Abstract

Background and Aims

Ecotypic differentiation has been explored in numerous plant species, but has been largely ignored in the Orchidaceae. Applying a specific germination protocol for widespread seed sources may be unreliable due to inherent physiological or genetic differences in localized populations. It is crucial to determine whether ecotypic differentiation exists for restoration and conservation programmes. Calopogon tuberosus var. tuberosus, a widespread terrestrial orchid of eastern North America, is a model species to explore ecotypic differences in germination requirements, as this species occupies diverse habitats spanning a wide geographical range.

Methods

Mature seeds were collected from south Florida, north central Florida, three locations in South Carolina, and the upper Michigan peninsula. Effects of three photoperiods (8/16, 12/12, 16/8 h L/D) were examined on asymbiotic in vitro seed germination and seedling development of C. tuberosus. Germination and early development was monitored for 8 weeks, while advanced development was monitored for an additional 8 weeks. In an additional experiment, asymbiotic seed germination and development was monitored for 8 weeks on six culture media (BM-1 terrestrial orchid medium, Knudson C, Malmgrem, half-strength MS, P723, and Vacin and Went). A tetrazolium test for embryo viability was performed.

Key Results

Short days promoted the highest germination among Florida populations, but few differences among photoperiods in other seed sources existed. Different media had little effect on the germination of Michigan and Florida populations, but germination of South Carolina seeds was higher on media with higher calcium and magnesium. Tetrazolium testing confirmed that South Carolina seeds exhibited low viability while viability was higher in Florida seeds. Seed germination and corm formation was rapid in Michigan seeds across all treatments. Michigan seedlings allocated more biomass to corms compared with other seed sources.

Conclusions

Rapid germination and corm formation may be a survival mechanism in response to a compressed growing season in northern populations. Ecotypic differentiation may be occurring based on seed germination and corm formation data.

INTRODUCTION

Ecotypic differentiation has recently been recognized as an important issue in several plant sciences including conservation, restoration and population genetics (Hufford and Mazer, 2003). Ecotypic differentiation enables species to survive diverse habitats and environmental conditions across its geographical range, but the specific functions they serve in ecosystems remain unclear (Seliskar et al., 2002). For this reason, using local plant material for restoration purposes or reintroductions may be necessary to maintain ecosystem health (Linhart, 1995). Introducing poorly adapted ecotypes into unsuitable habitats may lead to reduced plant population fitness (Hufford and Mazer, 2003; McKay et al., 2005).

Common garden studies are often utilized to detect local adaptation (Sanders and McGraw, 2005), but obtaining permits to collect and transplant protected, rare, threatened or endangered species is often difficult. Alternatively, studying the ecology and physiology of seed germination and seedling development from widespread populations may provide insight into ecotypic differentiation (Singh, 1973). Currently, little information exists on seed germination among geographically distinct orchid populations as well as orchid ecotypic differentiation. Studies of orchid seed germination ecology are needed to support reintroduction programmes that typically use seed germination as a propagation tool.

Calopogon tuberosus var. tuberosus is a terrestrial orchid of eastern North America from Florida to Canada and west to Texas, and occupies habitats including alkaline prairies, pine flatwoods, mesic roadsides, fens and sphagnum bogs (Luer, 1972). Goldman et al. (2004) defined three C. tuberosus geographic clines. Northern plants in glaciated areas are differentiated from southern plants by labellum apex shape, reduced flower size and reduced leaf and inflorescence height. South-west populations west of the Mississippi Embayment differ from those in the south-east by larger flowers and inflorescence heights. Morphological variation may be caused by environmental factors or cross-pollination avoidance with other Calopogon species (Goldman et al., 2004).

Given that C. tuberosus is a commonly recognized orchid in North America, information exists regarding ecology, pollination and seed germination for this species. However, seed germination information is often conflicting. Different environmental conditions for seed germination of C. tuberosus have been recommended ranging from complete darkness to light incubation (Stoutamire, 1974; Whitlow, 1996; Kauth et al., 2006). Likewise, different germination media have also been recommended (Henrich et al., 1981; Arditti et al., 1985; Anderson, 1990; Kauth et al., 2006).

Differences in germination and seedling development might be the result of local adaptation to specific environmental conditions. Attributing ecotypic differentiation to germination differences is difficult since seed source is rarely reported in C. tuberosus seed germination studies, and basing recommendations for seed germination of C. tuberosus on one population is tenuous. Evaluation of in vitro seed germination from diverse populations may clarify whether ecotypic differentiation occurs among C. tuberosus populations. In this paper, the effects of photoperiod and culture media on asymbiotic seed germination and seedling development are compared among widespread populations of C. tuberosus.

MATERIALS AND METHODS

Seed source

Intact seed capsules (slightly yellow in colour) of Calopogon tuberosus (L.) Britton, Sterns & Poggenb. var. tuberosus were collected before dehiscence approx. 2 months after peak flowering throughout summer 2006. Capsules were collected from the Florida Panther National Wildlife Refuge (Collier County, Florida, USA), Goethe State Forest (Levy County, Florida, USA), Ashmore Heritage Preserve (Greenville County, South Carolina, USA), Eva Chandler Heritage Preserve (Greenville County, South Carolina, USA), site ‘C’ near Eva Chandler Heritage Preserve (Greenville County, South Carolina, USA) and Carney Fen (Menominee County, Michigan, USA; Fig. 1). The populations from site ‘C’ and Eva Chandler occupy cataract bogs, which form when streams flow over granite out-cropping resulting in sphagnum-filled depressions (Porcher and Rayner, 2001); for further site-specific information see Table 1. Non-dehisced capsules were collected to reduce the potential for surface contamination of individual seeds. Upon collecting and receiving capsules, they were stored at 23 °C over silica desiccant for 2 weeks. After 2 weeks, seeds were removed from the capsules and stored over silica desiccant at –11 °C until use.

Fig. 1.

Habitat and location of Calopogon tuberosus populations used in the present study: (A) Calopogon tuberosus flower; (B) fen habitat on the upper Michigan peninsula; (C) cataract bog in South Carolina; (D) roadside habitat in north central Florida; (E) prairie habitat in south Florida; (F) population locations.

Fig. 1.

Habitat and location of Calopogon tuberosus populations used in the present study: (A) Calopogon tuberosus flower; (B) fen habitat on the upper Michigan peninsula; (C) cataract bog in South Carolina; (D) roadside habitat in north central Florida; (E) prairie habitat in south Florida; (F) population locations.

Table 1.

Location, habitat, and basic environmental conditions of Calopogon tuberosus seed sources used in the present study

Population location and (designation) Co-ordinates Habitat Soil designation and (composition) Long day Short day 
Panther Refuge (south Florida) 26°10′06″N, 81°21′51″W Alkaline prairie Ochopee fine sandy loam (fine sandy loam) 13 h 47 min 10 h 30 min 
Goethe State Forest (north central Florida) 29°09′18″N, 82°37′12″W Mesic roadside Smyrna fine sand (fine sand) 14 h 02 min 10 h 16 min 
Ashmore Preserve (South Carolina 1) 35°05′13″N, 82°34′46″W Lake bog Congaree (sphagnum/fine sandy loam) 14 h 30 min 9 h 49 min 
Site ‘C’ (South Carolina 2) 35°05′02″N, 82°35′51″W Cataract bog Ashe-Cleveland association (sphagnum/sandy loam) 14 h 30 min 9 h 49 min 
Chandler Preserve (South Carolina 3) 35°05′03″N, 82°36′27″W Cataract bog Ashe-Cleveland association (sphagnum/sandy loam) 14 h 30 min 9 h 49 min 
Carney fen (Michigan) 45°34′47″N, 87°39′38″W Fen Lupton-Cathro association (sphagnum/muck) 15 h 42 min 8 h 46 min 
Population location and (designation) Co-ordinates Habitat Soil designation and (composition) Long day Short day 
Panther Refuge (south Florida) 26°10′06″N, 81°21′51″W Alkaline prairie Ochopee fine sandy loam (fine sandy loam) 13 h 47 min 10 h 30 min 
Goethe State Forest (north central Florida) 29°09′18″N, 82°37′12″W Mesic roadside Smyrna fine sand (fine sand) 14 h 02 min 10 h 16 min 
Ashmore Preserve (South Carolina 1) 35°05′13″N, 82°34′46″W Lake bog Congaree (sphagnum/fine sandy loam) 14 h 30 min 9 h 49 min 
Site ‘C’ (South Carolina 2) 35°05′02″N, 82°35′51″W Cataract bog Ashe-Cleveland association (sphagnum/sandy loam) 14 h 30 min 9 h 49 min 
Chandler Preserve (South Carolina 3) 35°05′03″N, 82°36′27″W Cataract bog Ashe-Cleveland association (sphagnum/sandy loam) 14 h 30 min 9 h 49 min 
Carney fen (Michigan) 45°34′47″N, 87°39′38″W Fen Lupton-Cathro association (sphagnum/muck) 15 h 42 min 8 h 46 min 

Seed viability test

A seed viability test (Lakon, 1949) was performed on all populations by staining embryos with 2,3,5-triphenyl tetrazolium chloride (TTC). Seeds were scarified in an aqueous 5 % CaOCl2 solution for 0 min, 30 min, 1 h, 2 h or 3 h. Two replications of approx. 100 seeds each were used per treatment. After scarification, seeds were rinsed twice in distilled-deionized (dd) water and suspended in sterile water for 24 h in darkness at 23 ± 2 °C. Water was replaced with TTC and seeds were soaked for 24 h at 30 °C in darkness. After the TTC soak, embryos were scored as viable if any degree of red staining was observed.

Media and seed preparation

Media were prepared in 1000-ml batches, and the pH was adjusted to 5·7 with 0·1 n KOH prior to autoclaving for 40 min at 117·7 kPa and 121 °C. Aliquots (40 mL) of sterile medium were dispensed into square 100 × 15 mm Petri plates with a 36-cell bottom (Integrid™ Petri Dish; Becton Dickinson and Co., Franklin Lakes, NJ, USA). Mature seeds were surface sterilized in sterile scintillation vials for 3 min in a solution of 5 mL absolute ethanol, 5 mL 6 % NaOCl and 90 mL sterile dd water. Seeds were rinsed twice with sterile dd water after surface sterilization. Solutions were removed from the vials with sterile Pasteur pipettes. Seeds were then placed on the surface of the germination media with a sterile inoculating loop. The interior 16 cells of the Petri plates were used for subreplications to avoid uneven media drying at the edges. Petri plates were sealed with one layer of Nescofilm (Karlan Research Products, Santa Rosa, CA, USA). Seed germination and seedling development (Table 2) were monitored weekly for 8 weeks according to the six developmental stages described by Kauth et al. (2006).

Table 2.

Six stages of orchid seed development (from Kauth et al., 2006)

Stage Description 
Imbibed seed, swollen and greening still covered or partially covered by testa 
Enlarged seed without testa 
Protocorm with pointed shoot apex and rhizoids 
Protocorm with emerging leaf and developing rhizoids 
Seedling with one elongated leaf and one developing root 
Seedling with evident roots and two or more leaves 
Stage Description 
Imbibed seed, swollen and greening still covered or partially covered by testa 
Enlarged seed without testa 
Protocorm with pointed shoot apex and rhizoids 
Protocorm with emerging leaf and developing rhizoids 
Seedling with one elongated leaf and one developing root 
Seedling with evident roots and two or more leaves 

Photoperiod effects on asymbiotic germination and early seedling development

A 6 × 3 factorial design was used with six seed sources and three photoperiods including a short day (SD = 8/16 h L/D), neutral day (ND = 12/12 h L/D), long day (LD = 16/8 h L/D). PhytoTechnology Orchid Seed Sowing Medium (#P723; PhytoTechnology Laboratories, Shawnee Mission, KS, USA) was used based on previous success with C. tuberosus seed germination and development (Kauth et al., 2006). Ten replicate Petri plates with five randomly selected subreplications (48·5 ± 17·9 seeds) were used per seed source and photoperiod treatment. Culture vessels were placed under cool-white fluorescent lights (F96712, General Electric) at an average of 33·3 ± 7 (12/12 photoperiod), 31·6 ± 5 (8/16 photoperiod) and 31·6 ± 6 (16/8 photoperiod) μmol m−2 s−1, and incubated at 25 ± 0·4 °C.

Photoperiod effects on advanced in vitro seedling development

After 8 weeks, seedlings in the photoperiod experiment were transferred from Petri plates to PhytoTech culture boxes (95 × 95 × 100 mm) containing 100 mL P723 medium; seedlings were maintained in corresponding photoperiods. Ten seedlings were transferred to each culture box. After an additional 8 weeks and 16 weeks total, five culture vessels per treatment (50 total seedlings) were randomly selected. Seedling percentage biomass allocation was determined by dividing corm, root and shoot weights by the total seedling weight. Culture conditions were the same as previously described.

Asymbiotic germination media evaluation

A 6 × 6 factorial design with six germination media (Table 3) and six seed sources was used. Five media commercially prepared by PhytoTechnology Laboratories were used: BM-1 Terrestrial Orchid Medium (BM-1; #B141; van Waes and Debergh, 1986a), Knudson C Orchid Medium (KC; #K400; Knudson, 1946), Malmgren Modified Terrestrial Orchid Medium (MM; #M482; Malmgren, 1996), Orchid Seed Sowing Medium (#P723), and Vacin and Went Modified Orchid Medium (VW; #V895; Vacin and Went, 1949). Murashige and Skoog Medium in half-strength (MS; #M5524; Murashige and Skoog, 1962) was commercially prepared by Sigma-Aldrich (St Louis, MO, USA). BM-1 and VW were further supplemented with 0·1 % charcoal, KC was further supplemented with 0·1 % charcoal and 0·8 % TC® agar (PhytoTechnology Laboratories), MM was further supplemented with 0·8 % TC® agar, and 0·5MS was further supplemented with 0·1 % charcoal, 0·8 % TC agar, and organics found in P723. All media contained 2 % sucrose. Five replicate Petri plates with three randomly selected subreplications (62·6 ± 15·2 seeds) were used per treatment. Germination and development were monitored biweekly for 8 weeks. Culture vessels were placed under ND conditions, cool-white fluorescent lights at 33·3 ± 7 µmol m−2 s−1, and 25 ± 0·4 °C.

Table 3.

Comparative mineral salt content of asymbiotic orchid seed germination media: BM-1 Terrestrial Orchid Medium (BM-1), Knudson C (KC), Malmgren Modified Terrestrial Orchid Medium (MM), Murashige and Skoog (MS), PhytoTechnology Orchid Seed Sowing Medium (P723), Vacin and Went (VW)

 MM BM-1 P723 VW KC 0·5MS 
Macronutrients (mm      
 Ammonium   5·15 7·57 13·82 10·31 
 Calcium 0·73  0·75 1·93 2·12 1·50 
 Chlorine  0·0021 1·50  3·35 3·1 
 Magnesium 0·81 0·83 0·62 1·01 1·01 0·75 
 Nitrate   9·85 5·19 10·49 19·70 
 Potassium 0·55 2·20 5·62 7·03 5·19 10·89 
 Phosphate 1·03 2·20 0·31 3·77 1·84 0·63 
 Sulfate 0·92 1·10 0·71 8·71 8·69 0·86 
 Sodium 0·20 0·20 0·10 0·20  0·10 
Micronutrients (mm      
 Boron  161·7 26·7   50 
 Cobalt  0·105 0·026   0·053 
 Copper  0·10 0·025   0·5 
 Iron 100 100·2 50 100 90 50 
 Iodine   1·25   2·50 
 Manganese 10 147·9 25 30 30 50 
 Molybdenum  1·03 0·26   0·52 
 Zinc  34·8 9·22   14·95 
Organics (mg L−1      
 Biotin 0·05 0·05     
 Casein hydrolysate 400 500     
 Folic acid 0·5 0·5     
l-Glutamine  100     
 Glycine 2·0 2·0     
myo-Inositol 100 100 100   100 
 Nicotinic acid  5·0 1·0   1·0 
 Peptone   2000   2000 
 Pyridoxine HCl  0·5 1·0   1·0 
 Thiamine HCl  0·5 10   10 
 Total mineral salt concentration (mm4·35 6·98 24·72 35·54 46·72 48·01 
 Total inorganic N (mmn/a n/a 15·00 12·76 24·31 30·01 
 MM BM-1 P723 VW KC 0·5MS 
Macronutrients (mm      
 Ammonium   5·15 7·57 13·82 10·31 
 Calcium 0·73  0·75 1·93 2·12 1·50 
 Chlorine  0·0021 1·50  3·35 3·1 
 Magnesium 0·81 0·83 0·62 1·01 1·01 0·75 
 Nitrate   9·85 5·19 10·49 19·70 
 Potassium 0·55 2·20 5·62 7·03 5·19 10·89 
 Phosphate 1·03 2·20 0·31 3·77 1·84 0·63 
 Sulfate 0·92 1·10 0·71 8·71 8·69 0·86 
 Sodium 0·20 0·20 0·10 0·20  0·10 
Micronutrients (mm      
 Boron  161·7 26·7   50 
 Cobalt  0·105 0·026   0·053 
 Copper  0·10 0·025   0·5 
 Iron 100 100·2 50 100 90 50 
 Iodine   1·25   2·50 
 Manganese 10 147·9 25 30 30 50 
 Molybdenum  1·03 0·26   0·52 
 Zinc  34·8 9·22   14·95 
Organics (mg L−1      
 Biotin 0·05 0·05     
 Casein hydrolysate 400 500     
 Folic acid 0·5 0·5     
l-Glutamine  100     
 Glycine 2·0 2·0     
myo-Inositol 100 100 100   100 
 Nicotinic acid  5·0 1·0   1·0 
 Peptone   2000   2000 
 Pyridoxine HCl  0·5 1·0   1·0 
 Thiamine HCl  0·5 10   10 
 Total mineral salt concentration (mm4·35 6·98 24·72 35·54 46·72 48·01 
 Total inorganic N (mmn/a n/a 15·00 12·76 24·31 30·01 

Statistical nalysis

Germination percentages were calculated by dividing the number of germinated seeds by the total number of seeds with an embryo in each subreplication. The percentage of protocorms and seedlings in a developmental stage was calculated by dividing the number of seeds in a stage by the total number of seeds with an embryo. Germination counts were arcsine transformed to normalize variation. Germination and seedling development data were analysed using general linear model procedures and least square means at α = 0·05 in SAS v. 8·02.

RESULTS

Seed viability

For all populations except south Florida (no difference in pretreatment time), the highest percentage of viable embryos was observed after 3 h of calcium hypochlorite pretreatment. Maximum embryo viability for each population was as follows: 85·4 % south Florida; 66·7 % north central Florida; 25·0 % South Carolina 1; 38·1 % South Carolina 2; 42·1 % South Carolina 3; and 50·3 % Michigan.

Photoperiod effects on germination and early development

Total seed germination percentage (Fig. 2) was highest under SD conditions for north central Florida (60·2 %) and south Florida (48·5 %) populations. There was no difference in germination among the three photoperiods for Michigan seeds (11·6 %, 12·5 % and 9·9 %). Germination percentages in all South Carolina populations did not exceed 4 %.

Fig. 2.

Photoperiod effects on seed germination and subsequent development of Calopogon tuberosus from different populations after culture on P723 medium for 8 weeks: (A) upper Michigan peninsula population; (B) South Carolina population from Ashmore; (C) South Carolina population from site ‘C’; (D) South Carolina population from Eva Chandler; (E) north central Florida population; (F) South Florida population. Histobars within each seed source with the same letter are not significantly different (α = 0·05). See Table 2 for stages of germination and development.

Fig. 2.

Photoperiod effects on seed germination and subsequent development of Calopogon tuberosus from different populations after culture on P723 medium for 8 weeks: (A) upper Michigan peninsula population; (B) South Carolina population from Ashmore; (C) South Carolina population from site ‘C’; (D) South Carolina population from Eva Chandler; (E) north central Florida population; (F) South Florida population. Histobars within each seed source with the same letter are not significantly different (α = 0·05). See Table 2 for stages of germination and development.

Seeds from Michigan germinated and developed more quickly compared with other populations, with imbibition occurring 1 week after inoculation. By week 8 >95 % of the germinated protocorms in all photoperiods developed to stage 6. Protocorm development was similar among photoperiods (Fig. 2A).

South Carolina protocorm development was unpredictable. South Carolina 1 (Fig. 2B) and South Carolina 2 (Fig. 2C) protocorms developed slowly with <1 % developing to leaf-bearing stages. Only South Carolina 1 protocorms under ND developed to stage 6 while South Carolina 2 protocorms under both ND and LD conditions developed to stages 5 and 6. Development of South Carolina 3 (Fig. 2D) protocorms was more advanced than other South Carolina populations.

Seeds from north central Florida germinated quickly and corms formed after week 8. Greater than 16 % of the protocorms in each photoperiod developed to an advanced leaf-bearing stage (stage 6) by week 8 (Fig. 2E). Although germination of south Florida seeds was highest under SD conditions, the majority of seeds did not develop past the imbibition stage by week 8 (Fig. 2F). Fewer than 5 % of the south Florida seeds under SD conditions developed past imbibition after 8 weeks culture. Approximately 10 % of the seeds under both ND and LD conditions developed past imbibition. A low percentage of south Florida seedlings in all photoperiods developed to advanced leaf-bearing stages (stages 5 and 6).

Photoperiod effects on advanced seedling development

After 16 weeks culture, Michigan seedlings began to senesce while South Carolina and Florida seedlings continued to grow. Corm formation was limited in south Florida seedlings, while Michigan, South Carolina and north central Florida seedlings all formed corms (Fig. 3). Biomass allocation was similar among photoperiods within each seed source (Fig. 4). Maximum dry weight allocation to corms was observed in Michigan seedlings (Fig. 4A). Although north central Florida seedlings formed large corms, the percentage dry biomass allocation was more evenly distributed among shoots, corms and roots than other populations (Fig. 4C). The greatest seedling shoot biomass allocation was observed in South Carolina 3 and south Florida populations (Fig. 4B, D).

Fig. 3.

Effects of photoperiod on in vitro seedling development of Calopogon tuberosus from different populations after 16 weeks total culture (8 weeks in Petri dishes/8 weeks in PhytoTech culture boxes): (A–D) seedlings cultured under an 8/16 h L/D photoperiod; (E–H) seedlings cultured under a 12/12 h L/D photoperiod; (I–L) seedlings cultured under a 16/8 h L/D photoperiod; (A, E, I) South Florida seedlings; (B, F, J) North Central Florida seedlings; (C, G, K) South Carolina seedlings from Eva Chandler; (D, H, L) upper Michigan peninsula seedlings. Scale bars = 1 cm.

Fig. 3.

Effects of photoperiod on in vitro seedling development of Calopogon tuberosus from different populations after 16 weeks total culture (8 weeks in Petri dishes/8 weeks in PhytoTech culture boxes): (A–D) seedlings cultured under an 8/16 h L/D photoperiod; (E–H) seedlings cultured under a 12/12 h L/D photoperiod; (I–L) seedlings cultured under a 16/8 h L/D photoperiod; (A, E, I) South Florida seedlings; (B, F, J) North Central Florida seedlings; (C, G, K) South Carolina seedlings from Eva Chandler; (D, H, L) upper Michigan peninsula seedlings. Scale bars = 1 cm.

Fig. 4.

Percentage dry weight biomass allocation of Calopogon tuberosus seedlings after 16 weeks in vitro culture: (A) upper Michigan peninsula population; (B) South Carolina population from Eva Chandler; (C) north central Florida population; (D) south Florida population. Histobars represent the mean response of 50 seedlings ± s.e.

Fig. 4.

Percentage dry weight biomass allocation of Calopogon tuberosus seedlings after 16 weeks in vitro culture: (A) upper Michigan peninsula population; (B) South Carolina population from Eva Chandler; (C) north central Florida population; (D) south Florida population. Histobars represent the mean response of 50 seedlings ± s.e.

Media effects on germination and early development

Michigan seeds germinated and protocorms developed quickly on all media, but the highest germination percentages occurred on P723 (34·1 %). With the exception of KC and VW, over 90 % of the protocorms on all other media developed to stage 6 (Fig. 5A).

Fig. 5.

Effects of culture media on seed germination and early development of Calopogon tuberosus from different populations after 8 weeks culture under a 12/12 h L/D photoperiod: (A) upper Michigan peninsula population; (B) South Carolina population from Ashmore; (C) South Carolina population from site ‘C’; (D) South Carolina population from Eva Chandler; (E) north central Florida population; (F) south Florida population. Histobars within each seed source with the same letter are not significantly different (α = 0·05). For media abbreviations and formulae see Table 3. See Table 2 for stages of germination and development.

Fig. 5.

Effects of culture media on seed germination and early development of Calopogon tuberosus from different populations after 8 weeks culture under a 12/12 h L/D photoperiod: (A) upper Michigan peninsula population; (B) South Carolina population from Ashmore; (C) South Carolina population from site ‘C’; (D) South Carolina population from Eva Chandler; (E) north central Florida population; (F) south Florida population. Histobars within each seed source with the same letter are not significantly different (α = 0·05). For media abbreviations and formulae see Table 3. See Table 2 for stages of germination and development.

Seed germination for South Carolina 1 was highest on VW, but germination was only 4·9 % (Fig. 5B). No clear differences in germination were observed in South Carolina 2 (Fig. 5C), but germination on P723 was significantly lower than all other media. Germination on KC (39·7 %) and MS (30·4 %) was highest for South Carolina 3 seeds, while lowest germination occurred on P723 (Fig. 5D).

In both Florida populations, few differences in total germination existed among media; however, subsequent development differed greatly. For north central Florida, higher numbers of stage 4, 5 and 6 seedlings were observed on BM-1, MS, P723 and VW (Fig. 5E). The highest germination percentage of north central Florida seeds was observed on MM, but the majority of seeds remained in stage 1 after 8 weeks. The highest percentage of stage 4, 5 and 6 protocorms was observed on BM-1, P723 and VW for south Florida (Fig. 5F). Germination percentages were high on KC and MM for south Florida seeds, but no stage 6 seedlings developed within 8 weeks and considerably fewer stage 5 seedlings developed compared with all other media.

Media effects on corm development

Corm development on BM-1, MS and P723 was superior in all populations (Fig. 6). Seedling development of Michigan, South Carolina 3, and north central Florida seedlings was superior to other populations (Fig. 6). However, development of Michigan, South Carolina 3, and north central Florida seedlings differed markedly. Corm formation was more pronounced in seedlings from northern latitudes. Thus by week 8, no corm formation was observed in Florida seedlings while early and advanced corm formation was observed in South Carolina and Michigan seedlings, respectively.

Fig. 6.

Culture media effects on early seedling development of Calopogon tuberosus from different populations after culture for 8 weeks: (A–C) north central Florida seedlings; (D–F) South Carolina seedlings from Eva Chandler; (G–I) Upper Michigan peninsula seedlings; (A, D, G) seedlings cultured on BM-1 Terrestrial Orchid Medium; (B, E, H) seedlings cultured on P723 Orchid Seed Sowing Medium; (C, F, I) seedlings cultured on half-strength MS. Scale bars = 1 cm.

Fig. 6.

Culture media effects on early seedling development of Calopogon tuberosus from different populations after culture for 8 weeks: (A–C) north central Florida seedlings; (D–F) South Carolina seedlings from Eva Chandler; (G–I) Upper Michigan peninsula seedlings; (A, D, G) seedlings cultured on BM-1 Terrestrial Orchid Medium; (B, E, H) seedlings cultured on P723 Orchid Seed Sowing Medium; (C, F, I) seedlings cultured on half-strength MS. Scale bars = 1 cm.

DISCUSSION

Based upon differences in seed germination, seedling development and, particularly, corm development among C. tuberosus populations, further evidence for ecotypic differentiation beyond morphological variation is provided. Goldman et al. (2004) reported that morphological variation in C. tuberosus correlating to geographic location was likely to be caused by different selection pressures and abiotic factors, but these selective pressures were not specifically explored with respect to ecotypic differentiation.

Seed viability and quality

Differences in seed germination responses are often attributed to seed viability and quality. Comparisons of orchid seed germination among populations of the same species have been reported, but C. tuberosus has not been examined. Symbiotic germination and mycorrhizal specificity among populations rather than ecotypic differentiation were examined in these studies (Zettler and McInnis, 1992; Zettler and Hofer, 1998; Sharma et al., 2003). However, differences in seed germination and viability among populations were described which might be accounted for by ecotypic differentiation.

Population size and inbreeding depression may influence low seed germination of several C. tuberosus populations as well as differences in seed viability. Lower germination percentages in small populations of Platanthera integrilabia, compared with larger populations, were attributed to lower seed viability (Zettler and McInnis, 1992). Similarly, Platanthera clavellata seed germination differences were attributed to inbreeding depression (Zettler and Hofer, 1998). Reduction in pollinator numbers at different sites may lead to seed viability differences in C. tuberosus as reported for Platanthera leucophaea and P. praeclara (Bowles et al., 2002; Sharma et al., 2003).

Another plausible explanation regarding differences in seed viability may be self-pollination. Calopogon tuberosus is a non-rewarding/out-crosser pollinated by Bombus, Xylocopa and Megachile bees through deception (van der Pijl and Dodson, 1966; Thien and Marcks, 1972; Dressler, 1981). Self-pollination in C. tuberosus may be common as Firmage and Cole (1988) reported in Maine populations. Self-pollination in Calypso bulbosa, and probably C. tuberosus, was mediated by bumble bees since a mechanism for autogamy does not exist (Alexandersson and Agren, 2000). While fruit set is generally not affected by self-pollination, reduced seed viability or embryo production can be reduced (Tremblay et al., 2005). Low seed viability and germinability in certain C. tuberosus populations may be caused by higher levels of self-pollination; however, further investigation is warranted.

Differences in viability may be explained by varying degrees of testa permeability or hardness that warrants investigation. van Waes and Debergh (1986b) reported various optimal pretreatment times from 45 min to 16 h in calcium hypochlorite in 31 species of terrestrial orchids, thus differences in C. tuberosus viability are not surprising. Differences in the testa structures among C. tuberosus populations are likely since dry seeds among populations appear different (pers. obs.). Seeds from South Carolina have an opaque testa with rounded ends. Seeds from Michigan are long, narrow, and have tapered ends. Both Michigan and South Carolina seeds appear to have thick testas. Seeds from north central Florida are small and transparent, while seeds from south Florida contain large embryos and are also transparent. Because northern populations had lower viabilities, longer pretreatment in calcium hypochlorite may be required to weaken the less permeable testas.

The correlation between TTC determined seed viability and the corresponding observed percentage germination is often variable and species specific (St-Arnaud et al., 1992; Shoushtari et al., 1994; van Waes and Debergh, 1986a). Tetrazolium testing can overestimate viability because this test does not detect inactive enzymes that may become active during germination (Lauzer et al., 1994). For this reason, fluorescein diacetate (FDA) is used with results often correlating with germination (Pritchard, 1985; Vendrame et al., 2007). Lower germination percentages compared with viability may reflect non-optimal temperatures with seeds from northern climates requiring cooler temperatures in vitro or stratification to germinate; these concerns are currently being addressed in separate experiments. In addition, seeds that do not germinate in vitro may have an intrinsic dormancy mechanism. Embryo damage during surface sterilization is also a likely scenario that may have reduced germination.

Photoperiod

As far as is known, no other published articles exist that compare photoperiodic effects on North American orchid seed germination spanning several populations of the same species. For non-orchid ecotypes, however, photoperiod is reported to be an important factor on germination (Singh, 1973; Seneca, 1974; Probert et al., 1985).

Due to latitudinal differences in location, populations experience different seasonal variations in photoperiod, temperature regimes and growing season. Calopogon tuberosus flowers in early June to mid-July in the north and mid-May to early June in the south (Luer, 1972). In Florida, seed capsules dehisce and seeds are disbursed in July when photoperiods are approx. 13–14 h. Short-day conditions promoted the highest germination percentage for both Florida populations. At both Florida locations, the shortest natural photoperiods do not approach 8 h, but approx. 10 h. Whether Florida seeds are somewhat light sensitive during germination remains unclear without also conducting in situ germination studies.

Development of protocorms in all three photoperiods for north central Florida was very rapid compared with south Florida protocorms. A large percentage of south Florida seeds germinated only to the imbibition stage by week 8, perhaps due to the longer growing season in south Florida. In north central Florida, lower daily surface temperatures in winter can drop below freezing point, while low daily winter temperatures in south Florida rarely drop below 5 °C. The warmer conditions in south Florida may aid in slower protocorm development, assuming that temperatures within the soil where protocorms reside are also different. After 16 weeks culture, south Florida seedlings were small and did not form corms, while north central Florida seedlings were larger and readily formed corms.

Although total germination was low in Michigan seeds, seedling development and corm formation was more rapid than those from the southern populations. Imbibition occurred after 1 week, and corm initiation began by week 6. Regardless of photoperiod, after 16 weeks culture the large seedling corm : shoot : root ratios generated in seedlings from the Michigan population suggest that a high percentage of carbohydrates are allocated to corms. Rapid seed germination, seedling development and corm formation in northern populations may be indicative of a photoperiod-insensitive seedling developmental sequence that ensures rapid corm development during short northern growing seasons and increases winter survival. Kane et al. (2000) similarly reported more rapid corm development in more northern ecotypes of the wetland non-orchid species Sagittaria latifolia.

Media screen

P723 proved to be an adequate medium for germinating Florida and Michigan seeds, but discrepancies between germination and viability may have been caused by using a non-optimal medium for other seed sources. Abundant literature exists on mineral nutrition of orchid seeds, and how media composition influences germination and development (Curtis, 1947; Spoerl and Curtis, 1948; Raghavan, 1964; van Waes and Debergh, 1986a; Kauth et al., 2006). However, site-specific differences in soil nutrient availability could explain differences in germination and development as found in Dactylorhiza incarnata by Dijk and Eck (1995). Seedlings from coastal areas grew faster in vitro and were more tolerant of exogenous ammonium and nitrate compared with seedlings from inland populations. Coastal populations inhabit calcareous areas where high nutrient levels are found due to the introduction of fertilizers and poor drainage (Dijk and Eck, 1995).

Calopogon tuberosus from Eva Chandler Heritage Preserve in South Carolina is found in proximity to the rare Parnassia grandiflora, indicating high calcium and magnesium content in granite outcroppings (Porcher and Rayner, 2001). Although South Carolina 3 seed germination was low, higher germination occurred on VW and KC, which contain higher concentrations of both calcium and magnesium. Soil analysis from each C. tuberosus population may provide insight into differences in soil nutrient availability, and ultimately seed germination. Soil analysis from each C. tuberosus population may provide insight into differences in soil nutrient availability, and ultimately seed germination.

Conclusions

This study provides insight into physiological and developmental aspects that are important aspects for ecotypic differentiation. Based on in vitro seed germination studies, ecotypic differentiation may be occurring within C. tuberosus, evident by rapid germination and subsequent seedling development, as well as immediate corm formation in northern populations. Rapid corm development in northern plants may be a consequence of the relatively shorter growing season experienced by these populations. Conversely, southern plants display greater shoot biomass allocation and a slower tendency to form corms. Ecotypic differentiation does not extend only to distant populations (i.e. Florida and Michigan), but also within close proximity (north central and south Florida populations approx. 400 km apart).

In vitro seed germination is only one technique that can be utilized to differentiate ecotypes. Combining in vitro results with in situ data may provide more understanding into ecotypic differentiation since conditions experienced in the field differ from those in vitro. Other techniques should be integrated along with in vitro techniques such as in situ germination, cytological examination and genetic analysis, to gain a more complete understanding. These topics are being examined in subsequent studies.

ACKNOWLEDGEMENTS

We thank the following for collecting seed: Larry Richardson (Wildlife Biologist; Florida Panther National Wildlife Refuge); Jim Fowler (South Carolina populations); Kip Knudson (Carney Fen population). We also thank Mary Bunch (South Carolina Heritage Preserve Program) for issuing collection permits. Brand names are provided as references; the authors do not solely recommend or endorse these products. We also thank the US Fish and Wildlife-Florida Panther National Wildlife Refuge for assisting with partial financial support.

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