Chilling Relieves Corm Dormancy in Calopogon Tuberosus (Orchidaceae) from Geographically Distant Populations

Environmental and Experimental Botany 70 (2011) 283–288

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Environmental and Experimental Botany

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Chilling relieves corm dormancy in Calopogon tuberosus (Orchidaceae) from geographically distant populations

Philip J. Kauth a,∗, Michael E. Kane a, Wagner A. Vendrame b a Plant Restoration, Conservation, and Propagation Biotechnology Program, Environmental Horticulture Department, University of Florida, PO Box 110675, Gainesville, FL 32611, USA b Tropical Research and Education Center, University of Florida, 18905 SW 280th St, Homestead, FL 33031, USA article info abstract

Article history: Many plant species require a chilling period to commence regrowth from overwintering structures such Received 17 November 2009 as buds, corms, tubers, and rhizomes. While the effects of chilling have been thoroughly studied in a horti- Received in revised form 4 October 2010 cultural context, little information exists regarding the relationship between ecotypic differentiation and Accepted 6 October 2010 chilling requirements. Effects of chilling storage organs on shoot emergence of widespread orchid species has not been examined, and ecotypic differentiation in the Orchidaceae has also received little attention. Keywords: The effects of chilling on corm dormancy in Calopogon tuberosus, a widespread orchid of eastern North Corm America, were studied. Seeds were collected from south Florida, north central Florida, South Carolina, and Dormancy Ecotype Michigan, and germinated in vitro to produce plants. After 20 weeks in vitro culture, corms were removed Orchid from seedlings and chilled for 0, 2, 4, 6, and 8 weeks. Corms were subsequently planted in a soilless potting mix and placed under ex vitro conditions in an environmental growth chamber. Shoot emergence was monitored bi-weekly for 16 weeks, and shoot length, leaf number, leaf width, root number, root length, and corm diameter were measured after 16 weeks. Longer chilling periods broke corm dormancy more effectively than shorter chilling treatments regardless of population. Shoots of all populations sprouted rapidly on corms after 6 and 8 weeks chilling. In addition, a higher percentage of shoots sprouted on corms after 8 weeks chilling. After 16 weeks, north central Florida and South Carolina plantlets were larger than Michigan and south Florida plantlets. Differing chilling requirements among C. tuberosus populations may reﬂect ecotypic differentiation resulting from varying environmental conditions at each site. © 2010 Elsevier B.V. All rights reserved.

1. Introduction growth conditions are encountered (Garbisch et al., 1995; Rohde and Bhalerao, 2007), and remain dormant until favorable growth Calopogon tuberosus var. tuberosus is a terrestrial orchid native conditions are encountered the following growing season (Garbisch to eastern North America with a large distribution from Florida et al., 1995). In order to break dormancy, a chilling period is often to maritime Canada. Throughout its range, C. tuberosus occu- required (Rohde and Bhalerao, 2007). Longer chilling periods are pies a variety of habitats including bogs, fens, marl prairies, and often required to break dormancy in tubers and corms of temperate mesic roadsides. Both genetic and morphological variation have species, but extended periods often inhibit growth and develop- been reported recently (Goldman et al., 2004a,b; Trapnell et al., ment (Clark, 1995; Yanez˜ et al., 2005; Fukai et al., 2006). However, 2004), but whether ecotypes exist remains unclear. Recent stud- chilling period requirement may be different according to plant ies exploring the in vitro ecology of seed germination and seedling provenance in that southern species may require shorter chill peri- development determined that photoperiod, germination media, ods (Perry and Wang, 1960; Garbisch et al., 1995). and growing season length influenced the development of C. Chilling requirements as a function of ecotypic differentiation tuberosus ecotypes (Kauth et al., 2008; Kauth and Kane, 2009). have been explored in tree species (Perry and Wang, 1960; Kriebel Additionally, potential differences in the extent of corm dormancy and Wang, 1962), aquatic species (Garbisch et al., 1996), and for- among widespread C. tuberosus populations may be influenced by age grasses (Silsbury, 1961; Cooper, 1964; Eagles, 1967a,b; MacColl local adaptation. and Cooper, 1967). Acer rubrum ecotypes from Florida required Many temperate plant species form overwintering structures, no chilling to break dormancy, but longer chill periods were such as buds, tubers, rhizomes, and corms, before unfavorable required to break dormancy in more northern ecotypes (Perry and Wang, 1960). Acer saccharum ecotypes from Georgia and Tennessee required shorter chill periods to break dormancy than ecotypes ∗ Corresponding author. Tel.: +1 352 273 4864; fax: +1 352 392 1413. in Michigan and Ohio (Kriebel and Wang, 1962). In several forage E-mail address: pkauth@ufl.edu (P.J. Kauth). grass species, relative growth rate of Mediterranean populations

0098-8472/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2010.10.003 284 P.J. Kauth et al. / Environmental and Experimental Botany 70 (2011) 283–288 was higher at cooler temperatures compared to north European at 10 ± 0.3 ◦C for 2, 4, 6, and 8 weeks in complete darkness; a control populations that had a higher growth rate at warmer tempera- (no cold storage) was also used. Five culture vessels per seed source tures (Cooper, 1964). Prolonged chilling decreased both survival were allocated to each chilling period. and shoot growth of aquatic plant ecotypes from Florida (Garbisch The 20 culture boxes (five replications per each of the four popu- et al., 1996). lations) allocated to the five chilling treatments were subsequently C. tuberosus is a model orchid species to exam the relationship removed after the chilling period. Corms were subsequently between chilling period and corm dormancy in widespread popula- planted in a 9-cell pack containing Fafard 2 (Conrad Fafard, Inc., tions. Studying corm dormancy, chilling period necessary to break Agawam, MA, USA). Corms were planted in a randomized com- dormancy, and shoot emergence may provide insight into C. tubero- plete block design with block designated as the chill treatment so sus ecotypic differentiation. The objectives were to: (1) assess the that block 1 was the control, etc. Each seed source was allocated effect of chilling on the mechanism of corm dormancy and subse- to each block, and blocks were replicated five times. Corms were quent plantlet growth; (2) exam the response of four C. tuberosus buried approximately 1 cm below the soil line. Trays were placed populations to low temperature storage; and (3) examine ecotypic in a walk-in growth chamber under a 16/8 h L/D photoperiod at differentiation in C. tuberosus. Our hypotheses were: (1) all pop- 27 ± 2.2 ◦C and an average relative humidity of 85%. Four 400-W ulations will require a period of chilling to break the mechanism metal halide bulbs (Sylvania, Danvers, MA, USA) provided a light of corm dormancy; and (2) corms from more northern popula- level of 167 ␮mol m−2 s−1. Corms were watered as needed and as tions will require a longer chilling period compared to southern frequently as daily. populations. Shoot emergence date was recorded by the presence of the new shoot emerging from the soil. Every 2 weeks, starting upon emergence and continuing until week 16, shoot length was mea- 2. Materials and methods sured from the soil surface to the shoot apex. At the final data collection, leaf number, leaf width, shoot height, root number, root Seeds were collected from the following locations: Upper Penin- length, corm diameter, and axillary shoot formation were recorded. sula Michigan (Menominee County, Michigan, USA), upstate South Axillary shoots (Fig. 1) grow from storage organs to form new stor- Carolina (Greenville County, South Carolina, USA), north central age organs (Dixon and Pate, 1978; Hollick et al., 2001). Percent Florida (Levy County, Florida, USA), and south Florida (Collier shoot emergence and percent survival, noted by the presence of a County, Florida, USA). Seed capsules from all populations were corm beneath the soil surface, were recorded. Logistic regression collected before complete dehiscence and were stored at 23 ◦C was used to assess the affect of chilling treatment and popula- over silica gel for 2 weeks. Seeds were then removed from cap- tion on percent shoot emergence, percent survival, and percent sules, pooled by geographic population, and stored dry in the axillary shoot formation using the generalized linear mixed model dark at −11 ◦C until used. Further information about the envi- procedure (proc glimmix macro) in SAS v9.1. Least-square means ronmental conditions for each site is supplied in supplemental (lsmeans) were used to assess mean separation. Endpoint measure- Table 1. ment data were analyzed using the general linear procedure (proc Seeds were surface disinfected in sterile scintillation vials for glm), ANOVA (see supplemental Table 2), and least-square means 3 min in a solution of 5 mL absolute ethanol, 5 mL 6% NaOCl, in SAS v9.1. and 90 mL sterile distilled, deionized water. Seeds were rinsed with sterile dd water after surface sterilization. Solutions were 3. Results removed with sterile Pasteur pipettes. Seeds were transferred with a 10 ␮L sterile inoculating loop onto BM-1 Terrestrial Orchid 3.1. Effects of chilling on shoot emergence Medium (PhytoTechnology Laboratories, Shawnee Mission, KS) contained in 100 mm × 15 mm Petri plates. The medium was sup- The main effects (population and chilling period), and their plemented with 1% activated charcoal. Medium pH was adjusted interaction all significantly influenced the number of days to shoot to 5.7 with 0.1 N KOH prior to autoclaving for 40 min at 117.7 kPa emergence. The average number of days to shoot emergence post- and 121 ◦C. Ten replicate Petri plates with 30 mL medium each chilling was less under the longer chill periods of 6 and 8 weeks, were used for each seed source with approximately 100 seeds regardless of population (Fig. 2A). Corms subjected to the control or per plate. Cultures were placed in an environmental growth the 2-week chilling period exhibited the slowest shoot emergence. incubator (#I-35LL; Percival Scientific, Perry, IA, USA) under cool- South Carolina and south Florida corms chilled longer than 6 weeks white fluorescent lights in a 12 h photoperiod at 24.2 ± 0.2 ◦C and exhibited the quickest shoot emergence. Michigan corms required 40 ␮mol m−2 s−1. 4 weeks or longer for quickest shoot emergence, while shoots After 8 weeks culture, seedlings were transferred to larger emerged faster when north central Florida corms were chilled for culture vessels for further growth and development. Nine 8 weeks (Fig. 2A). seedlings were transferred to individual PhytoTech Culture Boxes Percent shoot emergence was highly influenced by the main (PhytoTechnology Laboratories, Shawnee Mission, KS) containing effect of chilling period, but the main effect of population and the 100 mL of BM-1 Terrestrial Orchid Medium. Five replicate vessels interaction between chilling treatment and population were not were prepared for each treatment and seed source combination for significant. Lower percent shoot emergence was observed when a total of 45 seedlings per treatment. A total of 25 vessels with a corms were chilled for shorter periods (Fig. 2B). Less than 20% shoot total of 225 seedlings were prepared for each seed source. A total of emergence was observed in unchilled corms and following the 2- 900 seedlings were transferred. Seedlings grew in vitro for another week chilling period among all populations. In fact, only one shoot 12 weeks, for a total of 20 weeks culture. Environmental conditions from South Carolina emerged in the control and only one shoot from were the same as described previously. north central Florida emerged in the 2-week chilling treatment. After the 20 weeks, shoots and roots on seedlings were removed Chilling periods longer than 6 weeks provided the highest percent so that only corms remained. The nine corms in each PhytoTech box shoot emergence in Michigan corms (Fig. 2B), while 8 weeks chilling were transferred to Sigma Phytatrays I (#P1552, Sigma–Aldrich, St. provided the highest shoot emergence for all other populations. Louis, MO) containing 100 mL of moist, sterilized vermiculite. Five Approximately 90% shoot emergence occurred on South Carolina, Phytatrays I (114 mm × 86 mm × 63.5 mm) were prepared for each north central Florida, and south Florida corms, compared to 78% for treatment. Cultures containing the corms were subsequently stored Michigan corms (Fig. 2B). P.J. Kauth et al. / Environmental and Experimental Botany 70 (2011) 283–288 285

Fig. 1. Ex vitro growth comparison of representative C. tuberosus plantlets. Plantlets represent average size after 16 weeks growth in a walk-in growth chamber. Plantlets were generated after chilled corms were planted under ex vitro conditions. (A) Michigan plantlet with axillary shoot formed between the original and new corm. (B) South Carolina plantlet with axillary shoot formed between the original and new corm. (C) North central Florida plantlet. (D) South Florida plantlet. Scale bars = 1 cm.

Fig. 2. Effects of chilling corms at 10 ◦C on shoot emergence after 16 weeks of ex vitro growth of C. tuberosus plantlets. (A) Mean number of days to shoot emergence post- chilling. (B) Percent shoot emergence recorded by the presence of a shoot emerged from the soil. (C) Percent survival measured by the presence of a corm beneath the soil. (D) Percent axillary shoot formation. Each histobar represents the mean response of ﬁve replications with nine plantlets for a total of 45 plantlets per treatment × population. Means with the same letter are not signiﬁcantly different at ˛ = 0.05. 286 P.J. Kauth et al. / Environmental and Experimental Botany 70 (2011) 283–288

Fig. 3. Effects of chilling corms at 10 ◦C on growth and development of C. tuberosus plantlets. Data were collected after 16 weeks ex vitro growth. Data from south Florida ecotype does not appear in the control due to shoot senescence prior to data collection endpoint. (A) Shoot length measured from the soil surface to the tip of the longest leaf. (B) Leaf number. (C) Leaf width measured at the widest point of the widest leaf. (D) Root number. (E) Root length of the longest root. (F) Corm diameter of the new corm measured horizontally at the widest point. Each histobar represents the mean response of ﬁve replications with nine plantlets for a total of 45 plantlets per treatment × population. Means with the same letter are not signiﬁcantly different at ˛ = 0.05.

3.2. Effects of chilling on corm survival lary shoot formation was prevalent in Michigan and South Carolina populations after 16 weeks (Fig. 2D). Axillary shoot formation was After 16 weeks, corm survival was high regardless of population higher on corms that were chilled for 6 or 8 weeks compared to or chilling period. Survival was measured by the presence of a viable shorter chilling periods in Michigan and South Carolina plantlets corm below the soil surface, and not the presence the emerged (Fig. 2D). No axillary shoots formed on north central Florida and shoot. The original corm often remained viable, but did not form a south Florida corms in the control, 2 weeks, and 4 weeks chilling shoot by the end of the experiment. Major differences in survival periods (Fig. 2D). Axillary shoot formation in both Florida popula- were not clearly evident according to population, chilling period, or tions was highest when corms were chilled for 6 weeks, while 8 their interaction. Corm survival was only different within Michigan weeks chilling suppressed formation. and south Florida populations. Survival of south Florida propagules was highest with 4–8 weeks chilling, while Michigan corm survival 3.4. Effects of chilling on shoot growth was highest after 2 weeks chilling (Fig. 2C). Near 100% survival was observed in South Carolina and north central Florida corms The main effects (population and chilling period) and their inter- regardless of treatment (Fig. 2C). action all significantly influenced shoot growth. No differences in shoot length occurred among chilling periods in the Michigan and 3.3. Effects of chilling on axillary shoot formation south Florida populations (Fig. 3A). Few differences were observed in South Carolina and north central Florida populations. Michigan Only the main effect of population significantly influenced axil- and south Florida shoots were generally the shortest of all popu- lary shoot formation, while chilling period and the interaction lations and north central Florida the highest (Fig. 3A). Shoots on between population and chilling period were not significant. Axil- south Florida ecotypes did emerge in the control treatment (Fig. 2), lary shoots were considered formed by the presence of a shoot but shoots dehisced by week 16 and no data was collected due to connecting the original corm to the new shoot (Fig. 1A, B). Axil- shoot-die back. P.J. Kauth et al. / Environmental and Experimental Botany 70 (2011) 283–288 287

The interaction between the population and chilling period and (Cooper, 1964; Eagles, 1967a,b; MacColl and Cooper, 1967). While main effect of chilling period did not significantly influence leaf few differences in chilling were found among populations, local number. However, population significantly influenced leaf devel- adaptation to different habitats and environmental conditions may, opment. Michigan had the highest leaf number after 16 weeks in part, explain the chilling requirement. followed by South Carolina and both Florida populations (Fig. 3B). Comparative influence of chilling storage organs such as tubers Less than two leaves were generally present on all plantlets regard- and corms resulting from ecotypic differentiation has received little less of population. attention. Chilling dormant buds of tree ecotypes has been investi- Leaf width was significantly affected by the interaction between gated previously (Perry and Wang, 1960; Kriebel and Wang, 1962; the main effects of population and chilling period as well as popula- Myking and Heide, 1995; Li et al., 2003, 2005), but correlating chill- tion alone. However, chilling period did not significantly influence ing response of buds with underground storage organs may be leaf width. Average leaf width was lowest on south Florida plantlets difficult due to location of plant parts. Regardless, chilling require- (Fig. 3C) while the widest leaves grew on north central Florida and ments should be considered in a restoration context if plants are South Carolina plantlets (Fig. 3C). No differences among treatments moved from their home-site since southern ecotypes may not be were observed on South Carolina and south Florida. Wider leaves cold-hardy (Garbisch et al., 1996). were observed in chilling periods longer than 4 weeks on Michi- Chilling of C. tuberosus corms followed a similar pattern to gan plantlets. Widest leaves were observed on north central Florida bud chilling in birch species (Betula pendula and Betula pubescens). plantlets from the control, 2 weeks, and 8 weeks chill period. Longer chilling treatments reduced days to bud break regardless of population latitude, and the number of days to bud break was 3.5. Effects of chilling on root growth more pronounced for southern ecotypes (Myking and Heide, 1995). Longer chilling periods increased shoot emergence and reduced the The main effects (population and chilling period) and their inter- number of days to shoot emergence regardless of chilling period action all significantly influenced root development. No differences in ecotypes of C. tuberosus. Longer chilling treatments had a more were observed in root number for both South Carolina and Michi- pronounced influence on emergence days of southern C. tubero- gan populations, but more roots were observed in shorter chilling sus ecotypes, yet corms from northern populations generally broke periods for both Florida populations (Fig. 3D). The highest root dormancy earlier than southern ecotypes. This may have been number was observed in the control treatment in north central caused by the southern ecotypes inability to initiate growth at Florida plantlets, but South Carolina plantlets had the greatest root 10 ◦C and a higher base temperature required for dormancy break number on average. (Myking and Heide, 1995). The main effects and their interaction all significantly influenced The requirement for a chill period longer than 6 weeks for the C. root length. Longer chilling periods promoted the longest roots on tuberosus Michigan population was expected. The long winters and Michigan plantlets (Fig. 3E). No difference was observed on South relatively constant temperatures below freezing require plants to Carolina plantlets. Chilling periods less than 8 weeks promoted maintain dormancy until environmental conditions are appropri- the longest roots on north central Florida plantlets. South Florida ate (Garbisch et al., 1996). No difference in shoot emergence was plantlets from the no chill treatment did not form roots, while few observed between the 6- and 8-week chilling period for Michigan differences were observed among chilling treatments (Fig. 3E). plants, and shoot emergence was lower than all other populations in the 8-week chilling treatment. Being subjected to longer winters, 3.6. Effects of chilling on corm development Michigan plants may require a chill period longer than 8 weeks for maximum shoot emergence. Population significantly influenced new corm development The longer chilling period required for shoot emergence while chilling period and the main effects interaction were not sig- on southern C. tuberosus ecotypes was surprising, but may be nificant. Few differences were observed among chilling treatments explained by temperature sensitivity. Winter temperatures in the within each population with the exception of South Carolina and south often exceed 17 ◦C, but temperatures may drop suddenly in north central Florida where the largest corms occurred in the con- subsequent days. South Florida temperatures do fall below 0 ◦C, trol and control/2 weeks chilling treatment, respectively (Fig. 3F). albeit for shorter periods than northern climates, reflecting the Regardless of treatment, smallest corms south Florida plantlets south Florida ecotype’s inability to grow at lower temperatures had the smallest corms, and new corms did not form on south (Myking and Heide, 1995). Longer chilling periods may ensure Florida plantlets in the control treatment. The largest corms grew Florida plants do not initiate shoot emergence until the threat of on South Carolina and north central Florida plantlets in the control freezing temperatures is surpassed (Garbisch et al., 1996). Early and 2 weeks chilling treatment, while Michigan and South Car- shoot dehiscence on south Florida shoots in the control treatment olina plantlets had the largest corms in the 4, 6, 8 week chilling may have been caused by an inadequate chilling period. Iwaya- treatments. Inoue et al. (1996) reported starch digestion and respiration rate was greater in tulip bulbs that received a chilling treatment. Sim- 4. Discussion ilarly, south Florida corms in the non-chilled treatment may not have had sufficient chilling that promoted starch digestion and This is the first study comparing the role of chilling in reliev- water movement to the developing shoot meristem (Iwaya-Inoue ing corm dormancy in an orchid species. The results supported et al., 1996). our first hypothesis that chilling will effectively break corm dor- An interesting morphological feature observed in Michigan and mancy. However, our second hypothesis was not supported. The South Carolina populations was the formation of an axillary shoot response of Florida corms to longer chilling periods was surpris- between the new and old corms. These axillary shoots, often ing. The results aid in our understanding of the ecological growth referred as droppers, grow downward to form replacement stor- strategy of C. tuberosus as well as ecotypic differentiation. age organs for the next season’s growth (Dixon and Pate, 1978; Previous research examining chilling of storage organs focused Hollick et al., 2001). However, the axillary shoots on C. tubero- on the effects of temperature and chilling period in relation to shoot sus corms were different because they grew upward toward the growth and flowering of horticultural crops (Clark, 1995; Kim et al., surface of the soil. This may be an ex situ phenomenon because 1996; González et al., 1998; Yanez˜ et al., 2005; Fukai et al., 2006), axillary shoots have not been seen on wild plants from any loca- but little information exists focusing on local adaptation to chilling tion (personal observation). Michigan and South Carolina plantlets 288 P.J. Kauth et al. / Environmental and Experimental Botany 70 (2011) 283–288 were more prone to form axillary shoots, and more axillary shoots Cooper, J.P., 1964. Climatic variation in forage grasses. I. Leaf development in climatic formed with longer chill periods regardless of plantlets population. races of Lolium and Dactylis. J. Appl. Ecol. 1, 45–61. Dixon, K.W., Pate, J.S., 1978. Phenology, morphology, and reproductive biology of The exact ecological role of droppers and storage organ axillary tuberous sundew, Drosera erthryorhiza Lindl. Aust. J. Bot. 26, 441–454. shoots are uncertain. Michigan and South Carolina ecotypes may Eagles, C.F., 1967a. The effect of temperature on vegetative growth in climatic races be more prone to forming axillary shoots to take advantage of a of Dactylis glomerata in controlled environments. Ann. Bot. 31, 31–39. Eagles, C.F., 1967b. Variation in the soluble carbohydrate content of climatic races shorter growing season by forcing new growth toward the soil sur- of Dactylis glomerata (Cocksfoot) at different temperatures. Ann. Bot. 31, 645– face. Because winter temperatures are more variable in Florida, 651. longer chilling requirements may influence axillary shoot growth Fukai, S., Kanechika, R., Hasegawa, A., 2006. Effect of low temperature on break- to avoid frost damage. Further investigation into the dynamics of ing dormancy and flowering of Arisaema sikokianum (Araceae). Sci. Hortic. 111, 97–100. axillary shoot formation is warranted. Garbisch, E.W., McIninch, S.M., Swartz, H.J., Salvaggio, G.J., 1995. Chilling responses Clear differences in growth and development of plantlets after for some herbaceous wetland plants. Wetland J. 7, 16–20. corm chilling were not evident. However, longer chilling periods Garbisch, E.W., McIninch, S.M., Swartz, H.J., Salvaggio, G.J., 1996. The effects of controlled chilling on five wetland herbaceous plant species. Wetland J. 8, 20– effectively broke dormancy of all populations. Several factors may 29. have influenced the inconsistent plantlets growth and develop- Goldman, D.H., Jansen, R.K., van den Berg, C., Leitch, I.J., Fay, M.F., Chase, M.W., ment results. Plantlet numbers were low in the control and shorter 2004a. Molecular and cytological examination of Calopogon (Orchidaceae, Epi- dendroideae): circumscription, phylogeny, polyploidy, and possible hybrid chill treatments, thus error was much larger creating fewer signif- speciation. Am. J. Bot. 91, 707–723. icant results. The few plantlets that did emerge and develop in the Goldman, D.H., van den Berg, C., Griffith, M.P., 2004b. Morphometric circumscription shorter chilling treatments had a longer time to develop compared of species and infraspecific taxa in Calopogon R. Br. (Orchidaceae). Plant Syst. Evol. 247, 37–60. to those in the longer chilling treatments contributing to the larger González, A., Banón,˜ S., Fernández, J.A., Franco, J.A., Casas, J.L., Ochoa, J., 1998. Flow- sized plantlets. ering responses of Gladiolus tristis (L.) after exposing corms to cold treatment. Chilling corms of C. tuberosus ecotypes effectively promoted Sci. Hortic. 74, 279–284. Hollick, P., Senaratna, T., McComb, J., Bunn, E., Dixon, K.W., 2001. Response to shoot emergence upon removal from the chilling treatments. The paclobutrazol of symbiotic mycorrhizal fungi and dropper (tuber stalk) forma- differential response to chilling length by C. tuberosus ecotypes may tion of host orchid seedlings. Plant Growth Regul. 00, 1–9. be in part due to temperature sensitivity, and the ability to suc- Iwaya-Inoue, M., Motooka, K., Ishida, N., Koizumi, M., Kano, H., 1996. Chilling cessfully utilize carbohydrate reserves after chilling. Additionally, effects for normal growth of tulip bulbs estimated by MRI. 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