpbot.2021.104742 . Kang H, Jaganathan GK, Han Y, Li J, Liu B. Revisiting the pericarp as a barrier restricting water entry/loss from cotyledons and embryonic axis of temperate desiccation-sensitive Quercus acorns. Planta. 2023;257:33. https://doi.org/10.1007/s00425-022-04061-4 . R Core Team. (2023). R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing. Retrieved from https://www.R-project.org/ Warton DI, Hui FKC. The arcsine is asinine: the analysis of proportions in ecology. Ecology. 2011;92:3–10. https://doi.org/10.1890/10-0340.1 . Hothorn T, Bretz F, Westfall P. Simultaneous inference in general parametric models. Biometrical J. 2008;50:346–63. https://doi.org/10.1002/bimj.200810425 . Xiao Z, Zhang Z, Krebs CJ. Long-term seed survival and dispersal dynamics in a rodent-dispersed tree: testing the predator satiation hypothesis and the predator dispersal hypothesis. J Ecol. 2013;101:1256–64. https://doi.org/10.1111/1365-2745.12113 . Fox JF. Adaptation of gray squirrel behavior to autumn germination by white oak acorns. Evolution. 1982;36:800–9. https://doi.org/10.1111/j.1558-5646.1982.tb05446.x . Cao L, Jansen PA, Wang B, Yan C, Wang Z, Chen J. Mutual cheating strengthens a tropical seed dispersal mutualism. Ecology. 2022;103:e03574. https://doi.org/10.1002/ecy.3574 . Baskin CC, Baskin JM. Seeds: ecology, biogeography, and evolution of dormancy and germination. 2nd ed. San Diego: Academic; 2014. Cornelissen JHC, Zhong Z, Werger M. Timing of germination in the subtropical chinese tree Castanopsis farqesii . Journal of Southwest China Normal University (Natural Science); 1994. Li Y, Yang X, Feng E, Zhao K, Zhang Z. Plant hormones mediate the interaction between oak acorn germination and rodent hoarding behaviour. New Phytol. 2024;242:2237–50. https://doi.org/10.1111/nph.19424 . Joët T, Ourcival JM, Capelli M, Dussert S, Morin X. Explanatory ecological factors for the persistence of desiccation-sensitive seeds in transient soil seed banks: Quercus ilex as a case study. Ann Bot. 2015;117:165–76. https://doi.org/10.1093/aob/mcv139 . IPCC. Sixth Assessment Report: Climate Change 2021:The Physical Science Basis. 2021. https://www.ipcc.ch/report/ar6/wg1/ Additional Declarations No competing interests reported. Supplementary Files Supplementaryfile.docx Cite Share Download PDF Status: Published Journal Publication published 05 Dec, 2025 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Revision requested 25 Sep, 2025 Reviews received at journal 24 Sep, 2025 Reviews received at journal 23 Sep, 2025 Reviews received at journal 22 Sep, 2025 Reviewers agreed at journal 15 Sep, 2025 Reviewers agreed at journal 14 Sep, 2025 Reviews received at journal 11 Sep, 2025 Reviewers agreed at journal 08 Sep, 2025 Reviewers agreed at journal 08 Sep, 2025 Reviewers invited by journal 08 Sep, 2025 Editor assigned by journal 08 Sep, 2025 Editor invited by journal 18 Aug, 2025 Submission checks completed at journal 14 Aug, 2025 First submitted to journal 14 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7259877","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":514856550,"identity":"dd2234c7-95e4-48af-924a-cea56fe76a46","order_by":0,"name":"Zhaoren Wang","email":"","orcid":"","institution":"Institute of Urban Environment , Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Zhaoren","middleName":"","lastName":"Wang","suffix":""},{"id":514856551,"identity":"657687e8-bed2-432f-a9c0-b685c48ed739","order_by":1,"name":"Lanyu Qin","email":"","orcid":"","institution":"Institute of Urban Environment , Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Lanyu","middleName":"","lastName":"Qin","suffix":""},{"id":514856552,"identity":"b2b5183b-53f5-4a4a-98f5-eacb89942bdd","order_by":2,"name":"Jerry M Baskin","email":"","orcid":"","institution":"University of Kentucky","correspondingAuthor":false,"prefix":"","firstName":"Jerry","middleName":"M","lastName":"Baskin","suffix":""},{"id":514856553,"identity":"30045435-ca89-4a3f-aafc-cd5c78243156","order_by":3,"name":"Carol C Baskin","email":"","orcid":"","institution":"University of Kentucky","correspondingAuthor":false,"prefix":"","firstName":"Carol","middleName":"C","lastName":"Baskin","suffix":""},{"id":514856554,"identity":"c8efd949-0779-4180-9014-c970d871ba10","order_by":4,"name":"Bomeng Wu","email":"","orcid":"","institution":"Institute of Urban Environment , Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Bomeng","middleName":"","lastName":"Wu","suffix":""},{"id":514856555,"identity":"69e137d7-5a28-48d4-9091-6bbe68fd36ab","order_by":5,"name":"Ye Tian","email":"","orcid":"","institution":"Institute of Urban Environment , Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ye","middleName":"","lastName":"Tian","suffix":""},{"id":514856556,"identity":"12d799fa-1c41-4488-ac56-149b4c6432bd","order_by":6,"name":"Zhenying Huang","email":"","orcid":"","institution":"Institute of Botany. Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Zhenying","middleName":"","lastName":"Huang","suffix":""},{"id":514856557,"identity":"f682283e-164a-4c47-b785-e14a90755f55","order_by":7,"name":"Shuanning Zheng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYHACNhAhx0ayFmPStSQ2EK1efnb7swcfd9Sm90kkH2D4UcMgb05Ii8GdA+mGM88cz22TSEtg7DnGYLiTkH0GEgnHpHnbjgG15Bgw8DYwJBgcIOSwGYlt0n/bjqWzSeR/YPxLjBaGG8ls0oxtNQlsEjkMzETZYnAjjU2yt+2AYRvPM4PDMsckDDcQdlj6M4mfbXXy8u3JDx++qbGRJ+wwCDgMJoGKJYhTDwR1RKscBaNgFIyCEQgADDM8PwaKH8AAAAAASUVORK5CYII=","orcid":"","institution":"Institute of Urban Environment , Chinese Academy of Sciences","correspondingAuthor":true,"prefix":"","firstName":"Shuanning","middleName":"","lastName":"Zheng","suffix":""}],"badges":[],"createdAt":"2025-07-31 08:38:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7259877/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7259877/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-025-07847-2","type":"published","date":"2025-12-05T15:58:05+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":91342230,"identity":"a5277325-92e2-457b-838c-ad2f6300b241","added_by":"auto","created_at":"2025-09-15 13:18:33","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":184137,"visible":true,"origin":"","legend":"\u003cp\u003eGermination process and seed structure/morphology of \u003cem\u003eC. kawakamii\u003c/em\u003eseeds. (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.\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7259877/v1/2afd6ad5088d2ec118287d21.jpeg"},{"id":91341993,"identity":"ba42e473-0421-4011-8960-cd1352f54dec","added_by":"auto","created_at":"2025-09-15 13:10:33","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":119803,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of desiccation (a) and temperature (b) on seed viability of \u003cem\u003eC. kawakamii\u003c/em\u003e. (a) The relationship between loss of moisture content (%) and seed survival (%). The red line in the graph indicates the fitted equation: Y=112.81-3.12x (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e=0.77,\u003cem\u003e P\u003c/em\u003e\u0026lt;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 10℃ and variable storage at 0-10, 5-15, and 10-20℃, respectively.\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7259877/v1/61671d3732c44803af1c3fad.jpeg"},{"id":91341995,"identity":"3247160e-3976-458c-a061-617d481b96c9","added_by":"auto","created_at":"2025-09-15 13:10:33","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":103802,"visible":true,"origin":"","legend":"\u003cp\u003eGermination characteristics (mean + SE) of newly 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 (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7259877/v1/2ffad96c8b9720f60578adb5.jpeg"},{"id":91342233,"identity":"dd16343f-646e-427d-ae38-bd31a42af7bb","added_by":"auto","created_at":"2025-09-15 13:18:33","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":125039,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of stratification environment and incubation temperature on emergence of radicle of \u003cem\u003eC. kawakamii\u003c/em\u003e. (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 (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7259877/v1/e1a3b6ccc78cdb77e6355fcf.jpeg"},{"id":91342004,"identity":"cb8dea38-a67b-4706-bda4-dfd2777cd4a7","added_by":"auto","created_at":"2025-09-15 13:10:33","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":158735,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of stratification and incubation temperature on germination of radicle and epicotyl of \u003cem\u003eC. kawakamii\u003c/em\u003e. (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℃.\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7259877/v1/7f53a014436c75b93d75311a.jpeg"},{"id":91342232,"identity":"a61ce893-6865-4b28-9219-5b5a9f362876","added_by":"auto","created_at":"2025-09-15 13:18:33","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":89169,"visible":true,"origin":"","legend":"\u003cp\u003eGermination dynamics of radicle and epicotyl dormancy-released seeds of \u003cem\u003eC. kawakamii\u003c/em\u003eover time. (a) Germination 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.\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7259877/v1/149115fcc9298dc93dcf6d74.jpeg"},{"id":91341998,"identity":"c1b54703-41f5-4baa-a4b0-8fb98b481fda","added_by":"auto","created_at":"2025-09-15 13:10:33","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":125439,"visible":true,"origin":"","legend":"\u003cp\u003eTemperature and germination dynamics of shoots and radicles of \u003cem\u003eC. kawakamii\u003c/em\u003e field soil seed bank over time (November 2022 to February 2023).\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-7259877/v1/6ed9d5217e24310ce7e9338e.png"},{"id":91343071,"identity":"0c2490ab-21aa-4689-a074-e3bdc7ee34b6","added_by":"auto","created_at":"2025-09-15 13:26:33","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":246179,"visible":true,"origin":"","legend":"\u003cp\u003ePutative pattern of germination of radicle and epicotyl in \u003cem\u003eC. kawakamii\u003c/em\u003e seeds. RPD: radicle physiological dormancy; RND: radicle non-dormancy; EPD: epicotyl physiological dormancy; END: epicotyl non-dormancy.\u003c/p\u003e","description":"","filename":"image8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7259877/v1/fbdcfd88a47d70ee12697b90.jpeg"},{"id":97723913,"identity":"fa622b6f-cbc5-40ad-8a5c-ccf9991df77f","added_by":"auto","created_at":"2025-12-08 16:09:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1947210,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7259877/v1/636596f9-5b4e-40c6-ad5e-4d146abae64d.pdf"},{"id":91343398,"identity":"3d91fa90-8763-4f38-b144-78c624b50cf0","added_by":"auto","created_at":"2025-09-15 13:34:33","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1844971,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-7259877/v1/dccd5ef6bb697e067e84da15.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Recalcitrant seeds with physiological epicotyl dormancy may limit seedling recruitment of an endangered subtropical oak species","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe transition from seed to seedling is a high-risk period in the life-cycle of most plant species [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], and it is crucial for local adaptation of species and population restoration [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The endangered species \u003cem\u003eCastanopsis kawakamii\u003c/em\u003e Hay. (Fagaceae) is a Tertiary relict subtropical species confined to southeastern China and Vietnam [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Population aging and seedling scarcity have been reported across its distributional range [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The \u003cem\u003eCastonopsis kawakamii\u003c/em\u003e National Forest Park in China is the largest remaining natural population of \u003cem\u003eC. kawakamii\u003c/em\u003e, yet many mature/aging trees exhibit trunk rot, resulting in hollow trunks [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Despite abundant seed production, seedlings and saplings are scarce, raising concerns about the mechanisms restricting regeneration from seeds [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. One potential factor limiting recruitment of seedlings is that seeds of \u003cem\u003eC. kawakamii\u003c/em\u003e are desiccation sensitive (recalcitrant) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], and we have observed that up to 4 months are required for complete germination, i.e. emergence of both root and shoot.\u003c/p\u003e\u003cp\u003eBased on their response to desiccation, seeds are categorized as orthodox (\u0026lt;\u0026thinsp;7% MC), intermediate (10\u0026ndash;12% MC), or recalcitrant (20\u0026ndash;30% MC) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Unlike orthodox seeds, which undergo dehydration tolerance at maturity, recalcitrant seeds are desiccation and low-temperature sensitive [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Also, recalcitrant seeds lose viability at a relatively high MC, making them vulnerable to environmental stress [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Low temperatures can further accelerate loss of viability of recalcitrant seeds [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], but some recalcitrant seeds require cold (c. 0 to 10 C) moist stratification to break PD [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDesiccation sensitivity has been demonstrated in seeds of various oak species (Fagaceae) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This desiccation sensitivity of seeds can restrict the geographical distribution of species and has important implications for conservation and population restoration efforts [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOccurring epicotyl PD has been observed in \u003cem\u003eCastanopsis chinensis\u003c/em\u003e, \u003cem\u003eC. purpurella\u003c/em\u003e, and \u003cem\u003eC. sclerophylla\u003c/em\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], resulting in a delay in timing of epicotyl emergence. Thus, we assume that seeds of \u003cem\u003eC. kawakamii\u003c/em\u003e also have epicotyl PD and exhibit a delay between root and shoot emergence. Epicotyl PD occurs in various genera such as \u003cem\u003eBrownea\u003c/em\u003e [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], \u003cem\u003eLecythis\u003c/em\u003e [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], \u003cem\u003eHumboldtia\u003c/em\u003e, \u003cem\u003eYunnanopilia\u003c/em\u003e [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], \u003cem\u003eChionanthus\u003c/em\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], and oak species [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eEarly studies on oak species [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] demonstrated that epicotyl PD in \u003cem\u003eQuercus robur\u003c/em\u003e can be released by cold stratification, ensuring that shoot emergence aligns with optimal conditions for seedling establishment and early growth in spring [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Seeds of species in sections \u003cem\u003eQuercus\u003c/em\u003e, \u003cem\u003eLobatae\u003c/em\u003e, and \u003cem\u003eProtoquercus\u003c/em\u003e of Fagaceae typically exhibit PD [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Radicles of \u003cem\u003eQ. alba\u003c/em\u003e, \u003cem\u003eQ. prinus\u003c/em\u003e, and \u003cem\u003eQ. robur\u003c/em\u003e (in section \u003cem\u003eQuercus\u003c/em\u003e) have little or no PD, and they emerge immediately upon dispersal [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, the epicotyl has PD, which is broken by cold stratification during winter [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In seeds of some species such as those of \u003cem\u003eQ. cerris\u003c/em\u003e [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and \u003cem\u003eQ. nigra\u003c/em\u003e [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] in sections \u003cem\u003eCerris\u003c/em\u003e and \u003cem\u003eLobatae\u003c/em\u003e, respectively, both the radicle and epicotyl have PD, which is broken by cold stratification during winter.\u003c/p\u003e\u003cp\u003eSeeds 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 \u003cem\u003eCyclobalanopsis chungii\u003c/em\u003e [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], \u003cem\u003eHumboldtia laurifolia\u003c/em\u003e, \u003cem\u003eCynometra cauliflora\u003c/em\u003e [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], \u003cem\u003ePaeonia ostia\u003c/em\u003e [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and many oak species [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], comprehensive studies on their dormancy-breaking and germination requirements and maintenance of seed viability are limited [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study, we hypothesize that the combined effects of recalcitrance and epicotyl PD in acorns (hereafter seeds) of \u003cem\u003eC. kawakamii\u003c/em\u003e in the \u003cem\u003eCastonopsis kawakamii\u003c/em\u003e National Forest Park could significantly reduce seedling establishment due to lack of 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. It is important to gain a good understanding of the requirements for plant regeneration from seeds, in view of the changes in temperature and precipitation patterns that may occur in the natural habitat of this species due to global warming.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStudy site and seed collection\u003c/h2\u003e\u003cp\u003e\u003cem\u003eCastanopsis kawakamii\u003c/em\u003e National Forest Park (26\u0026deg;8\u0026prime;~26\u0026deg;13\u0026prime;N, 117\u0026deg;25\u0026prime;~117\u0026deg;30\u0026prime;E) is the world largest population of \u003cem\u003eC. kawakamii\u003c/em\u003e Hay. (Fagaceae). Average annual precipitation in this region is 1,819.68 mm (1952\u0026ndash;2019), with the majority of it occurring from April to June (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). 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\u0026deg;C, with January reaching a low of 2.9\u0026deg;C. The plant samples were identified by Prof. Zhenying Huang, a botanist at the Institute of Botany, Chinese Academy of Sciences. 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 \u003cem\u003eCastanopsis kawakamii\u003c/em\u003e 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. The voucher specimens of the plant materials have been deposited at the National Park Research Center, Sanming University. The seed dispersal and dormancy-breaking periods of \u003cem\u003eC. kawakamii\u003c/em\u003e overlap with the dry, cold season, which is not favorable for the long-term field storage of recalcitrant seeds.\u003c/p\u003e\u003cp\u003eSeed yield of \u003cem\u003eC. kawakamii\u003c/em\u003e in the study area is high. Freshly matured seeds were collected in \u003cem\u003eCastanopsis kawakamii\u003c/em\u003e National Forest Park, specifically within a mountainous experimental area measuring approximately 500 \u0026times; 500 meters, encompassing the foothill, slope, and summit regions. The seeds naturally fall during rainfall upon maturation, and we collected the newly 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 (seeds that fell on November 15), mid (seeds that fell on December 15), and late (seeds that fell on January 15 of the following year). The size and weight of 100 randomly selected seeds were measured for each collection date. Some seeds were used for anatomical observations, while others were used in germination experiments.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eEffect of Dehydration and Low Temperature on Seed Viability\u003c/h3\u003e\n\u003cp\u003e\u003cb\u003eSeed Sensitivity to desiccation.\u003c/b\u003e Newly matured seeds were randomly sampled and divided into seven treatment groups, with four replicates per group, totaling 28 samples. Each sample contained 25 seeds. After weighing, seeds were embedded in dry silica gel for dehydration, and their MC was determined by weighing every 12 h until seed weight no longer decreased. Seed viability was assessed using 0.4% TTC (triphenyltetrazolium chloride) 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.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSeed Sensitivity to Low Temperature.\u003c/b\u003e Newly matured seeds were randomly sampled and divided into 24 portions (6 temperature treatments \u0026times; 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 \u0026times; 10 cm \u0026times; 10 cm deep). These boxes were placed in incubators at constant temperatures of 0, 4, and 6\u0026deg;C and at fluctuating temperatures of 10/0, 5/15, and 10/20\u0026deg;C. Seed viability was assessed using TTC on the 15th and 30th days to evaluate the effects of low temperatures on seed viability.\u003c/p\u003e\n\u003ch3\u003eSeed Dormancy Release Experiment\u003c/h3\u003e\n\u003cp\u003e\u003cb\u003eInitial Germination Experiment.\u003c/b\u003e Freshly-collected seeds for each dispersal period were randomly selected and divided into 4 treatment groups\u0026times;3 periods\u0026times;4 replicates, 48 samples with 25 seeds per replicate. The germination experiments were conducted at four temperature regimes: 5/15\u0026deg;C (representing the average monthly minimum temperature of 6\u0026deg;C and the average monthly maximum temperature of 15\u0026deg;C in January, February, and December; 12h light/12h dark), 10/20\u0026deg;C (representing the climatic temperatures in March, April, and November), 15/25\u0026deg;C (representing the climatic temperatures in May and October), and 20/30\u0026deg;C (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 \u0026ge;\u0026thinsp;5 mm. The incubation period lasted for 30 days, and number of seeds with an emerged radicle was recorded daily.\u003c/p\u003e\u003cp\u003e\u003cb\u003eKind of Seed Dormancy.\u003c/b\u003e (1) \u003cem\u003eEffect of Cold Stratification on Breaking Radicle Dormancy\u003c/em\u003e: In the \"Seed Sensitivity to Low Temperature\" experiment, seed mortality was high (97.02% after 1 month) at 4\u0026deg;C; therefore, we conducted the cold stratification treatments at 5/15\u0026deg;C and 6\u0026deg;C. The alternation temperature regime of 5/15\u0026deg;C simulated the ambient daily minimum and maximum temperature, respectively, from December to February, while 6\u0026deg;C 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\u0026deg;C, 10/20\u0026deg;C, 15/25\u0026deg;C, and 20/30\u0026deg;C. (2) \u003cem\u003eBreaking Epicotyl dormancy\u003c/em\u003e: Since epicotyl emergence was not observed in response to cold stratification, we incubated seeds with an emerged radicle at 6\u0026deg;C, 5/15\u0026deg;C, and 15/25\u0026deg;C (12h dark/12h light, simulating average spring temperatures) for an additional 60 days. This extended period of incubation was used to determine the effects of cold stratification at 5/15\u0026deg;C and warm stratification at 15/25\u0026deg;C on the release of epicotyl dormancy after the radicle had emerged.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDifferences in dormancy across different maturation periods\u003c/b\u003e. To investigate dormancy characteristics of seeds dispersed at different times, we used seed lots that matured and fell in November 2022, December 2022, January 2023, and February 2023. To evaluate radicle and epicotyl dormancy, we employed three stratification treatments: winter, field cold stratification (starting on November 30), cold stratification at 6\u0026deg;C, and warm stratification at 25\u0026deg;C. Following stratification for 1 month, seeds were incubated at 5/15\u0026deg;C and 15/25\u0026deg;C (12h dark/12h light) for 30 days, and radicle and epicotyl emergence were checked daily.\u003c/p\u003e\u003cp\u003e\u003cb\u003eField Soil Seed Bank Germination Experiment.\u003c/b\u003e 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\u0026times;4 replicates\u0026times;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 \u003cem\u003eC. kawakamii\u003c/em\u003e, free from human interference, to create an artificial soil seed bank. The bags were placed flat on the ground with the seeds evenly spread and pressed down to ensure contact with the soil. The artificial soil seed bank was enclosed with wire mesh to prevent predation. This setup was established in late October 2022, and the temperature of the test site was continuously monitored using a temperature recorder. Emergence of radicles and shoots were monitored monthly November 2022 to March 2023.\u003c/p\u003e\n\u003ch3\u003eStatistical analyses\u003c/h3\u003e\n\u003cp\u003eAll data analyses were done with R software [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The response variables were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE or mean\u0026thinsp;+\u0026thinsp;SE (non-transformed data appear in all figures). A general linear model (GLM) with family\u0026thinsp;=\u0026thinsp;binomial (\u0026lsquo;logit\u0026rsquo;) model [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] was used to test whether different experimental treatments, including incubation temperature, storage condition, and length of stratification time as variables, radicle/epicotyl emergence as response variables, significances on emergence followed by multiple contrasts with Tukey\u0026rsquo;s HSD tests using the \u0026lsquo;glht\u0026rsquo; function of the \u0026lsquo;multcomp\u0026rsquo; package, level of significance was set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e"},{"header":"Result","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eMorphology of Seed Germination\u003c/h2\u003e\u003cp\u003eSeeds of \u003cem\u003eC. kawakamii\u003c/em\u003e are irregularly spherical, with an average mass of 2.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 g (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE, n\u0026thinsp;=\u0026thinsp;100) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The pericarp is leathery and densely covered with retrorse hairs on the inner surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). The two cotyledons account for 99.16% of total embryo mass, while the embryo axis comprises only 0.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11% and was clearly differentiated except for the radicle (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eUnder suitable conditions, the radicle emerges, after which the hypocotyl elongates (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The radicle then elongates, and lateral roots develop (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, e). After epicotyl dormancy is broken, the cotyledon petioles elongate, pushing the upper hypocotyl out of the seed. The epicotyl(shoot) emerges at the junction of the two cotyledonary petioles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, S2).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSeed Recalcitrance\u003c/h3\u003e\n\u003cp\u003eSeeds of \u003cem\u003eC. kawakamii\u003c/em\u003e are highly sensitive to dehydration and low temperatures. There was a significant negative correlation between seed viability and water loss (R\u0026sup2;= 0.77, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). 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 \u0026le;\u0026thinsp;4\u0026deg;C (freezing point) for 15 or more days resulted in significant seed mortality. Storing seeds at 6\u0026deg;C for 30 days increased mortality by 10% compared to storing them for 15 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Excessively low winter temperatures (e.g., 0/10\u0026deg;C) caused high (84.7%) seed mortality, while prolonged exposure to moderately low temperatures (e.g., 5/15\u0026deg;C for 30 days, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) lead to 17.6% mortality. However, at 10/20\u0026deg;C \u003cem\u003eC. kawakamii\u003c/em\u003e seeds can be stored for an extended period with nearly 100% of them remaining viabile.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eInitial Germination Characteristics and Seed Coat Permeability\u003c/h3\u003e\n\u003cp\u003eMost early-dispersed seeds were dormant, and radicle emergence was \u0026le;\u0026thinsp;5% for seeds at all temperatures (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). For mid-dispersed (15 December) seeds, emergence at 20/30\u0026deg;C was slightly above 5%, but there were no significant differences in germination across the different temperatures (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, radicle emergence percentages of seeds dispersed late in the season (15 January) differed significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) at 10/20\u0026deg;C, 15/25\u0026deg;C, and 20/30\u0026deg;C, with percentages increasing as temperatures increased.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAt 5/15\u0026deg;C, radicle emergence was nil for seeds from all dispersal periods. At 10/20\u0026deg;C, only a small number of late-dispersed seeds (\u0026lt;\u0026thinsp;5%) exhibited radicle emergence, with no significant difference compared to seeds from other dispersal periods (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). At 15/25\u0026deg;C, a few mid-dispersed seeds also began to exhibit radicle emergence (\u0026lt;\u0026thinsp;5%), but the percentage was significantly lower than that of the late-dispersal seeds (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). At 20/30\u0026deg;C, radicle emergence of early-dispersed seeds was the lowest (\u0026lt;\u0026thinsp;5%) and did not differ significantly from mid-dispersal seeds, while late-dispersed seeds exhibited the highest emergence (\u0026gt;\u0026thinsp;30%) and differed significantly from early- and mid-dispersed seeds (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Thus, radicle emergence follows the pattern: late-dispersed seeds\u0026thinsp;\u0026gt;\u0026thinsp;mid-dispersed seeds\u0026thinsp;\u0026gt;\u0026thinsp;early-dispersed seeds, suggesting that radicle dormancy becomes shallower as the dispersal period progresses.\u003c/p\u003e\u003cp\u003eMC of newly-matured seeds was 46.7%, and after 100 hours 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Initial MC of seeds with the seed coat removed was 49.6%, and after 100 hours 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.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eSeed Dormancy Release\u003c/h2\u003e\u003cp\u003eCold stratification (5/15\u0026deg;C) for 2 to 8 weeks effectively broke radicle dormancy of \u003cem\u003eC. kawakamii\u003c/em\u003e seeds (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). As duration of the cold stratification period (5/15\u0026deg;C) increased, radicle emergence steadily increased, with higher incubation temperatures leading to higher percentages (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). When the stratification period was less than 2 weeks, radicle emergence was minimal at 5/15\u0026deg;C. However, after 2 weeks of cold stratification, radicle emergence at 15/25\u0026deg;C was significantly higher than that of non-stratified seeds, with no significant difference from that at 20/30\u0026deg;C (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;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\u0026deg;C. At 20/30\u0026deg;C, radicle emergence for seeds receiving 8 weeks of cold stratification was about 100%.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSimilarly, the longer the field storage period, the higher the percentage of seeds with radicle emergence (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). After 2 weeks of field storage, the percentage of seeds with radicle emergence at 10/20\u0026deg;C was slightly higher than that of seeds incubated at 20/30\u0026deg;C, although the difference was non-significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). After 4 weeks of field storage root emergence at 20/30\u0026deg;C increased to approximately 50% and was significantly higher than that of seeds incubated at 10/20\u0026deg;C. As field storage time increased to 8 and 12 weeks, radicle emergence of seeds incubated at 20/30\u0026deg;C was nearly 100%, while it was only \u003cem\u003eca\u003c/em\u003e. 60% for control seeds at 10/20\u0026deg;C. High-temperature stratification (25℃) was ineffective in breaking radicle dormancy (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, f).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe percentage of seeds with radicle emergence following cold stratification at 6℃ (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, e) was higher than that of seeds stored in the field or at 25℃ (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, d and Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, f). After 2 months of cold stratification, radicle emergence at 15/25℃ reached 79.0%, while epicotyl emergence was \u0026lt;\u0026thinsp;2% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee), indicating that cold stratification broke radicle but not epicotyl dormancy of \u003cem\u003eC. kawakamii\u003c/em\u003e seeds. After warm stratification at 25℃, neither the radicle nor the epicotyl emerged (\u0026lt;\u0026thinsp;2%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee).\u003c/p\u003e\u003cp\u003eWhen seeds with an emerged radicle were incubated at 6\u0026deg;C for 4 months, a few epicotyls emerged (1.1%), beginning after 40 days of incubation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Additionally, epicotyl emergence was higher at 15/25℃ (38.8%) than that 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℃.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eSeed germination under field condition\u003c/h2\u003e\u003cp\u003eBetween January and February 2023, as temperatures gradually decreased in the field, the percentage of seeds with an emerged radicle began to increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). 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.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). 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.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe confirmed that freshly matured seeds of \u003cem\u003eC. kawakamii\u003c/em\u003e 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 \u003cem\u003eQuercus robur\u003c/em\u003e [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], epicotyl PD of \u003cem\u003eC. kawakamii\u003c/em\u003e remains insensitive to environmental cues until the root system is established. Our results support the hypothesis that environmental conditions required for radicle and shoot emergence impose significant constraints on seedling recruitment in the endangered species \u003cem\u003eC. kawakamii\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eIn addition to the long dormancy-breaking period, seeds of \u003cem\u003eC. lawakamii\u003c/em\u003e are recalcitrant. Mortality of \u003cem\u003eC. kawakamii\u003c/em\u003e 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 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Oak species have evolved mechanisms to delay dehydration, balancing rapid germination with habitat dependency [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Seed dispersal of \u003cem\u003eC. kawakamii\u003c/em\u003e occurs from November to the following February, during which time the habitat is relatively dry from October to January. It seems that delayed seed dispersal to after January would help ensure a continued water supply from the mother plant for the seeds. 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 \u003cem\u003eC. kawakami\u003c/em\u003e acorns still lose viability during overwintering.\u003c/p\u003e\u003cp\u003eDue to PD, radicle emergence in most \u003cem\u003eC. kawakamii\u003c/em\u003e 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 reduces seed susceptibility to rodent predation [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn red oak species, which typically inhabit temperate climates, radicle emergence is delayed until spring, as in \u003cem\u003eC. kawakamii\u003c/em\u003e, reducing the risk of seedling mortality during the dry/cold winter. In \u003cem\u003eC. fargesii\u003c/em\u003e that inhabits a dry winter climate, radicle emergence also is delayed until spring, but this increases the chance of rodent predation [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Recent studies have suggested that rodents prefer to eat seeds with an emerged radicle immediately, because the stored nutrients in the acorns would be translocated from the cotyledons to the expanding root after radicle emergence [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], suggesting that the radicle-emerged seeds also face a high risk of predation. Our study suggests that the delay of radicle emergence from \u003cem\u003eC. kawakamii\u003c/em\u003e 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 [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Thus, seed mortality also should be considered when assessing adaptability, particularly in oak species whose seed are generally sensitive to desiccation.\u003c/p\u003e\u003cp\u003eA 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 \u003cem\u003eC. kawakamii\u003c/em\u003e exhibits epicotyl dormancy. Anatomical observations revealed that from seed maturation to the development of secondary roots following radicle emergence, the shoot apical meristem remained undifferenced. 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.\u003c/p\u003e\u003cp\u003eAlthough \u003cem\u003eC. kawakamii\u003c/em\u003e 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 [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], the mild climate in the \u003cem\u003eC. kawakamiii\u003c/em\u003e 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 \u003cem\u003eC. kawakamii\u003c/em\u003e habitat. Increased temperatures and decreased rainfall in recent decades in subtropical southeastern China, especially in winter [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], pose severe future threats to the seedling establishment of \u003cem\u003eC. kawakamii\u003c/em\u003e.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eDesiccation and low temperature sensitivity of \u003cem\u003eC. kawakamii\u003c/em\u003e seeds and their requirement for cold (winter) and warm (spring) stratification for complete germination, i.e. emergence of both the radicle and epicotyl. 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 soil moisture (\u0026gt;\u0026thinsp;35% seed MC) by burying seeds to buffer against surface extremes in winter, and (3) applying post-radicle artificial warm stratification (15/25\u0026deg;C, ~\u0026thinsp;30 days) to break epicotyl dormancy, to achieve effective population recovery.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cp\u003e All experimental studies on the plants in this study were conducted in compliance with relevant national, institutional, and international guidelines.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cp\u003eThe authors declare that they do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eData statement\u003c/h2\u003e\u003cp\u003eAll data generated or analyzed during this study are included in this manuscript. Further inquiries can be directed to the corresponding author.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis 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).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eS.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.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors thank equipment provided by Administrative Committee of Castanopsis kawakamii National Forest Park and Sanming University (Sanming city, Fujian province, China) experimental platforms for the assistance for this study. Special thanks are extended to undergraduate students Huijing Jin, Shunan, Yang and Jiaxing Jing from the School of Economics and Management, Sanming University, for their contributions to sample collection and processing.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analyzed during this study are included in this manuscript. Further inquiries can be directed to the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHarper JL. Population biology of plants. London, UK: Academic; 1977.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang Z, Baskin JM, Baskin CC, Liu G, Ye X, Yang X, Huang Z. Soil salinity regulates spatial-temporal heterogeneity of seed germination and seedbank persistence of an annual diaspore-trimorphic halophyte in northern China. BMC Plant Bio. 2024;24:604. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12870-024-05307-x\u003c/span\u003e\u003cspan address=\"10.1186/s12870-024-05307-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDonohue K, Casas RRD, Burghardt L. Germination, post germination adaptation, and species ecological ranges. Ann Rev Ecol Syst. 2010;41:293\u0026ndash;319. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1146/annurev-ecolsys-102209-144715\u003c/span\u003e\u003cspan address=\"10.1146/annurev-ecolsys-102209-144715\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXia K, Daws MI, Peng LL. Climate drives patterns of seed traits in \u003cem\u003eQuercus\u003c/em\u003e species across China. New Phytol. 2022;234:1629\u0026ndash;38. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/nph.18103\u003c/span\u003e\u003cspan address=\"10.1111/nph.18103\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang Z, Baskin JM, Baskin CC, Yang X, Liu G, Ye X, Huang Z, Cornelissen JHC. Great granny still ruling from the grave: Phenotypical response of plant performance and seed functional traits to salt stress affects multiple generations of a halophyte. J Ecol. 2022;110:117\u0026ndash;28. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/1365-2745.13789\u003c/span\u003e\u003cspan address=\"10.1111/1365-2745.13789\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHuang C, Chang Y, Bruce B. Fagaceae. In: Chun Woonyong \u0026amp; Huang Chengchiu, eds. Fl. Reipubl. Popularis Sin. 1998;22:1-332. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.iplant.cn/info/Fagaceae?t=foc\u003c/span\u003e\u003cspan address=\"https://www.iplant.cn/info/Fagaceae?t=foc\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu JF, He ZS, Hong W, Zheng S, Wang Z. Conservation ecology of endangered plant \u003cem\u003eCastanopsis kawakamii\u003c/em\u003e. J Beijing Forestry Univ. 2011;33:136\u0026ndash;43. (in Chinese with English abstract). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://j.bjfu.edu.cn/en/article/id/9662\u003c/span\u003e\u003cspan address=\"http://j.bjfu.edu.cn/en/article/id/9662\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHong WJ, Zeng SJ, Ma DW, Wang P, Lai Z, Zhong W, Liao Y, Yang L, Zhuang X. Community characteristics with the population of \u003cem\u003eCastanopsis kawakamii\u003c/em\u003e in Lianhuashan-Baipenzhu Nature Reserve, Guangdong. Forestry Environ Sci. 2016;32:10\u0026ndash;6. (in Chinese with English abstract).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu JF. Studies on structure and dynamics characteristics of \u003cem\u003eCastanopsis kawakamii\u003c/em\u003e Hayata population[M]. Beijing Forestry Univ. 2004. (in Chinese with English abstract).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHe ZS, Liu FJ, Zheng SQ, Su S, Hong W, Wu Z, Xu D, Wu C. Studies on the seeds dispersal and seedlings regeneration in gaps and understory of \u003cem\u003eCastanopsis kawakamii\u003c/em\u003e natural forest. J Trop Subtrop Bot. 2012;20:506\u0026ndash;12. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://api.semanticscholar.org/CorpusID:87572748\u003c/span\u003e\u003cspan address=\"https://api.semanticscholar.org/CorpusID:87572748\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRoberts EH. Predicting the storage life of seeds. Seed Sci Technol. 1973;1:499\u0026ndash;514.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePritchard HW. Classification of seed storage \u0026lsquo;types\u0026rsquo; for ex-situ conservation in relation to temperature and moisture. In: Havens EO, Maunder K, editors. Ex-Situ Plant Conservation. Supporting Species Survival in the Wild;Guerrant. Washington, DC, USA: M., Eds.;Island; 2004. pp. 139\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMasaki T, Wijenayake PR. How long do tree seeds survive in soil seedbanks? A multi-species comparison in an old-growth deciduous temperate forest. J Res. 2024;29:374\u0026ndash;82. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/13416979.2024.2327979\u003c/span\u003e\u003cspan address=\"10.1080/13416979.2024.2327979\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBerjak P, Pammenter NW. Implications of the lack of desiccation tolerance in recalcitrant seeds. Front Plant Sci. 2014;5:123. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fpls.2013.00478\u003c/span\u003e\u003cspan address=\"10.3389/fpls.2013.00478\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHong TD, Ellis RH. Contrasting seed storage behaviour among different species of Meliaceae. Seed Sci Technol. 1998;26:77\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu X, Xiao Y, Wang Y, Wang R, Huang R, Liang H, Jiang Y, Jiang Y. Seed germination ecology of endangered plant \u003cem\u003eHorsfieldia hainanensis\u003c/em\u003e Merr. In China. BMC Plant Biol. 2024;24: 486. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12870-024-05208-z\u003c/span\u003e\u003cspan address=\"10.1186/s12870-024-05208-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePritchard HW, Steadman KJ, Nash JV, Jones C. Kinetics of dormancy release and the high temperature gemination response in \u003cem\u003eAesculus hippocastanum\u003c/em\u003e seeds. J Exp Bot. 1999;50:1507\u0026ndash;14. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/jxb/50.338.1507\u003c/span\u003e\u003cspan address=\"10.1093/jxb/50.338.1507\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRodr\u0026iacute;guez-Calcerrada J, Sancho-Knapik D, Martin-StPaul NK, Limousin JM, McDowell NG, Gil-Pelegr\u0026iacute;n E. Drought-induced oak decline\u0026mdash;factors involved, physiological dysfunctions, and potential attenuation by forestry practices. In: Gil-Pelegr\u0026iacute;n E, Peguero-Pina JJ, Sancho-Knapik D, editors. Oaks physiological ecology exploring the functional diversity of genus Quercus L. Cham: Springer International Publishing; 2017. pp. 419\u0026ndash;51. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/978-3-319-69099-5_13\u003c/span\u003e\u003cspan address=\"10.1007/978-3-319-69099-5_13\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWalters C, Berjak P, Pammenter N, Kennedy K, Raven P. Preservation of recalcitrant seeds. Science. 2013;339:915\u0026ndash;6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/science.123093\u003c/span\u003e\u003cspan address=\"10.1126/science.123093\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi J, Jaganathan GK, Kang H, Liu B. The co-occurrence of physiological and epicotyl physiological dormancy in three desiccation-sensitive \u003cem\u003eCastanopsis\u003c/em\u003e (Fagaceae) acorns from China with specific reference to the embryonic axis position. Forests. 2023;14:2330. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/f14122330\u003c/span\u003e\u003cspan address=\"10.3390/f14122330\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJaganathan GK, Phartyal SS. A classification system for germination in desiccation-sensitive Fagaceae acorns: with particular focus on physiological and epicotyl dormancy. Bot J Linn Soc. 2025;207:197\u0026ndash;207. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/botlinnean/boae049\u003c/span\u003e\u003cspan address=\"10.1093/botlinnean/boae049\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJayasuriya KMGG, Wijetunga ASTB, Baskin JM, Baskin CC. Physiological epicotyl dormancy and recalcitrant storage behaviour in seeds of two tropical Fabaceae (subfamily Caesalpinioideae) species. AoB Plants. 2012;2012:pls044. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/aobpla/pls044\u003c/span\u003e\u003cspan address=\"10.1093/aobpla/pls044\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFlores EM. Seed biology, pp. 13\u0026ndash;124 in Vozzo JA, editor Tropical tree seed manual. USDA Forest Service Agriculture Handbook 721, 2002. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/aob/mch046\u003c/span\u003e\u003cspan address=\"10.1093/aob/mch046\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang G, Yang L, Wang Y, Shen S. Physiological epicotyl dormancy and its alleviation in seeds of \u003cem\u003eYunnanopilia longistaminea\u003c/em\u003e: the first report of physiological epicotyl dormancy in China. PeerJ. 2017;5:e3435. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.7717/peerj.3435\u003c/span\u003e\u003cspan address=\"10.7717/peerj.3435\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRong Z, Yi Y, Shen X, Deng S, Xu L, Li J, Mou J, Deng Z. Breaking deep epicotyl physiological dormancy in \u003cem\u003eChionanthus hionanthus\u003c/em\u003e retusus (Oleaceae) seeds. Seed Sci Technol. 2024;52:67\u0026ndash;77. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.15258/sst.2024.52.1.07\u003c/span\u003e\u003cspan address=\"10.15258/sst.2024.52.1.07\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMcCartan SA, Jinks RL, Barsoum N. Using thermal time models to predict the impact of assisted migration on the synchronization of germination and shoot emergence of oak (\u003cem\u003eQuercus robur\u003c/em\u003e L). Ann For Sci. 2015;72:479\u0026ndash;87. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s13595-014-0454-5\u003c/span\u003e\u003cspan address=\"10.1007/s13595-014-0454-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBaskin JM, Baskin CC. The great diversity in kinds of seed dormancy: a revision of the Nikolaeva-Baskin classification system for primary seed dormancy. Seed Sci Res. 2021;31:249\u0026ndash;77. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1017/S096025852100026X\u003c/span\u003e\u003cspan address=\"10.1017/S096025852100026X\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHopper GM, Smith DW, Parrish DJ. Germination and seedling growth of northern red oak: effects of stratification and pericarp removal. For Sci. 1985;31:31\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/forestscience/31.1.31\u003c/span\u003e\u003cspan address=\"10.1093/forestscience/31.1.31\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWigston DL. Epicotyl dormancy in \u003cem\u003eQuercus robur\u003c/em\u003e L. Quart J For. 1987;81:110\u0026ndash;2.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSun X, Song Y, Ge B, Dai X, Kozlowski G. Intermediate epicotyl physiological dormancy in the recalcitrant seed of \u003cem\u003eQuercus chungii\u003c/em\u003e F.P.Metcalf with the elongated cotyledonary petiole. Forests. 2021;12:263. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/f12030263\u003c/span\u003e\u003cspan address=\"10.3390/f12030263\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFarmer R. Epicotyl dormancy in white and chestnut Oaks. For Sci. 1977;23:329\u0026ndash;32. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/forestscience/23.3.329\u003c/span\u003e\u003cspan address=\"10.1093/forestscience/23.3.329\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJastrzębowski S, Ukalska J, Walck JL. Does the lag time between radicle and epicotyl emergences in acorns of pedunculate oak (\u003cem\u003eQuercus robur\u003c/em\u003e L.) depend on the duration of cold stratification and poststratification temperatures? Modelling with the sigmoidal growth curves approach. Seed Sci Res. 2021;31:105\u0026ndash;115. 1 https://doi.org/0.1017/S096025852100009X.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMacchia F, Cavallaro V, Vita F, Sburlino G. Acorn dormancy and aridity as factors of \u003cem\u003eQuercus Cerris\u003c/em\u003e L. distribution. In: Teller A, Mathy P, Jeffers JNR, editors. Responses of forest ecosystems to environmental changes. Netherlands, Dordrecht: Springer; 1992. pp. 633\u0026ndash;4. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/978-94-011-2866-7_91\u003c/span\u003e\u003cspan address=\"10.1007/978-94-011-2866-7_91\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHawkins TS. The influence of dormancy break requirements on germination and viability responses to winter submergence in acorns of three bottomland red oak (Sect. Lobatae) species. For Sci. 2019;65:556\u0026ndash;61. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/forsci/fxz028\u003c/span\u003e\u003cspan address=\"10.1093/forsci/fxz028\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang K, Ji Y, Yao L, Liu H, Zhang Y, Baskin JM, Baskin CC, Zhang L, Xu C, Tao J, Prinzing A. Paternal intergenerational plasticity in the plant species \u003cem\u003ePaeonia ostii\u003c/em\u003e: Implications for parental fitness and offspring performance. Funct Ecol. 2024;38:832\u0026ndash;47. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/1365-2435.14506\u003c/span\u003e\u003cspan address=\"10.1111/1365-2435.14506\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang K, Pan H, Baskin CC, Baskin JM, Xiong Z, Cao W, Yao L, Tang B, Zhang C, Tao J. Epicotyl morphophysiological dormancy in seeds of \u003cem\u003ePaeonia ostii\u003c/em\u003e (Paeoniaceae): seasonal temperature regulation of germination phenology. Environ Exp Bot. 2022;194:104742. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.envexpbot.2021.104742\u003c/span\u003e\u003cspan address=\"10.1016/j.envexpbot.2021.104742\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKang H, Jaganathan GK, Han Y, Li J, Liu B. Revisiting the pericarp as a barrier restricting water entry/loss from cotyledons and embryonic axis of temperate desiccation-sensitive Quercus acorns. Planta. 2023;257:33. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00425-022-04061-4\u003c/span\u003e\u003cspan address=\"10.1007/s00425-022-04061-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eR Core Team. (2023). R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing. Retrieved from \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.R-project.org/\u003c/span\u003e\u003cspan address=\"https://www.R-project.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWarton DI, Hui FKC. The arcsine is asinine: the analysis of proportions in ecology. Ecology. 2011;92:3\u0026ndash;10. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1890/10-0340.1\u003c/span\u003e\u003cspan address=\"10.1890/10-0340.1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHothorn T, Bretz F, Westfall P. Simultaneous inference in general parametric models. Biometrical J. 2008;50:346\u0026ndash;63. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/bimj.200810425\u003c/span\u003e\u003cspan address=\"10.1002/bimj.200810425\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXiao Z, Zhang Z, Krebs CJ. Long-term seed survival and dispersal dynamics in a rodent-dispersed tree: testing the predator satiation hypothesis and the predator dispersal hypothesis. J Ecol. 2013;101:1256\u0026ndash;64. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/1365-2745.12113\u003c/span\u003e\u003cspan address=\"10.1111/1365-2745.12113\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFox JF. Adaptation of gray squirrel behavior to autumn germination by white oak acorns. Evolution. 1982;36:800\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1558-5646.1982.tb05446.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1558-5646.1982.tb05446.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCao L, Jansen PA, Wang B, Yan C, Wang Z, Chen J. Mutual cheating strengthens a tropical seed dispersal mutualism. Ecology. 2022;103:e03574. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/ecy.3574\u003c/span\u003e\u003cspan address=\"10.1002/ecy.3574\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBaskin CC, Baskin JM. Seeds: ecology, biogeography, and evolution of dormancy and germination. 2nd ed. San Diego: Academic; 2014.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCornelissen JHC, Zhong Z, Werger M. Timing of germination in the subtropical chinese tree \u003cem\u003eCastanopsis farqesii\u003c/em\u003e. Journal of Southwest China Normal University (Natural Science); 1994.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi Y, Yang X, Feng E, Zhao K, Zhang Z. Plant hormones mediate the interaction between oak acorn germination and rodent hoarding behaviour. New Phytol. 2024;242:2237\u0026ndash;50. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/nph.19424\u003c/span\u003e\u003cspan address=\"10.1111/nph.19424\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJo\u0026euml;t T, Ourcival JM, Capelli M, Dussert S, Morin X. Explanatory ecological factors for the persistence of desiccation-sensitive seeds in transient soil seed banks: \u003cem\u003eQuercus ilex\u003c/em\u003e as a case study. Ann Bot. 2015;117:165\u0026ndash;76. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/aob/mcv139\u003c/span\u003e\u003cspan address=\"10.1093/aob/mcv139\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIPCC. Sixth Assessment Report: Climate Change 2021:The Physical Science Basis. 2021. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ipcc.ch/report/ar6/wg1/\u003c/span\u003e\u003cspan address=\"https://www.ipcc.ch/report/ar6/wg1/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Castanopsis kawakamii, cold/warm stratification, endangered plant, epicotyl dormancy, germination phenology, seed recalcitrance, soil seedbank","lastPublishedDoi":"10.21203/rs.3.rs-7259877/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7259877/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eCastanopsis kawakamii\u003c/em\u003e 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 \u003cem\u003eC. kawakamii\u003c/em\u003e were collected from the largest population, \u003cem\u003eCastanopsis kawakamii\u003c/em\u003e National Nature Reserve, to assess viability under drying and temperature treatments and to determine environmental cues for radicle and epicotyl emergence. Seeds of \u003cem\u003eC. kawakamii\u003c/em\u003e rapidly lost viability under low temperature and at a seed moisture content (MC)\u0026thinsp;\u0026lt;\u0026thinsp;35%. Cold stratification (5/15\u0026deg;C) or field winter temperatures broke radicle dormancy, but epicotyl physiological dormancy (PD) persisted and required\u0026thinsp;~\u0026thinsp;30 days of warm stratification (15/25\u0026deg;C) 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. Almost all 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.\u003c/p\u003e","manuscriptTitle":"Recalcitrant seeds with physiological epicotyl dormancy may limit seedling recruitment of an endangered subtropical oak species","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-15 13:10:28","doi":"10.21203/rs.3.rs-7259877/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-25T06:16:41+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-24T11:41:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-23T22:51:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-23T02:21:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"30169831519182619634497399821722113512","date":"2025-09-15T18:29:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"334982286103231572095070288876234089549","date":"2025-09-14T15:38:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-11T09:42:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"182967418895991441139649019600334116491","date":"2025-09-08T23:57:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"262979872809078002437275169900956695374","date":"2025-09-08T12:18:57+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-08T09:40:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-08T05:28:22+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-08-18T07:33:20+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-15T02:22:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-08-15T02:19:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"aae0bd1a-b540-4f04-a149-7f4bcc0e462f","owner":[],"postedDate":"September 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-08T16:02:46+00:00","versionOfRecord":{"articleIdentity":"rs-7259877","link":"https://doi.org/10.1186/s12870-025-07847-2","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2025-12-05 15:58:05","publishedOnDateReadable":"December 5th, 2025"},"versionCreatedAt":"2025-09-15 13:10:28","video":"","vorDoi":"10.1186/s12870-025-07847-2","vorDoiUrl":"https://doi.org/10.1186/s12870-025-07847-2","workflowStages":[]},"version":"v1","identity":"rs-7259877","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7259877","identity":"rs-7259877","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}