asymbiotic germination of calopogon tuberosus

Asymbiotic Germination Response to Photoperiod and Nutritional Media in SixPopulations of Calopogon tuberosus var. tuberosus (Orchidaceae): Evidence for

Ecotypic Differentiation

PHILIP J. KAUTH1,*, MICHAEL E. KANE1, WAGNER A. VENDRAME3 and

CARRIE REINHARDT-ADAMS2

1Plant Restoration, Conservation and Propagation Biotechnology Program, 2Restoration and Plant EcologyProgram, Environmental Horticulture Department, University of Florida, PO Box 110675, Gainesville, FL 32611,

USA and 3Tropical Research and Education Center, University of Florida, 18905 SW 280th Street, Homestead,FL 33031-3314, USA

Received: 25 April 2008 Returned for revision: 8 July 2008 Accepted: 30 July 2008 Published electronically: 30 August 2008

† Background and Aims Ecotypic differentiation has been explored in numerous plant species, but has been largelyignored in the Orchidaceae. Applying a specific germination protocol for widespread seed sources may be unreliabledue to inherent physiological or genetic differences in localized populations. It is crucial to determine whether eco-typic differentiation exists for restoration and conservation programmes. Calopogon tuberosus var. tuberosus, awidespread terrestrial orchid of eastern North America, is a model species to explore ecotypic differences in germi-nation 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 asym-biotic in vitro seed germination and seedling development of C. tuberosus. Germination and early development wasmonitored for 8 weeks, while advanced development was monitored for an additional 8 weeks. In an additionalexperiment, 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 tetra-zolium test for embryo viability was performed.† Key Results Short days promoted the highest germination among Florida populations, but few differences amongphotoperiods in other seed sources existed. Different media had little effect on the germination of Michigan andFlorida populations, but germination of South Carolina seeds was higher on media with higher calcium and mag-nesium. Tetrazolium testing confirmed that South Carolina seeds exhibited low viability while viability washigher 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 compressedgrowing season in northern populations. Ecotypic differentiation may be occurring based on seed germinationand corm formation data.

Key words: Asymbiotic germination, corm development, Calopogon tuberosus, ecotypic differentiation, native orchid,orchid seed germination, seedling development.

INTRODUCTION

Ecotypic differentiation has recently been recognized as animportant issue in several plant sciences including conser-vation, restoration and population genetics (Hufford andMazer, 2003). Ecotypic differentiation enables species tosurvive diverse habitats and environmental conditionsacross its geographical range, but the specific functionsthey serve in ecosystems remain unclear (Seliskar et al.,2002). For this reason, using local plant material forrestoration purposes or reintroductions may be necessaryto maintain ecosystem health (Linhart, 1995). Introducingpoorly adapted ecotypes into unsuitable habitats may leadto reduced plant population fitness (Hufford and Mazer,2003; McKay et al., 2005).

Common garden studies are often utilized to detect localadaptation (Sanders and McGraw, 2005), but obtainingpermits to collect and transplant protected, rare, threatened

or endangered species is often difficult. Alternatively, studyingthe ecology and physiology of seed germination and seedlingdevelopment from widespread populations may provideinsight into ecotypic differentiation (Singh, 1973). Currently,little information exists on seed germination amonggeographically distinct orchid populations as well as orchidecotypic differentiation. Studies of orchid seed germinationecology are needed to support reintroduction programmesthat typically use seed germination as a propagation tool.

Calopogon tuberosus var. tuberosus is a terrestrial orchidof eastern North America from Florida to Canada and westto Texas, and occupies habitats including alkaline prairies,pine flatwoods, mesic roadsides, fens and sphagnumbogs (Luer, 1972). Goldman et al. (2004) defined threeC. tuberosus geographic clines. Northern plants in glaciatedareas are differentiated from southern plants by labellumapex shape, reduced flower size and reduced leaf andinflorescence height. South-west populations west of theMississippi Embayment differ from those in the south-east

* For correspondence. E-mail [email protected]

# The Author 2008. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.

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Annals of Botany 102: 783–793, 2008

doi:10.1093/aob/mcn163, available online at www.aob.oxfordjournals.org

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by larger flowers and inflorescence heights. Morphologicalvariation may be caused by environmental factors orcross-pollination avoidance with other Calopogon species(Goldman et al., 2004).

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

Differences in germination and seedling developmentmight be the result of local adaptation to specific environ-mental conditions. Attributing ecotypic differentiation togermination differences is difficult since seed source israrely reported in C. tuberosus seed germination studies,and basing recommendations for seed germination ofC. tuberosus on one population is tenuous. Evaluation ofin vitro seed germination from diverse populations mayclarify whether ecotypic differentiation occurs amongC. tuberosus populations. In this paper, the effects of photo-period and culture media on asymbiotic seed germinationand seedling development are compared among widespreadpopulations of C. tuberosus.

MATERIALS AND METHODS

Seed source

Intact seed capsules (slightly yellow in colour) of Calopogontuberosus (L.) Britton, Sterns & Poggenb. var. tuberosus werecollected before dehiscence approx. 2 months after peak flow-ering throughout summer 2006. Capsules were collected fromthe 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’ nearEva Chandler Heritage Preserve (Greenville County, SouthCarolina, USA) and Carney Fen (Menominee County,Michigan, USA; Fig. 1). The populations from site ‘C’ andEva Chandler occupy cataract bogs, which form whenstreams flow over granite out-cropping resulting insphagnum-filled depressions (Porcher and Rayner, 2001);for further site-specific information see Table 1.Non-dehisced capsules were collected to reduce the potentialfor surface contamination of individual seeds. Upon collect-ing and receiving capsules, they were stored at 23 8C oversilica desiccant for 2 weeks. After 2 weeks, seeds wereremoved from the capsules and stored over silica desiccantat –11 8C until use.

Seed viability test

A seed viability test (Lakon, 1949) was performed on allpopulations by staining embryos with 2,3,5-triphenyl tetra-zolium chloride (TTC). Seeds were scarified in an aqueous

5 % CaOCl2 solution for 0 min, 30 min, 1 h, 2 h or 3 h. Tworeplications of approx. 100 seeds each were used per treat-ment. After scarification, seeds were rinsed twice indistilled-deionized (dd) water and suspended in sterilewater for 24 h in darkness at 23+ 2 8C. Water was replacedwith TTC and seeds were soaked for 24 h at 30 8C in dark-ness. After the TTC soak, embryos were scored as viable ifany degree of red staining was observed.

Media and seed preparation

Media were prepared in 1000-ml batches, and the pH wasadjusted to 5.7 with 0.1 N KOH prior to autoclaving for40 min at 117.7 kPa and 121 8C. Aliquots (40 mL) ofsterile medium were dispensed into square 100 � 15 mmPetri plates with a 36-cell bottom (IntegridTM Petri Dish;Becton Dickinson and Co., Franklin Lakes, NJ, USA).Mature seeds were surface sterilized in sterile scintillationvials for 3 min in a solution of 5 mL absolute ethanol,5 mL 6 % NaOCl and 90 mL sterile dd water. Seeds wererinsed twice with sterile dd water after surface sterilization.Solutions were removed from the vials with sterile Pasteurpipettes. Seeds were then placed on the surface of the ger-mination media with a sterile inoculating loop. The interior16 cells of the Petri plates were used for subreplications toavoid uneven media drying at the edges. Petri plates weresealed with one layer of Nescofilm (Karlan ResearchProducts, Santa Rosa, CA, USA). Seed germination andseedling development (Table 2) were monitored weeklyfor 8 weeks according to the six developmental stagesdescribed by Kauth et al. (2006).

Photoperiod effects on asymbiotic germination and earlyseedling development

A 6 � 3 factorial design was used with six seed sourcesand three photoperiods including a short day (SD ¼ 8/16 hL/D), neutral day (ND ¼ 12/12 h L/D), long day (LD ¼ 16/8 h L/D). PhytoTechnology Orchid Seed Sowing Medium(#P723; PhytoTechnology Laboratories, Shawnee Mission,KS, USA) was used based on previous success withC. tuberosus seed germination and development (Kauthet al., 2006). Ten replicate Petri plates with five randomlyselected subreplications (48.5+ 17.9 seeds) were used perseed source and photoperiod treatment. Culture vesselswere placed under cool-white fluorescent lights (F96712,General Electric) at an average of 33.3+ 7 (12/12 photo-period), 31.6+ 5 (8/16 photoperiod) and 31.6+ 6 (16/8photoperiod) mmol m22 s21, and incubated at 25+ 0.4 8C.

Photoperiod effects on advanced in vitro seedling development

After 8 weeks, seedlings in the photoperiod experimentwere transferred from Petri plates to PhytoTech cultureboxes (95 � 95 � 100 mm) containing 100 mL P723medium; seedlings were maintained in correspondingphotoperiods. Ten seedlings were transferred to eachculture box. After an additional 8 weeks and 16 weekstotal, five culture vessels per treatment (50 total seedlings)were randomly selected. Seedling percentage biomass

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allocation was determined by dividing corm, root and shootweights by the total seedling weight. Culture conditionswere 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 mediacommercially prepared by PhytoTechnology Laboratories

were used: BM-1 Terrestrial Orchid Medium (BM-1;#B141; van Waes and Debergh, 1986a), Knudson C OrchidMedium (KC; #K400; Knudson, 1946), MalmgrenModified 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 SkoogMedium in half-strength (MS; #M5524; Murashige andSkoog, 1962) was commercially prepared by Sigma-Aldrich

FI G. 1. Habitat and location of Calopogon tuberosus populations used in the present study: (A) Calopogon tuberosus flower; (B) fen habitat on theupper Michigan peninsula; (C) cataract bog in South Carolina; (D) roadside habitat in north central Florida; (E) prairie habitat in south Florida;

(F) population locations.

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(St Louis, MO, USA). BM-1 and VW were further sup-plemented with 0.1 % charcoal, KC was furthersupplemented with 0.1 % charcoal and 0.8 % TCw agar(PhytoTechnology Laboratories), MM was further sup-plemented with 0.8 % TCw agar, and 0.5MS was further sup-plemented with 0.1 % charcoal, 0.8 % TC agar, and organicsfound in P723. All media contained 2 % sucrose. Five repli-cate Petri plates with three randomly selected subreplications(62.6+15.2 seeds) were used per treatment. Germinationand development were monitored biweekly for 8 weeks.Culture vessels were placed under ND conditions, cool-whitefluorescent lights at 33.3+7 mmol m22 s21, and 25+0.4 8C.

Statistical nalysis

Germination percentages were calculated by dividing thenumber of germinated seeds by the total number of seedswith an embryo in each subreplication. The percentage ofprotocorms and seedlings in a developmental stage was cal-culated by dividing the number of seeds in a stage by thetotal number of seeds with an embryo. Germinationcounts were arcsine transformed to normalize variation.Germination and seedling development data were analysedusing general linear model procedures and least squaremeans at a ¼ 0.05 in SAS v. 8.02.

RESULTS

Seed viability

For all populations except south Florida (no difference inpretreatment time), the highest percentage of viable

embryos was observed after 3 h of calcium hypochloritepretreatment. Maximum embryo viability for each popu-lation was as follows: 85.4 % south Florida; 66.7 % northcentral Florida; 25.0 % South Carolina 1; 38.1 % SouthCarolina 2; 42.1 % South Carolina 3; and 50.3 % Michigan.

Photoperiod effects on germination and early development

Total seed germination percentage (Fig. 2) was highestunder SD conditions for north central Florida (60.2 %)and south Florida (48.5 %) populations. There was nodifference in germination among the three photoperiodsfor Michigan seeds (11.6 %, 12.5 % and 9.9 %).Germination percentages in all South Carolina populationsdid not exceed 4 %.

Seeds from Michigan germinated and developed morequickly compared with other populations, with imbibitionoccurring 1 week after inoculation. By week 8 .95 % ofthe germinated protocorms in all photoperiods developedto stage 6. Protocorm development was similar amongphotoperiods (Fig. 2A).

South Carolina protocorm development was unpredict-able. South Carolina 1 (Fig. 2B) and South Carolina 2(Fig. 2C) protocorms developed slowly with ,1 % devel-oping to leaf-bearing stages. Only South Carolina 1 proto-corms under ND developed to stage 6 while SouthCarolina 2 protocorms under both ND and LD conditionsdeveloped to stages 5 and 6. Development of SouthCarolina 3 (Fig. 2D) protocorms was more advanced thanother South Carolina populations.

Seeds from north central Florida germinated quicklyand corms formed after week 8. Greater than 16 % of theprotocorms in each photoperiod developed to an advancedleaf-bearing stage (stage 6) by week 8 (Fig. 2E).Although germination of south Florida seeds was highestunder SD conditions, the majority of seeds did notdevelop past the imbibition stage by week 8 (Fig. 2F).Fewer than 5 % of the south Florida seeds under SD con-ditions developed past imbibition after 8 weeks culture.Approximately 10 % of the seeds under both ND and LDconditions developed past imbibition. A low percentageof south Florida seedlings in all photoperiods developedto advanced leaf-bearing stages (stages 5 and 6).

TABLE 1. Location, habitat, and basic environmental conditions of Calopogon tuberosus seed sources used in the presentstudy

Population location and(designation) Co-ordinates Habitat Soil designation and (composition) Long day Short day

Panther Refuge (south Florida) 2681000600N, 8182105100W Alkalineprairie

Ochopee fine sandy loam (fine sandyloam)

13 h 47 min 10 h 30 min

Goethe State Forest (northcentral Florida)

2980901800N, 8283701200W Mesicroadside

Smyrna fine sand (fine sand) 14 h 02 min 10 h 16 min

Ashmore Preserve (SouthCarolina 1)

3580501300N, 8283404600W Lake bog Congaree (sphagnum/fine sandy loam) 14 h 30 min 9 h 49 min

Site ‘C’ (South Carolina 2) 3580500200N, 8283505100W Cataract bog Ashe-Cleveland association (sphagnum/sandy loam)

14 h 30 min 9 h 49 min

Chandler Preserve (SouthCarolina 3)

3580500300N, 8283602700W Cataract bog Ashe-Cleveland association (sphagnum/sandy loam)

14 h 30 min 9 h 49 min

Carney fen (Michigan) 4583404700N, 8783903800W Fen Lupton-Cathro association (sphagnum/muck)

15 h 42 min 8 h 46 min

TABLE 2. Six stages of orchid seed development (fromKauth et al., 2006)

Stage Description

1 Imbibed seed, swollen and greening still covered or partiallycovered by testa

2 Enlarged seed without testa3 Protocorm with pointed shoot apex and rhizoids4 Protocorm with emerging leaf and developing rhizoids5 Seedling with one elongated leaf and one developing root6 Seedling with evident roots and two or more leaves


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Photoperiod effects on advanced seedling development

After 16 weeks culture, Michigan seedlings began tosenesce while South Carolina and Florida seedlings contin-ued to grow. Corm formation was limited in south Floridaseedlings, while Michigan, South Carolina and northcentral Florida seedlings all formed corms (Fig. 3).Biomass allocation was similar among photoperiodswithin each seed source (Fig. 4). Maximum dry weight allo-cation to corms was observed in Michigan seedlings(Fig. 4A). Although north central Florida seedlingsformed large corms, the percentage dry biomass allocationwas more evenly distributed among shoots, corms and rootsthan other populations (Fig. 4C). The greatest seedlingshoot biomass allocation was observed in South Carolina3 and south Florida populations (Fig. 4B, D).

Media effects on germination and early development

Michigan seeds germinated and protocorms developedquickly on all media, but the highest germination percen-tages occurred on P723 (34.1 %). With the exception ofKC and VW, over 90 % of the protocorms on all othermedia developed to stage 6 (Fig. 5A).

Seed germination for South Carolina 1 was highest onVW, but germination was only 4.9 % (Fig. 5B). No clear

differences in germination were observed in SouthCarolina 2 (Fig. 5C), but germination on P723 wassignificantly lower than all other media. Germination onKC (39.7 %) and MS (30.4 %) was highest for SouthCarolina 3 seeds, while lowest germination occurred onP723 (Fig. 5D).

In both Florida populations, few differences in total ger-mination existed among media; however, subsequent devel-opment differed greatly. For north central Florida, highernumbers of stage 4, 5 and 6 seedlings were observed onBM-1, MS, P723 and VW (Fig. 5E). The highest germina-tion percentage of north central Florida seeds was observedon MM, but the majority of seeds remained in stage 1 after8 weeks. The highest percentage of stage 4, 5 and 6 proto-corms was observed on BM-1, P723 and VW for southFlorida (Fig. 5F). Germination percentages were high onKC and MM for south Florida seeds, but no stage 6 seed-lings developed within 8 weeks and considerably fewerstage 5 seedlings developed compared with all other media.

Media effects on corm development

Corm development on BM-1, MS and P723 was superiorin all populations (Fig. 6). Seedling development ofMichigan, South Carolina 3, and north central Floridaseedlings was superior to other populations (Fig. 6).

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 10.31Calcium 0.73 0.75 1.93 2.12 1.50Chlorine 0.0021 1.50 3.35 3.1Magnesium 0.81 0.83 0.62 1.01 1.01 0.75Nitrate 9.85 5.19 10.49 19.70Potassium 0.55 2.20 5.62 7.03 5.19 10.89Phosphate 1.03 2.20 0.31 3.77 1.84 0.63Sulfate 0.92 1.10 0.71 8.71 8.69 0.86Sodium 0.20 0.20 0.10 0.20 0.10

Micronutrients (mM)Boron 161.7 26.7 50Cobalt 0.105 0.026 0.053Copper 0.10 0.025 0.5Iron 100 100.2 50 100 90 50Iodine 1.25 2.50Manganese 10 147.9 25 30 30 50Molybdenum 1.03 0.26 0.52Zinc 34.8 9.22 14.95

Organics (mg L21)Biotin 0.05 0.05Casein hydrolysate 400 500Folic acid 0.5 0.5L-Glutamine 100Glycine 2.0 2.0myo-Inositol 100 100 100 100Nicotinic acid 5.0 1.0 1.0Peptone 2000 2000Pyridoxine HCl 0.5 1.0 1.0Thiamine HCl 0.5 10 10Total mineral salt concentration (mM) 4.35 6.98 24.72 35.54 46.72 48.01Total inorganic N (mM) n/a n/a 15.00 12.76 24.31 30.01


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However, development of Michigan, South Carolina 3, andnorth central Florida seedlings differed markedly. Cormformation was more pronounced in seedlings from northernlatitudes. Thus by week 8, no corm formation was observedin Florida seedlings while early and advanced cormformation was observed in South Carolina and Michiganseedlings, respectively.

DISCUSSION

Based upon differences in seed germination, seedlingdevelopment and, particularly, corm development amongC. tuberosus populations, further evidence for ecotypicdifferentiation beyond morphological variation is provided.Goldman et al. (2004) reported that morphologicalvariation in C. tuberosus correlating to geographic locationwas likely to be caused by different selection pressures and

abiotic factors, but these selective pressures were notspecifically explored with respect to ecotypicdifferentiation.

Seed viability and quality

Differences in seed germination responses are oftenattributed to seed viability and quality. Comparisons oforchid seed germination among populations of the samespecies have been reported, but C. tuberosus has not beenexamined. Symbiotic germination and mycorrhizal speci-ficity among populations rather than ecotypic differentiationwere examined in these studies (Zettler and McInnis, 1992;Zettler and Hofer, 1998; Sharma et al., 2003). However,differences in seed germination and viability among popu-lations were described which might be accounted for byecotypic differentiation.

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FI G. 2. Photoperiod effects on seed germination and subsequent development of Calopogon tuberosus from different populations after culture on P723medium 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 (a ¼ 0.05). See Table 2 for stages of germination and development.


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South Florida

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FI G. 3. Effects of photoperiod on in vitro seedling development of Calopogon tuberosus from different populations after 16 weeks total culture (8 weeksin 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 seed-

lings; (C, G, K) South Carolina seedlings from Eva Chandler; (D, H, L) upper Michigan peninsula seedlings. Scale bars ¼ 1 cm.

Photoperiod Photoperiod

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FI G. 4. Percentage dry weight biomass allocation of Calopogon tuberosus seedlings after 16 weeks in vitro culture: (A) upper Michigan peninsulapopulation; (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.


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Population size and inbreeding depression may influencelow seed germination of several C. tuberosus populationsas well as differences in seed viability. Lower germina-tion percentages in small populations of Platantheraintegrilabia, compared with larger populations, wereattributed to lower seed viability (Zettler and McInnis,1992). Similarly, Platanthera clavellata seed germinationdifferences were attributed to inbreeding depression(Zettler and Hofer, 1998). Reduction in pollinatornumbers at different sites may lead to seed viabilitydifferences in C. tuberosus as reported for Platanthera

leucophaea and P. praeclara (Bowles et al., 2002;Sharma et al., 2003).

Another plausible explanation regarding differences inseed viability may be self-pollination. Calopogon tuberosusis a non-rewarding/out-crosser pollinated by Bombus,Xylocopa and Megachile bees through deception (van derPijl and Dodson, 1966; Thien and Marcks, 1972;Dressler, 1981). Self-pollination in C. tuberosus may becommon as Firmage and Cole (1988) reported in Mainepopulations. Self-pollination in Calypso bulbosa, and prob-ably C. tuberosus, was mediated by bumble bees since a

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FI G. 5. Effects of culture media on seed germination and early development of Calopogon tuberosus from different populations after 8 weeks cultureunder a 12/12 h L/D photoperiod: (A) upper Michigan peninsula population; (B) South Carolina population from Ashmore; (C) South Carolina populationfrom site ‘C’; (D) South Carolina population from Eva Chandler; (E) north central Florida population; (F) south Florida population. Histobars within eachseed source with the same letter are not significantly different (a ¼ 0.05). For media abbreviations and formulae see Table 3. See Table 2 for stages of

germination and development.


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mechanism for autogamy does not exist (Alexanderssonand Agren, 2000). While fruit set is generally not affectedby self-pollination, reduced seed viability or embryo pro-duction can be reduced (Tremblay et al., 2005). Low seedviability and germinability in certain C. tuberosus popu-lations may be caused by higher levels of self-pollination;however, further investigation is warranted.

Differences in viability may be explained by varyingdegrees of testa permeability or hardness that warrantsinvestigation. van Waes and Debergh (1986b) reportedvarious optimal pretreatment times from 45 min to 16 h incalcium hypochlorite in 31 species of terrestrial orchids,thus differences in C. tuberosus viability are not surprising.Differences in the testa structures among C. tuberosuspopulations are likely since dry seeds among populationsappear different (pers. obs.). Seeds from South Carolinahave an opaque testa with rounded ends. Seeds fromMichigan are long, narrow, and have tapered ends. BothMichigan and South Carolina seeds appear to have thicktestas. Seeds from north central Florida are small andtransparent, while seeds from south Florida contain largeembryos and are also transparent. Because northernpopulations had lower viabilities, longer pretreatment incalcium hypochlorite may be required to weaken the lesspermeable testas.

The correlation between TTC determined seed viabilityand the corresponding observed percentage germinationis often variable and species specific (St-Arnaud et al.,

1992; Shoushtari et al., 1994; van Waes and Debergh,1986a). Tetrazolium testing can overestimate viabilitybecause this test does not detect inactive enzymes thatmay become active during germination (Lauzer et al.,1994). For this reason, fluorescein diacetate (FDA) is usedwith results often correlating with germination (Pritchard,1985; Vendrame et al., 2007). Lower germination percen-tages compared with viability may reflect non-optimaltemperatures with seeds from northern climates requiringcooler temperatures in vitro or stratification to germinate;these concerns are currently being addressed in separateexperiments. In addition, seeds that do not germinatein vitro may have an intrinsic dormancy mechanism.Embryo damage during surface sterilization is also alikely scenario that may have reduced germination.

Photoperiod

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

Due to latitudinal differences in location, populationsexperience different seasonal variations in photoperiod,temperature regimes and growing season. Calopogontuberosus flowers in early June to mid-July in the north

0·5MSP723BM-1

NorthCentralFlorida

SouthCarolina

Michigan

FI G. 6. Culture media effects on early seedling development of Calopogon tuberosus from different populations after culture for 8 weeks: (A–C) northcentral Florida seedlings; (D–F) South Carolina seedlings from Eva Chandler; (G–I) Upper Michigan peninsula seedlings; (A, D, G) seedlings culturedon 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 ¼ 1 cm.


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and mid-May to early June in the south (Luer, 1972).In Florida, seed capsules dehisce and seeds are disbursedin July when photoperiods are approx. 13–14 h.Short-day conditions promoted the highest germination per-centage for both Florida populations. At both Floridalocations, the shortest natural photoperiods do not approach8 h, but approx. 10 h. Whether Florida seeds are somewhatlight sensitive during germination remains unclear withoutalso conducting in situ germination studies.

Development of protocorms in all three photoperiods fornorth central Florida was very rapid compared with southFlorida protocorms. A large percentage of south Floridaseeds germinated only to the imbibition stage by week 8,perhaps due to the longer growing season in southFlorida. In north central Florida, lower daily surface temp-eratures in winter can drop below freezing point, while lowdaily winter temperatures in south Florida rarely drop below5 8C. The warmer conditions in south Florida may aid inslower protocorm development, assuming that temperatureswithin the soil where protocorms reside are also different.After 16 weeks culture, south Florida seedlings weresmall and did not form corms, while north central Floridaseedlings were larger and readily formed corms.

Although total germination was low in Michigan seeds,seedling development and corm formation was more rapidthan those from the southern populations. Imbibitionoccurred after 1 week, and corm initiation began by week6. Regardless of photoperiod, after 16 weeks culture thelarge seedling corm : shoot : root ratios generated in seed-lings from the Michigan population suggest that a highpercentage of carbohydrates are allocated to corms. Rapidseed germination, seedling development and corm for-mation in northern populations may be indicative of aphotoperiod-insensitive seedling developmental sequencethat ensures rapid corm development during short northerngrowing seasons and increases winter survival. Kane et al.(2000) similarly reported more rapid corm development inmore northern ecotypes of the wetland non-orchid speciesSagittaria latifolia.

Media screen

P723 proved to be an adequate medium for germinatingFlorida and Michigan seeds, but discrepancies betweengermination and viability may have been caused by usinga non-optimal medium for other seed sources. Abundantliterature exists on mineral nutrition of orchid seeds, andhow media composition influences germination anddevelopment (Curtis, 1947; Spoerl and Curtis, 1948;Raghavan, 1964; van Waes and Debergh, 1986a; Kauthet al., 2006). However, site-specific differences in soilnutrient availability could explain differences in germina-tion and development as found in Dactylorhiza incarnataby Dijk and Eck (1995). Seedlings from coastal areasgrew faster in vitro and were more tolerant of exogenousammonium and nitrate compared with seedlings frominland populations. Coastal populations inhabit calcareousareas where high nutrient levels are found due to theintroduction of fertilizers and poor drainage (Dijk andEck, 1995).

Calopogon tuberosus from Eva Chandler HeritagePreserve in South Carolina is found in proximity to therare Parnassia grandiflora, indicating high calcium andmagnesium content in granite outcroppings (Porcher andRayner, 2001). Although South Carolina 3 seed germina-tion was low, higher germination occurred on VW andKC, which contain higher concentrations of both calciumand magnesium. Soil analysis from each C. tuberosus popu-lation may provide insight into differences in soil nutrientavailability, and ultimately seed germination. Soil analysisfrom each C. tuberosus population may provide insightinto differences in soil nutrient availability, and ultimatelyseed germination.

Conclusions

This study provides insight into physiological anddevelopmental aspects that are important aspects forecotypic differentiation. Based on in vitro seed germinationstudies, ecotypic differentiation may be occurring withinC. tuberosus, evident by rapid germination and subsequentseedling development, as well as immediate corm for-mation in northern populations. Rapid corm developmentin northern plants may be a consequence of the relativelyshorter growing season experienced by these populations.Conversely, southern plants display greater shoot biomassallocation and a slower tendency to form corms. Ecotypicdifferentiation 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 canbe utilized to differentiate ecotypes. Combining in vitroresults with in situ data may provide more understandinginto ecotypic differentiation since conditions experiencedin the field differ from those in vitro. Other techniquesshould be integrated along with in vitro techniques suchas in situ germination, cytological examination andgenetic analysis, to gain a more complete understanding.These topics are being examined in subsequent studies.

ACKNOWLEDGEMENTS

We thank the following for collecting seed: LarryRichardson (Wildlife Biologist; Florida Panther NationalWildlife Refuge); Jim Fowler (South Carolina populations);Kip Knudson (Carney Fen population). We also thank MaryBunch (South Carolina Heritage Preserve Program) forissuing collection permits. Brand names are provided asreferences; the authors do not solely recommend orendorse these products. We also thank the US Fish andWildlife-Florida Panther National Wildlife Refuge forassisting with partial financial support.

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