Southeastern Naturalist C.A. Adams, O.C. Adejumo, M. Jahan, and K.W. Montgomery 2017 Vol. 16, No. 4 488 2017 SOUTHEASTERN NATURALIST 16(4):488–502 Seed Dormancy and Germination Ecology of Calycanthus floridus L., a Species with Threatened Status in Kentucky Christopher A. Adams1,*, Olamide C. Adejumo1, Moondil Jahan1, and Kevin W. Montgomery1 Abstract - The seed germination ecology of Calycanthus floridus (Eastern Sweetshrub) has not been formally investigated. The purposes of this study were to determine the type of seed dormancy found in the species, the most effective method of breaking this dormancy, and the environmental conditions producing maximum germination. We employed a variety of standard treatments to determine the specific type(s) of dormancy present, as well as treatments to determine effective dormancy-breaking mechanisms. We determined that seeds possessed physical dormancy (PY) imposed by a water-impermeable seed coat. Mechanical scarification was the most effective method of breaking PY; submersion in acid was also moderately effective. Seed germination following scarification can occur over a wide range of temperatures. There is no light requirement for seed germination. This study represents the first case of physical dormancy reported for the genus and only the second for the family. Introduction The Calycanthaceae is a plant family of 2 (Johnson 1997, Weakley 2015) or 3 genera (Bingtao and Bartholemew 2008, Kubitzki, 1993) and ~9 species found in temperate areas worldwide (Bingtao and Bartholemew 2008, Johnson 1997, Kubitzki 1993, Weakley 2015). In North America, the family is represented by the single genus Calycanthus (Johnson 1997). Gleason and Cronquist (1991) recognized 1 species in North America, Calycanthus floridus L. (Eastern Sweetshrub). Weakley (2015) recognized 3 species, Eastern Sweetshrub and C. brockianus Ferry & Ferry (Georgia Sweetshrub), in the eastern US and C. occidentalis Hook. & Arn. (Western Sweetshrub) in the western US. Nicely (1965) stated that as many as 6 species have been recognized in the east and southeast, but he concluded that these should all be considered as varieties or ecological variants of Eastern Sweetshrub. Johnson (1997) recognized only 2 species (Eastern Sweetshrub and Western Sweetshrub) within the US, stating that Georgia Sweetshrub could represent a triploid variation of particular Eastern Sweetshrub populations. In Kentucky, where we conducted this study, the genus is represented only by Eastern Sweetshrub (Kartesz 2015), a species of mesic forest slopes, hillsides, and stream banks (Jones 2005, KSNPC 2015, Weakley 2015). Two varieties of the species are recognized: Calycanthus floridus var. glaucus and C. floridus var. floridus (Weakley 2015). Jones (2005), however, reported that only C. floridus var. glaucus naturally occurs in the state. The species is ranked as threatened and 1Department of Biology, Berea College, Berea, KY 40404. *Corresponding author - Christopher_Adams@berea.edu. Manuscript Editor: Robert Carter Southeastern Naturalist 489 C.A. Adams, O.C. Adejumo, M. Jahan, and K.W. Montgomery 2017 Vol. 16, No. 4 occurs only in several southern counties (Campbell and Medley 2012, KSNPC 2015). C. floridus var. floridus may grow sporadically within the state, but these records represent established, scattered populations of escaped cultivated plants (Campbell and Medley 2012). Very little information is available in the published literature on seed dormancy and germination within the family. The Calycanthaceae is a very small family, with only 3 genera (Calycnathus, Chimonanthus, and Idiospermum) and fewer than 10 species (Bingtao and Bartholomew 2008, Kubitzki 1993); thus, our study provides valuable information on the germination ecology of the family, primarily to those interested in the evolution of seed dormancy and its different forms. Idiospermum australiense (Diels) S.T. Blake (Ribbonwood) is a rare, tropical, woody species with recalcitrant seeds (Franks and Drake 2003). Seeds of the species have physiological dormancy at maturity (Baskin and Baskin 2014, Edwards et al. 2001). Tang and Tian (2010) reported that seeds of Chimonanthus praecox (L.) Link (Wintersweet) possess combinational dormancy, i.e., there are both endogenous and exogenous components to dormancy: the embryos are physiologically dormant—a period of cold stratification is required to break embryo dormancy (Baskin and Baskin 2014, Tang and Tian 2010)—and the seed coat is impermeable to water (Baskin and Baskin 2014). One of the 2 recognized Calycanthus species (Johnson 1997) has been investigated. The seeds of Western Sweetshrub reportedly possess non-deep physiological dormancy (Baskin and Baskin 2014) and require 90 d of cold stratification to break dormancy (Emery 1988). Thus, all species of the Calycanthaceae examined thus far have some form of dormancy preventing mature seeds from germinating. Seeds of Eastern Sweetshrub have a large embryo consisting of little to no endosperm with the cotyledons folded around the hypocotyl (Eames 1961, Nicely 1965). In the classification system of Baskin and Baskin (2014), Eastern Sweetshrub possesses an embryo described as “folded”. In his monograph on the family, Nicely (1965) reported germinating seeds within 2–3 weeks following scarification. This result hints at the likelihood that the seeds possess at least physical dormancy; the seed coat is impermeable to water, preventing the seed from making metabolic advancement towards germination (Bewley and Black 1994). Seeds that display physical dormancy alone simply require the seed coat to become permeable to water for the embryo to begin growing, which results in germination (Baskin and Baskin 2014). The observations of Nicely stand in contrast to Western Sweetbush, which is reported to exhibiit physiological, but not physical, dormancy (Emery 1988). Thus, it is possible that seeds of Eastern Sweetbush could be physically dormant, physiologically dormant, or possess a combination of both (i.e., combinational dormancy). Physical dormancy (PY) in seeds is typically broken through some form of scarification, a process that produces a water-permeable seed coat, allowing the embryo to imbibe (Baskin and Baskin 2014). Two of the most common forms of scarification are abrasion of the seed coat (i.e., mechanical) and exposure to acid (i.e., chemical) (Baskin and Baskin 2014). Mechanical and acid scarification both Southeastern Naturalist C.A. Adams, O.C. Adejumo, M. Jahan, and K.W. Montgomery 2017 Vol. 16, No. 4 490 correspond to natural phenomena that produce permeable seed coats, allowing for the breaking of dormancy and subsequent germination. Nicely (1965) stated that some bird species consume fruits and seeds of Calycanthus spp. Miller and Miller (2005) referred to seeds of Calycanthus spp. as being “animal-dispersed”, which could include consumption of seeds. It might also refer to animals chewing through fruit coats, allowing seeds to be gravity dispersed. Scarification through abrasion could occur following dispersal. C.A. Adams has observed fruits that have been gnawed on, resulting in holes through which seeds easily can fall down to the soil when the fruits are gently shaken. Kubitzki (1993) maintained that, due to toxicity, seeds are not consumed by animals, but, rather, are gravity-dispersed only. If that is correct, then abrasion following dispersal would be the more likely method of breaking PY. Physiological dormancy (PD) is present in seeds of many US species and is the most common type among species in temperate climates throughout the world (Baskin and Baskin 2014). Laboratory studies have repeatedly confirmed that the processes of cold and/or warm stratification are usually effective in breaking seed dormancy in most species, allowing for subsequent germination (Baskin and Baskin 2014, Bewley 1997, Bewley and Black 1994). The particular stratification process used to break physiological seed dormancy in seeds with fully developed embryos, such as Eastern Sweetbush, depends upon the phenology of seed dispersal: dormant seeds dispersed in late spring or early summer typically require warm stratification (simulating summer environmental conditions), while dormant seeds dispersed in autumn typically require cold stratification (simulating winter environmental conditions) (Baskin and Baskin 2014). As seeds overcome dormancy, they are able to germinate when ambient temperatures are favorable to support germination: autumn for spring/summer-dispersed seeds or spring for autumn-dispersed seeds (Baskin and Baskin 2014). Baskin and Baskin (2004) define combinational dormancy as a combination of both the presence of a water-impermeable seed coat and a physiologically dormant embryo. The respective dormancies are broken via scarification and a treatment that removes the physiological barrier to germination for the embryo, e.g., stratification or dry storage (Baskin and Baskin 2014). For most species, the seed coat must become permeable to water first, and only then can the physiological component be broken (Baskin and Baskin 2014). It has been noted, however, that, at least for a few species, physiological dormancy can be broken by dry storage prior to the breaking of physical dormancy (Baskin and Baskin 2004). Fruits of Eastern Sweetbush are mature in mid- to late autumn and most fruits dehisce from the parent plant by late autumn/early winter. In these seasons, temperatures are typically too low to promote germination for seeds of most species, even if they are nondormant at maturity. Thus, most seeds are subjected to a winter of cold stratification, which would most likely break physiological dormancy, if present. In the case of the study species, however, it has been observed that a small number of fruits (an average of 6 per plant) persist on the plant through the fall and winter. They dehisce in the next year, often in the spring, when new flowers are Southeastern Naturalist 491 C.A. Adams, O.C. Adejumo, M. Jahan, and K.W. Montgomery 2017 Vol. 16, No. 4 produced. Seeds dispersed at this time experience the warm stratification regimes of late spring and summer. Thus, the possibility of seeds requiring a warm-stratifi - cation period to break physiological dormancy, if present, cannot be ruled out. The purposes of this study were to determine: (1) the type of dormancy found in seeds of Eastern Sweetbush, (2) the most effective method(s) of breaking this dormancy, and (3) the germination conditions that produce maximum germination. We propose the following hypotheses: (1) seeds of Eastern Sweetbush possess physical dormancy, and (2) traditional methods of scarification should alleviate the seeds of this germination inhibitor and allow embryos to emerge from the seed. Furthermore, the experimental design also allowed the detection of combinational dormancy, if present in the seeds. Methods Seed collection We collected mature seeds from 8 stems of Eastern Sweetbush growing in a mesic deciduous forest in southern Kentucky. We did not determine whether each stem represented a unique plant or was part of a largely clonal colony (i.e., had the same genotype as other stems). Our collection included ~60 mature fruits from the 8 stems. We collected mature fruits (each containing an average of 22 seeds) in mid- November 2015, ~1 month after the first frost of the autumn season. At the time of collection, the fibrous hypanthium of each fruit had turned a dark brown, indicating fruit (and thus seed) maturity (Baskin and Baskin 2014). Seeds were removed from each hypanthium (average seed weight = 0.21 g), mixed together in a plastic pan, and allowed seeds to air-dry for 5 days. We commenced seed germination studies in early December 2015 and seed dormancy-breaking studies in early May 2016. We stored the seeds used in the dormancy-breaking studies dry in a paper bag on a laboratory shelf. Preliminary research conducted a year prior confirmed that dry storage of seeds over an 8-month period did not result in any change in dormancy state (C.A. Adams, unpubl. data). Seed imbibition We conducted the imbibition study 1 week after air-drying was complete. We prepared 2 treatments: non-scarified and scarified seeds. We randomly chose 30 seeds for each treatment and nicked each seed in the scarified treatment with a razor blade to ensure that the seed coat had become permeable to water; scarification occurred prior to the exposing of seeds to the moist sand. For both treatments, we placed seeds on moist quartz-sand in plastic Petri dishes and added 3 mL of distilled water to each dish. Seeds remained on the moist substrate for 12 h, which was the duration of the study. Prior to scarification, we used an electronic balance to determine the initial weight of each seed in both treatments, then calculated an average seed weight for each treatment and placed the seeds on the moist sand in each of the 2 Petri dishes. Every hour, we removed all seeds from the Petri dishes, dried their surfaces with paper towels, and recorded their weight using an electronic balance. After Southeastern Naturalist C.A. Adams, O.C. Adejumo, M. Jahan, and K.W. Montgomery 2017 Vol. 16, No. 4 492 weighing, we returned the seeds to their respective treatments; the same procedure was followed each hour for a total of 12 consecutive hours. We calculated average seed weight every hour and we used the percentage of mass increase for each hour to construct an imbibition curve. Seed dormancy-breaking studies A wide variety of laboratory methods have been used to break PY in seeds of various species (Baskin and Baskin 2014). To determine which mechanism(s) might be most effective in breaking physical dormancy, we exposed seeds to various scarification methods. All treatments, except for the fire treatments, consisted of 3 replicates of 30 seeds each. Fire treatments consisted of 1 replicate of 90 seeds each. We placed all seeds subjected to scarification treatments on moist quartz-sand in plastic Petri dishes and wrapped them in clear plastic film to retard water loss during incubation and/or stratification. We incubated the seeds in a 14-h photoperiod of around 40 μmol m-2s-1, 400–700 nm, of cool white fluorescent light at a 24-h constant temperature of 25 °C. We chose this temperature as the general incubation temperature because it was observed that scarified seeds stored on a moist substrate at room temperature germinated at relatively high temperatures (C.A. Adams, unpubl. data) This temperature is also optimal for the germination of seeds of many species in a temperate climate (Baskin and Baskin 2014). Non-scarified seeds served as controls for each treatment. Mechanical scarification. We employed 2 treatments to mechanically scarify seeds. In 1 treatment, we nicked the coat of each seed in every replicate with a razor blade near its chalazal end. In the other treatment, we rubbed coarse-grade sandpaper on the upper and lower surfaces of the seeds simultaneously for 15– 20 seconds. Freeze/thaw cycles. We cycled seeds placed on moist quartz-sand in Petri dishes between 0 °C and 25 °C over the course of 60 d; seeds remained at each temperature, in light, for 5 days before being moved to the other regime (0°C → 25°C → 0°C → 25°C ...). At the conclusion of the 60-d treatment, we moved seeds to 25 °C to incubate for 30 d to determine if dormancy had been broken. Dry storage. Fresh seeds that had dried on a lab bench for 5 days were transferred to a brown paper bag, stored on an office shelf for 6 months, and placed into incubation. This was a new treatment, distinct from a similar procedure conducted a year prior in a preliminary study, as described in the seed collection methodology. Fire. We employed 2 separate treatments to test the possibility that fire would break physical dormancy. In the first treatment, we placed 90 seeds on dry soil in a metal tray, scattered ~5 cm of dry leaves over the surface of the soil to cover the seeds, ignited the leaves with a match, and allowed them to burn. We removed the seeds and placed them on moist quartz-sand in Petri dishes for evaluation of breaking dormancy. In the second treatment, we buried 90 seeds ~2 cm under the soil surface in a metal tray, scattered ~5 cm of dry leaves over the soil surface, and burned the leaves. We exhumed the seeds and placed them on moist sand in Petri dishes for evaluation. Southeastern Naturalist 493 C.A. Adams, O.C. Adejumo, M. Jahan, and K.W. Montgomery 2017 Vol. 16, No. 4 High heat. We conducted 6 separate treatments to test the efficacy of heat in breaking physical dormancy. In the first 3 treatments, we placed seeds on dry sand in metal trays in drying ovens at 60 °C, 70 °C, and 80 °C, respectively, for 30 mins. Seeds were then transferred to the previously described incubation regime. In the second 3 treatments, we placed seeds on moist sand in metal trays in drying ovens at 60 °C, 70 °C, and 80 °C, respectively, for 30 min. Seeds were then moved to the previously described incubation regime. Acid Scarification. We submerged seeds in concentrated (95–97%) sulfuric acid for treatment-time intervals of 15 sec, 30 sec, 1min, and 2 min. We placed the 3 replicates for each of the 4 treatments individually into a loose-leaf tea strainer, immersed it in the acid for the particular treatment time, and transferred the seeds to the previously described incubation regime. Percussion. Following the protocol of Hamly (1932), we placed seeds, in 3 replicates of 30 seeds each, into a glass bottle, shook it 3 times per second for 5 minutes and transferred seeds to the previously described incubation regime. Germination studies Germination temperature. All treatments consisted of 3 replicates of 30 seeds each. We placed seeds on moist quartz-sand in plastic Petri dishes wrapped in clear plastic film to retard water loss during incubation and/or stratification and incubated them in a 14-h photoperiod of around 40 μmol m-2s-1, 400–700 nm, of cool-white fluorescent light at 24-h constant temperatures of 35 °C, 30 °C, 25 °C, 20 °C, 15 °C, or 5 °C, depending upon the specific treatment, for 30 d. We chose the temperatures that correspond to monthly mean maximum temperatures in Kentucky (Wallis 1977). During incubation, we assessed seeds every 5 d for radicle emergence, at which time water was added to the dishes to keep the sand moist. Our criterion for germination was emergence of the radicle from the seed. We tested for viability all seeds that remained ungerminated at the end of each treatment period by examining them under a dissecting microscope to determine if the embryo was firm and white (indicating they were viable) or soft and brown (indicating they were nonviable). Very few (less than 10%) ungerminated seeds contained viable embryos. Testing for physiological dormancy. We exposed seeds to either a warm- or cold-stratification treatment to determine if Eastern Sweetbush has combinational dormancy. Cold-stratified seeds were incubated, in light, at 5 °C for 4, 8, and 12 weeks, to simulate the winter season in Kentucky, then moved to 25 °C in light for 30 d. Warm-stratified seeds were incubated, in light, at 35 °C for 4, 8, and 12 weeks, to simulate the summer season in Kentucky, then moved to 25 °C in light incubator for 30 d. We assessed seeds every 5 days for the presence of radicle emergence. In contrast to the cold-stratification treatment, we monitored seeds exposed to warm stratification for germination during the stratification treatment. Scarified seeds can germinate at 35 °C, so it was necessary to monitor germination at regular intervals before moving seeds to the cooler temperature following stratifi cation. To further confirm the presence of combinational dormancy, we also exposed seeds to gibberellic acid treatments. Gibberellic acid-3 (GA3) promotes seed germination and can allow seeds to overcome a physiological component to dormancy Southeastern Naturalist C.A. Adams, O.C. Adejumo, M. Jahan, and K.W. Montgomery 2017 Vol. 16, No. 4 494 (Bewley and Black 1994). We placed scarified seeds into Petri dishes on filter paper and added 4-mL aliquots of 1000 ppm, 750 ppm, or 500 ppm GA3 to 3 replicate dishes for each treatment. Dishes were wrapped in clear plastic film, placed on a laboratory bench for 30 h, then washed with distilled water, dried with towels, transferred to Petri dishes with moist sand, and incubated at 25 °C, in light, for 30 d. Testing for a light requirement. We employed 2 treatments to determine whether light was necessary for germination: (1) scarified seeds incubated in light, and (2) scarified seeds incubated in darkness at 25 °C. We wrapped the Petri dishes containing seeds for dark incubation in 2 layers of aluminum foil. We checked seeds every 5 d in the dark using a green “safe” light (Walck et al. 2000) to prevent exposure to any photosynthetically active radiation. We placed the “light” treatment replicates into the incubator with only a clear plastic wrap covering. Statistical analyses We converted germination data to percentages based on number of viable seeds and calculated means and standard errors for each percentage. A Shapiro–Wilk test confirmed that the data were normally distributed; thus, no transformation was required, and we compared means by 1-way analyses of variance (ANOVA) and Fisher’s least-significant–difference tests (LSD, P < 0.5). Results Seed imbibition Non-scarified seeds did not take up water; average mass increase was less than 1% after 12 h (Fig. 1). In contrast, for scarified seeds, average mass increase was 24% mid-way (i.e., 6 h) through the experiment, and 36% at the conclusion of the study, indicating that seeds were taking up water. Scarification clearly produced an opening for water to enter the seed that was not present prior to the abrasion of the seed coat. Seeds did not take up water without scarification, indicating that their seed coats were impermeable to water. Figure 1. Imbibition curve for 30 non-scarified (■) and 30 mechanically scarified (●) Eastern Sweetbush seeds. One hundred percent of the mechanically scarified seeds imbibed, but only 3% of nonscarified seeds imbibed. Southeastern Naturalist 495 C.A. Adams, O.C. Adejumo, M. Jahan, and K.W. Montgomery 2017 Vol. 16, No. 4 Seed dormancy-breaking studies We tested a variety of scarification methods to determine which method was most effective in producing water-permeable seed coats and obtained mixed results (Table 1). Mechanical scarification was the most effective treatment—90% of seeds scarified by razor blade and 81% by sandpaper germinated. The only other moderately effective treatment was immersion in acid, but of the 4 treatments, none produced higher than a 42% seed-germination rate; germination varied from 23% to 42%. Although the germination rates for acid-scarified seeds were not high, all treatments produced significantly more germination than the non-scarified control. The freeze/thaw treatment and the percussion treatment yielded 8% and 17% germination, respectively. These rates were very low but statistically significantly different from the control group. All other treatments apparently were ineffective in producing the seed-coat permeability that allows germination. No other treatment produced a significantly higher germination percentage compared to the control. The highest germination rate among this group of treatments was for the buried seeds over which we lit a fire on the soil surface (5%), which was not statistically significantly different from the control. No more than 5 seeds germinated in any of these treatments. Germination studies Mechanically scarified seeds germinated over a broad range of incubation temperatures (Fig. 2). Seeds incubated at 35 °C, 30 °C, 25 °C, and 20 °C following scarification germinated to 76%, 82%, 92%, and 86%, respectively. The 15 °C regime was only moderately effective, with 38% germination. Cold or warm stratification treatments did not increase overall germination percentages, except for 1 treatment (Table 2). Scarified seeds that received a warm-stratification treatment prior to incubation did not germinate to Table 1. Mean germination percentages (± SE) of Eastern Sweetbush seeds exposed to various physical dormancy-breaking mechanisms. Following the treatment, seeds were incubated, in light, at 25 °C for 30 days. Three replicates of 30 seeds each were used for each treatment. Reported means were recorded on the 30th day of incubation. Means followed by the same letter are not significantly different based on Fisher’s protected LSD at P < 0.05. Treatment Germination % Control (no treatment) 3.0 ± 0.2a Mechanical Scarification Razor blade 90.2 ± 3b Sandpaper 81.4 ± 4c Acid Submersion 15 seconds 35.5 ± 4e 30 seconds 42.3 ± 3e 1 minute 30.6 ± 4e 2 minute 22.8 ± 2d Freeze/thaw cycle 8.3 ± 2d Fire (seeds on soil surface) 0.0 Fire (seeds buried in soil) 5.3 ± 1a Treatment Germination % High Heat With water 60 °C 0.0 70 °C 2.2 ± 0.2a 80 °C 0.0 Without water 60 °C 0.0 70 °C 0.0 80 °C 3.3 ± 0.4a Dry storage 4.2 ± 0.8a Percussion 16.8 ± 4d Southeastern Naturalist C.A. Adams, O.C. Adejumo, M. Jahan, and K.W. Montgomery 2017 Vol. 16, No. 4 496 statistically higher percentages than seeds that received no stratification. None of the specific treatments produced higher than 88% germination; the control group (no stratification) produced ~87% germination. Cold stratification yielded Table 2. Mean germination percentages (± SE) of Eastern Sweetbush seeds incubated at 25 °C following mechanical scarification and various periods of cold (5 °C) or warm stratification (35 °C): 0, 4, 8, and 12 weeks. Reported means were recorded on the 30th day of incubation. Means followed by the same letter are not significantly different based on Fisher ’s protected LSD at P < 0.05. Stratification regime Percent germination 0 weeks Cold 88.4 ± 2a Warm 86.6 ± 4a 4 weeks Cold 89.4 ± 3a Warm 88.4 ± 2a 8 weeks Cold 94.4 ± 3b Warm 86.2 ± 4a 12 weeks Cold 87.6 ± 4a Warm 87.2 ± 3a Figure 2. Mean (± SE) percent germination of freshly-matured Eastern Sweetbush seeds mechanically scarified (using a razor blade) and incubated, in light, at various temperatures: 35 °C (○), 30 °C (■), 25 °C (□), 20 °C (●), 15 °C (Δ), and 5 °C (▲). Three replicates of 30 seeds per Petri dish were used for each treatment. Southeastern Naturalist 497 C.A. Adams, O.C. Adejumo, M. Jahan, and K.W. Montgomery 2017 Vol. 16, No. 4 similar results. Only 1 treatment (8 weeks at 5 °C) produced a significantly different result from the control group (94% vs. 88%), a difference of only 5 seeds out of 90 (85 seeds vs. 80 seeds). The other cold stratification treatments did not produce significantly different germination percentages compared to the control group. Exposure to various concentrations of gibberellic acid did not produce significantly higher germination percentages than the control (Table 3). Seeds exposed to 500 ppm and 750 ppm GA3 had germination rates of 87% and 90%, respectively, while seeds in the control showed germination of 89%. The 1000-ppm treatment actually showed decreased germination over the duration of the incubation regime (80%), which was significantly different from the control. In our trials to assess light requirement for germination, the overall germination percentage for scarified seeds incubated in darkness was not sig nificantly different from the those in the light-exposed control group (Fig. 3). The germination rate for seeds incubated in light was 92% and 88% for those incubated in darkness. Figure 3. Mean (± SE) percent g e r m i n a t i o n of f r e s h l y - matured Eastern Sweetbush seeds mechanically scarified (using a razor blade) and incubated, in light (●) or darkness (▲), at 25 °C for 30 d. Three replicates of 30 seeds per Petri dish were used for each treatment. Table 3. Mean germination percentages (± SE) of Eastern Sweetbush seeds incubated at 25 °C following mechanical scarification and imbibition in gibberellic acid (GA3) solution. Three replicates of 30 seeds each were used for each treatment. Reported means were recorded on the 30th day of incubation. Means followed by the same letter are not significantly different based on Fisher’s protected LSD at P < 0.05. Treatment Percent germination Scarification only 89.4 ± 3a Scarification + 500 ppm GA3 86.6 ± 4a Scarification + 750 ppm GA3 90.2 ± 3a Scarification + 1000 ppm GA3 80.4 ± 3b Southeastern Naturalist C.A. Adams, O.C. Adejumo, M. Jahan, and K.W. Montgomery 2017 Vol. 16, No. 4 498 Discussion Prior to this study, the only information in the published literature regarding seed germination ecology for the genus Calycanthus is for the California endemic, Western Sweetbush (Johnson 1997). Emery (1988) reported that the seeds possess physiological dormancy, a mechanism that inhibits the embryo and prevents radicle emergence (Bewley and Black 1994). Species within the same genus often have the same type of dormancy (Baskin and Baskin 2014), so it is reasonable to expect that seeds of the southeastern species, Eastern Sweetbush, might have PD as well. Preliminary work revealed that mature seeds of Eastern Sweetbush have water- impermeable seed coats, suggesting that these seeds are physically dormant (Baskin and Baskin 2014). Seeds exposed to water, at room temperature, for a period of 8–12 h with no increase in weight over this time frame are described as having water-impermeable seed coats (Bansal et al. 1980). The imbibition curve produced in this study clearly shows that non-scarified seeds do not take up water. However, scarified seeds did take up water and demonstrated a substantial increase in mass over the 12-h imbibition period; thus, demonstrating that seeds of Eastern Sweetbush possess physical dormancy. In terms of correlating embryo size/shape to dormancy, it might be expected that Eastern Sweetbush seeds possess PY. Seeds of most plant families that contain taxa with PY produce large embryos (Baskin and Baskin 2014), and the findings in this study are consistent with general patterns. The embryo in Eastern Sweetbush seeds is a large type referred to as “folded”, and the demonstrated PY is consistent with general patterns. The seeds of the Western Sweetbush apparently do not possess PY. Physical dormancy, however, is known within the Calycanthaceae: seeds of Wintersweet possess PY (Tang and Tian 2010) in addition to physiological dormancy of the embryo. Thus, despite the apparent absence in its closest relative, it should not be surprising that PY is found in seeds of Eastern Sweetbush. Despite the expression of physiological dormancy in the western congener and combinational dormancy within the family, embryos in most seeds of Eastern Sweetbush do not appear to be physiologically dormant. The vast majority of seeds germinated without any kind of PD-breaking treatment, thus there is no physiological component to dormancy for most propagules. Approximately 2–3% of mature seeds will germinate without any kind of scarification treatment. This result suggests 2 major possibilities: either the individual seeds have seed coats that developed with permeability to water or that these seeds were not fully mature when collected and PY had not developed. Baskin and Baskin (2014) reported that, in many species, if a seed is collected before it completely dries prior to full maturation, then the seed coat would have not developed water impermeability. In either case, there seem to be only a very small number of seeds in any population that do not possess PY. Most Eastern Sweetbush seeds possess PY and not combinational dormancy. We also examined mechanisms to break PY. The most effective laboratory method of breaking PY was mechanical scarification. Either abrading seeds with sandpaper or nicking the seed coat with a razor blade produced significantly higher germination Southeastern Naturalist 499 C.A. Adams, O.C. Adejumo, M. Jahan, and K.W. Montgomery 2017 Vol. 16, No. 4 percentages compared to the non-scarified control. In nature, some sort of abrasion to the seed coat, following dispersal, should produce seed coats that are permeable to water and able to germinate under favorable environmental conditions. Seeds of Eastern Sweetbush are easily carried by water (Chambert and James 2009) following dispersal; thus, abrasion could occur during transport by water. The limited published literature available suggests that the most common natural means of dispersal is via gravity, aided by the activity of animals, and the most likely cause of dormancy break is abrasion due to water transport (Kubtizki 1993, Miller and Miller 2005, Chambert and James 2009). The next most-effective method of breaking PY was exposure to sulfuric acid. Seeds submerged in concentrated sulfuric acid for various time periods produced significantly higher germination percentages than the control. In nature, an analogous scenario would be passage of a seed through an animal’s digestive tract; thus, if animals do consume Eastern Sweetbush fruits, as suggested by some (Miller and Miller 2005, Nicely 1965), stomach acid seems to be a likely agent for producing water-permeable seed coats. The other scarification methods were only mildly effective or ineffective in breaking PY. Percussion generated the 3rd-highest percentage of germination of all treatments. Only 15 seeds experienced dormancy break using this method; however, we tested only 1 form of treatment. It is possible that an increased period of percussion or more vigorous percussion might result in the breaking of PY in a higher number of seeds. The only other PY-breaking method that produced results significantly different from the control was the freeze/thaw cycling treatment. The overall percent germination, however, was still very low. No other treatment produced significantly higher germination than the nonscarified control. Some of these results, however, are inconclusive. For example, exposure of seeds to high heat occurred at only 3 temperatures over the same time period. For species where high heat can break PY, the effective temperature and duration of exposure can vary greatly (Baskin and Baskin 2014). In the case of Sida spp. (fanpetals), exposure to temperatures of 70–90 °C for either 12- or 24-h intervals are required to break PY (Chawan 1971), while Trifolium pratense L. (Red Clover) seeds become water permeable after only 4 min at 104 °C (Rincker 1954). To conclusively state that dry heat does or does not break PY, a more extensive study would need to be conducted using a broader range of temperatures and a broader use of heat-exposure durations. Similarly, more expansive studies should be conducted to determine whether high heat (wet), dry storage, and freeze/thaw cycles are effective in breaking PY in seeds of Eastern Sweetbush. Fire, which is known to create permeability in the seed coats of many species (Baskin and Baskin 2014) was not effective in breaking dormancy, and, in fact, was detrimental to seed health. We found that direct exposure to a surface fire was lethal to seeds. No germination occurred following the burning of leaf litter on top of exposed seeds. Most of the fire-exposed seeds began to decay within just a few days, and we observed 100% seed mortality within 10 days (embryos were almost completely liquefied); thus, direct exposure to fire results in rapid seed mortality. Southeastern Naturalist C.A. Adams, O.C. Adejumo, M. Jahan, and K.W. Montgomery 2017 Vol. 16, No. 4 500 Even seeds buried a few centimeters below the soil surface experienced high mortality following fire; within 10 d of the burn, 75% of the buried seeds perished. Unless seeds are buried relatively deeply within the soil, it is very likely that few will survive a surface fire. Once Eastern Sweetbush seeds have overcome physical dormancy, they will germinate under a wide range of environmental conditions. There is no light requirement for germination; therefore, nondormant seeds could germinate if completely shaded or buried. Nondormant seeds will germinate over a broad range of temperatures, provided that the embryo can imbibe water. The lower temperature limit for germination appears to be around 15 °C, at which at least a few seeds germinate, suggesting that, in nature, nondormant seeds could germinate during any season of the year except winter. No scarified seeds germinated in temperatures less than 15 °C. Conclusions This study confirms that seeds of Eastern Sweetbush possess physical dormancy. Seeds that have broken PY can germinate over a broad range of temperatures, predominantly those that occur in spring and summer. Seeds will germinate in light or darkness. This is the first report on seeds of a species in the Calycanthaceae where PY is the dormancy class solely possessed by the majority of seeds, as well as the first report on the seed germination ecology of Eastern Sweetbush. Although we conducted our study using seeds collected from a single location, there is currently no evidence for changes in the dormancy state of seeds with PY within a species’ range (Ferreras et al. 2017); thus, we expect that Eastern Sweetbush seeds throughout the southeast possess PY. Our results provide information on the seeddormancy status for Eastern Sweetbush, the only species in the genus Calycanthus for which it was previously unknown. Acknowledgments We thank L. Ballou, A. Wentworth, J. 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