Journal of the Torrey Botanical Society 136(4), 2009, pp. 433–444

In vitro ecology of tuberosus var. tuberosus () seedlings from distant populations: implications for assessing ecotypic differentiation1 Philip J. Kauth2 and Michael E. Kane Restoration, Conservation, and Propagation Biotechnology Program, Environmental Horticulture Department, University of , Gainesville, FL 3261

KAUTH,P.J.AND M. E. KANE (Plant Restoration, Conservation, and Propagation Biotechnology Program, Environmental Horticulture Department, University of Florida, Gainesville, FL 32611). In vitro ecology among four populations of Calopogon tuberosus var. tuberosus (Orchidaceae): implications for ecotypic differentiation. J. Torrey Bot. Soc. 136: 000–000. 2009.—In vitro culture techniques can be used to study the unique growth habits of as well as the ecological factors that influence seedling growth and development (i.e., in vitro ecology) such as adaptation to local environmental conditions. The in vitro seedling ecology of Calopogon tuberosus var. tuberosus from Michigan, South Carolina, and Florida was studied with emphasis on timing of corm formation and biomass allocation. In vitro seedling growth and development were monitored for 20 weeks. Corm formation was most rapid in Michigan seedlings, but was progressively delayed in southern populations. Similarly, biomass allocation to corms was highest in Michigan seedlings while south Florida seedlings exhibited the lowest corm biomass allocation. Shoot senescence in vitro also began earlier in more northern populations. The rapid corm formation and biomass allocation in seedlings from more northern populations represents an adaptive response to a shorter growing season. The relative differences in corm formation, biomass allocation, and shoot senescence in C. tuberosus seedlings suggest that in vitro common garden studies are useful to assess the degree of ecotypic differentiation among populations for a wide range of ecological factors. Additionally, these in vitro techniques can be transferred to numerous species worldwide. Key words: biomass allocation, common garden study, corm, ecotype, orchid.

Introduction. Widely distributed plant spe- stability since non-locally adapted ecotypes cies have evolved the ability to survive broad can reduce plant population fitness (Linhart environmental conditions leading to local and Grant 1996, Hufford and Mazer 2003, adaptation to biotic and abiotic conditions McKay et al. 2005). (Linhart 1995, Joshi et al. 2001, Sanders and Local adaptation has been studied in McGraw 2005). Local adaptation in plants numerous species through common garden was first examined using common garden and reciprocal transplant experiments (Nuis- studies by Turesson (1922), who first used mer and Gandon 2008). Common garden the term ecotype, and by Clausen et al. (1941) studies test local adaptation and fitness of using reciprocal transplant studies. The im- individuals from local or distant habitats in a portance of using appropriate ecotypes for common environment. Common garden stud- conservation and restoration studies has been ies may more efficiently test the genetic recently highlighted. Using locally adapted contribution to fitness while minimizing envi- plant material for restoration purposes may be ronmental impacts on fitness. Transplant necessary to maintain ecosystem function and studies may better estimate environmental variation since individuals are transplanted to habitats with environmental conditions not 1 We thank Larry Richardson (Wildlife Biologist; experienced in the natural habitat (Nuismer Florida Panther National Wildlife Refuge), Jim Fowler (South Carolina population), and Kip and Gandon, 2008). Local adaptation can be Knudson (Michigan population) for collecting studied by examining performance of ecotypes seeds. We also thank Mary Bunch (South Carolina under different photoperiods (Howe et al. Heritage Preserve Program). We also thank the U.S. 1995, Kurepin et al. 2007), temperatures Fish and Wildlife-Florida Panther National Wildlife Refuge for assisting with partial financial support. (Seneca 1972, Probert et al. 1985), and soil Brand names are provided as references only and we regimes (Grzes´ 2007, Sambatti and Rice 2007). do not solely recommend these products. Differences in biomass allocation have also 2 Author for correspondence. E-mail: pkauth@ been proposed as an important aspect of ufl.edu Received for publication February 2, 2009, and in ecotypic differentiation. Northern ecotypes of revised form August 27, 2009. Spartina alterniflora allocated more biomass

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Journal of the Torrey Botanical Society tbot-136-04-02.3d 30/11/09 14:03:40 433 Cust # 09-RA-013R1 434 JOURNAL OF THE TORREY BOTANICAL SOCIETY [VOL.136 to underground organs including roots and in vitro ecology studies can be used to rhizomes (Gallagher 1983, Gallagher and correlate environmental and genetic variables Howarth 1987, Gross et al. 1991). Greater that affect plant growth and development in biomass allocation to underground organs in vitro with ecological factors affecting growth northern ecotypes of several species was due to and development in situ. In vitro ecology a shorter growing season (Potvin 1986, Sa- could also be used to assess ecotypic differen- wada et al. 1994, Kane et al. 2000, Liancourt tiation for habitat restoration and plant and Tielbo¨rger 2009) and a higher allocation reintroduction programs by conducting in of carbohydrate reserves to overwintering vitro common garden studies under controlled structures (Mooney and Billings 1960). Bio- environmental conditions. Since this use of in mass allocation has also been correlated with vitro ecology is a new area of research its various reproductive strategies in ecotypes. validity must be verified. Ecotypes found in fields or areas of younger Calopogon tuberosus var. tuberosus (L.) succession allocated more biomass to repro- Britton, Sterns, & Poggenberg is a terrestrial ductive organs than those in wooded habitats orchid native to eastern , and that allocated more biomass to vegetative occupies diverse habitats such as wet prairies, structures (Abrahamson 1975, 1979). Marsh pine flatwoods, roadsides, fens, and sphagnum plants that occupied areas of greater distur- bogs. Based on morphological variation, bance allocated more biomass and carbohy- Goldman et al. (2004) defined three specific drate reserves to underground storage organs geographic areas for C. tuberosus: northern (Sun et al. 2001, Pen˜as-Fronteras et al. 2009). plants in glaciated areas, southwest plants west Common garden and transplant studies can of the Mississippi Embayment, and southeast be performed in greenhouses, growth cham- plants east of the Mississippi River and south bers, natural habitats, and outdoor plots of the glaciated zone. However, Goldman et (Gallagher et al. 1988, Howe et al. 1995, al. (2004) did not classify C. tuberosus Majerowicz et al. 2000, Suzuki 2008), but ecotypes, but stated that variation in C. obtaining permits to collect and transplant tuberosus could be caused by environmental protected, rare, threatened, or endangered conditions. Further ecotypic differentiation species, as many orchids are, is difficult. Seeds has not been previously explored in C. can be used to produce mature plants for tuberosus. Additionally, little information ex- common garden and transplant studies. While ists on ecotypic differentiation of orchids. this may be an effective method for quick- Although morphological and genetic variation growing species, orchids often require four or exists in C. tuberosus, all plants throughout its more years to flower from initial seed germi- range form corms. Differences in biomass nation (Stoutamire 1964). Additionally, in situ allocation among C. tuberosus populations orchid seed germination is difficult and time have been previously reported (Kauth et al. consuming since germination is often low 2008). However, a detailed timecourse com- (Brundrett et al. 2003, Zettler et al. 2005, Diez parison for C. tuberosus seedling development 2007). Alternatively, in vitro techniques can be has not been reported, and little information used to study environmental requirements for exists regarding the influence of storage organ orchid seed germination (Kauth et al. 2008) as biomass allocation on ecotypic differentiation. well as seedling growth and development (Dijk Evaluation of in vitro seedling development and Eck 1995). from several Calopogon tuberosus populations Many in vitro culture techniques can be from diverse geographic sources might clarify grouped under the discipline of in vitro the extent of ecotypic differentiation across its ecology. In vitro ecology has been previously range. In this study, the in vitro ecology of C. defined to include environmental and exoge- tuberosus seedlings was studied in relation to nous factors (i.e., temperature, light, gas corm formation, biomass allocation, and phase, culture media) that affect in vitro geographic source. Additionally, our goal is growth and development (Hughes 1981, Wil- to confirm the effectiveness of using an in vitro liams 2007). Here, we further define in vitro common garden study to aid in differentiating ecology to include the evaluation and use of in C. tuberosus ecotypes. vitro culture techniques to identify, propagate, evaluate, and select plant genotypes and Materials and Methods. SEED COLLECTION. ecotypes for ecological purposes. Specifically, Seeds were collected throughout summer 2007

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c from the following locations (Table 1): upper peninsular Michigan (Menominee County, Michigan), Blue Ridge Escarpment (Green-

(mm) ville County, South Carolina), north central Florida (Levy County, Florida), and Florida

Total precipitation Panther National Wildlife Refuge (Collier County, Florida). Seed capsules from at least C)

u three parent plants in each population were ( b collected before complete dehiscence and stored at 23uC over silica gel for two weeks. Seeds were then removed from capsules, pooled by geographic source, and stored in complete darkness at 211uC until used. 12.8 (Dec.) 4.9 740 C).

u SEED AND GERMINATION MEDIUM PREPARA- 2 TION. Seeds were surface disinfected in sterile scintillation vials for 3 minutes in a solution of 5 ml absolute ethanol, 5 ml 6% NaOCl, and 90 ml sterile distilled-deionized (dd) water. Seeds were rinsed with sterile dd water after

(hrs) Average monthly air temperature surface sterilization, and solutions were re- seed sources used in the present study. b moved with sterile Pasteur pipettes. Seeds were transferred with a sterile inocu- lating loop to BM-1 Terrestrial Orchid Medi- tuberosus Day length Max Min Max Min Mean

C) and first fall frost (below 0 um (PhytoTechnology Laboratories, Shawnee u var.

d Mission, KS, USA) in 100 3 15 mm Petri , c, plates (Fisher Scientific, Pittsburgh, PA, USA). BM-1 medium was selected based on season Growing prior Calopogon tuberosus germination and seedling development performance (Kauth et (m)

a al. 2008b). The medium was supplemented with 1% activated charcoal. Medium pH was

Calopogon tuberosus adjusted to 5.7 with 0.1 N KOH prior to autoclaving for 40 minutes at 117.7 kPa and 121uC. Ten replicate Petri plates with 30 ml medium each were used for each seed source with approximately 100 seeds per plate. Cul- tures were placed in an environmental growth chamber (#I-35LL; Percival Scientific, Perry, N Wet prairie 4 365 13.8 10.5 32.1 (Jun.) 11.9 (Jan.) 22.1 1450 N Cataract bog 500 210 14.5 9.8 34.2 (Jun.) 0.3 (Jan.) 16.5 800 N Northern fen 240 125 15.7 8.8 24.4 (Jul.) W W W

N Mesic roadside 12 270 14.1 10.3 33.4 (Jul.)IA, 7.8 (Jan.) USA) 21.7 under 1400 cool-white fluorescent lights W 0 0 0 0 0 0 0 0 in a 12/12 hr photoperiod at 24.2 6 0.2uCand 06 28 47 51 19 38 9 9 9 9 9 9 a light level of 40 mmol m22 s21. 0918 3712 10 04 34 21 36 39 u u u u u u u u Population Coordinates Habitat Elev. 82 81 82 87 SEEDLING TRANSFER AND DATA COLLECTION. After 6 weeks culture seedlings were trans- ferred from Petri plates to PhytoTech Culture Boxes (PhytoTechnology Laboratories) con- taining 100 ml of BM-1 medium. Medium was prepared as described previously. Uniform- sized seedlings with developing leaves were then transferred to individual culture boxes. Three PhytoTech Culture Boxes with nine seedlings each were prepared per seed source Photoperiod and temperature data from Weather Underground, Inc. Growing season length is the number of days between the last spring frost (above 0 Elevational data from Google Earth. Data from the 2007 Climatological Data Annual Summary.

Population (weather center) for each week. A total of 21 PhytoTech Table 1. Location, habitat, and environmental conditions of a b c d South Florida (Naples) 26 North Central Florida (Ocala) 29 South Carolina (Greenville) 35 Michigan (Escanaba) 45 Culture Boxes were prepared per seed source.

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FIG. 1. In vitro seedling development of Calopogon tuberosus from distinct geographic sources. Note the delayed corm formation in southern populations. Shoot die-back was characterized by yellowing and browning of leaves. A–D. Seedlings after 8 weeks culture. E–H. Seedlings after 12 weeks culture. I–L. Seedlings after 16 weeks culture. M–P. Seedlings after 20 weeks culture. A, E, I, M. Michigan seedlings. Scale bars 5 1 cm.

Cultures were completely randomized within Results. CORM FORMATION. Corm formation the growth chamber under the same condi- differed significantly by population (F 5 tions previously described. 73.86, P , 0.0001), week (F 5 70.54, P , Data were collected bi-weekly on three 0.0001), as well as population by week (F 5 replicate PhytoTech Culture Boxes containing 15.12, P , 0.0001). Corm formation on nine seedlings each per seed source. Data were Michigan seedlings was evident by week 8, taken on 27 seedlings per seed source each week 10 on South Carolina seedlings, week 14 week. Data for week 10 South Carolina on north central Florida seedlings, and week seedlings were collected on two replications 18 on south Florida seedlings (Fig. 1, Ta- due to contamination of one replicate. The ble 2). Initial mean corm diameter on Michi- following data were collected: shoot length, gan and South Carolina seedlings was similar root number, root length, corm diameter, and until week 16 (Table 2). Mean corm diameter dry weight. Shoot, root, and corm dry weights on Michigan seedlings did not change signif- were measured after tissues were dried for 24 h icantly after week 14. Mean corm diameter at 60uC. Seedling percent biomass allocation was similar in Michigan and south Florida was determined by dividing corm, root, and seedlings, but south Florida seedlings contin- shoot weights by the total seedling weight. ued to grow after week 20 while Michigan Shoot length, root number, root length, corm seedlings were fully dormant (pers. obs). Mean diameter, and biomass data were statistically corm diameter was largest on South Carolina analyzed using general linear procedures, and north central Florida seedlings at week 20. ANOVA, and Tukey’s HSD test at a 5 0.05 in SAS 9.1 (SAS Institute 2003). Regression SHOOT LENGTH. Shoot lengths were signifi- and Pearson’s correlation analyses were per- cantly different among populations (F 5 formed on corm biomass allocation and 340.78, P , 0.0001), week (F 5 340.78, P , growing season length reported in Table 1. 0.0001), and population by week (F 5 45.76, P Corm biomass allocation data were arcsine , 0.0001). Initial shoot lengths on Michigan transformed prior to regression analysis. and South Carolina seedlings were larger than

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both Florida populations (Table 2). After week 12, mean shoot length on Michigan 1.05d 0.35c 4.42b 3.66b 2.23c 2.71c 7.25b 6 6 seedlings were the shortest of all seedlings. 6 6 6 6 6 Shoot growth on Michigan seedlings did not significantly increase from week 8 to 16, but did decrease significantly there after. Similarly, shoot growth did not increase significantly on South Carolina seedlings from week 12 to 20. 8.85a 52.4 6.2a 41.8 5.01a 29.3 1.32b2.99b 9.56 22.8 0.69b 5.04 9.13a 90.7 Shoots on north central Florida seedlings were 6 6 6 6 6 6 6 the largest by week 14, and growth continued to increase until week 18. Shoot growth on south Florida seedlings was initially small, and only north central Florida seedlings exceeded mean shoot length of south Florida seedlings at week 20. Shoot length (mm) 3.69b 137.2 3.16b 111.1 1.43b 74.4 1.54a2.07a 22.4 40.0 0.55a 10.6 4.62c 131.9 Shoot senescence, characterized by yellow- 6 6 6 6 6 6 6 ing and browning of leaves, began on Michi- seedlings from four populations after 20 weeks in vitro gan seedlings after 16 weeks culture, and by week 20 almost 100% of shoots were senesced (Fig. 1M). Shoot senescence was delayed in

tuberosus southern populations. Shoot senescence on 1.66d 48.0 1.49c 49.9 1.22c 50.0 1.06d 52.2 0.91c 5A0.2 0.95c 29.0 0.88a 13.8 South Carolina seedlings did not occur until var. weekly comparison within populations) are not significantly different 6 6 6 6 6 6 6 24 weeks culture, 32 weeks culture on north

5 central Florida seedlings, and 38 weeks on south Florida seedlings (pers. obs.).

ROOT LENGTH AND NUMBER. Root length 0.22b 2.2 0.16c 6.0 was significantly influenced by population (F 6 6 5 161.91, P , 0.0001), week (F 5 89.62, P ,

Calopogon tuberosus 0.0001), and population by week (F 5 12.61, P , 0.0001). Root elongation was similar in Michigan, South Carolina, and north central Florida seedlings after 8 weeks culture (Ta- ble 3). By week 14, mean root length was 0.23a 2.59 0.23a 2.34 0.24a 0 11.7 0.16b 0 14.9 longest on north central Florida seedlings, 6 6 6 6 while few differences were observed in south Florida and South Carolina roots. Mean root length on Michigan seedlings was generally the among population comparisons; uppercase shortest. Root length increased in south 5 Florida and north central Florida seedlings

Corm diameter (mm) throughout the experiment. Mean root length 0.16a 5.48 0.15a 4.49 0.12a 3.59 0.09a0.15a 0 2.05 0 14.7 0.08b 0 0 14.4 0.05. decreased on Michigan seedlings after week 18 6 6 6 6 6 6 5 due to root die-back, which was characterized a by shriveling and browning of roots. After 20 weeks culture, roots were longest on north central Florida seedlings. Population (F 5 238.57, P , 0.0001), week 0.16b 4.76 0.12b 4.18 0.10b 3.90 0.08a 2.18 0.09a 2.75 0.09a 1.78 0.07a 0 0 0 13.4 SE)cormdiameterandshootlengthmeasurementsof ABAB A AAABB B AB C AA B BC AAAACAAA (F 5 59.24, P , 0.0001), and population by DACED 6 6 6 6 6 BC6 CD A A D C 6 6 AB C D A A C C CD Dweek (F 5 18.34, P , 0.0001) all A significantly B DE D Michigan South Carolina North Central Florida South Florida Michigan South Carolina North Central Florida South Florida influenced root number. Root number on north central Florida seedlings increased significantly, and by week 16 they contained the highest number of roots (Table 3). Root number on Michigan seedlings was initially Table 2. Mean ( Week 20 2.77 Week 18 2.72 Week 16 2.60 Week 12 2.11 Week 14 2.37 Week 10 1.73 Week 8 1.42 culture. Means with the same letter (lowercase according to Tukey’s HSD test at similar to South Carolina and north central

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Florida seedlings, but by week 14 Michigan seedlings had the lowest root number. 0.13b 0.22b 0.10c 0.17b 0.15b 0.11c 0.09b 6 6 6 6 6 6 6 BIOMASS ALLOCATION. ANOVA results re- vealed that percent biomass allocation to shoots, corms, and roots differed significantly among populations (Table 4). Corm biomass allocation was inversely related to latitude with the highest allocation being observed on 0.30a 2.59 0.38a 2.63 0.27a 2.22 0.16a 2.15 0.13a 1.37 0.12b 0.56 0.06a 0.33 Michigan seedlings. Approximately 97% bio- 6 6 6 6 6 6 6 mass was allocated to corms in Michigan seedlings by week 20, which was significantly higher than the 77% to corms in South Carolina seedlings, 53% to corms in north

Root Number central Florida seedlings, and 7% to corms in south Florida seedlings (Fig. 2). Greater corm 0.15b 4.11 0.13b 4.85 0.15b 3.85 0.13a 3.00 0.10a 2.56 0.09a 2.33 0.09a 1.04 biomass allocation was evident on Michigan 6 6 6 6 6 6 6 seedlings by week 8, and continued through- out the experiment (Fig. 2C). Corm biomass allocation on South Carolina seedlings was significantly greater than both Florida popu- lations, and north central Florida greater than 0.12c 2.67 0.12c 2.85 0.14d 2.89 0.12c 2.74 0.11b 2.52 0.11c 2.83 0.10a 1.33 south Florida with the exception of week 10 6 6 6 6 6 6 6 seedlings from four populations after 20 weeks in vitro culture. Means

weekly comparison within populations) are not significantly different and 12 when corms were not present (Fig. 2C).

5 Percent shoot biomass allocation generally declined among populations throughout the

tuberosus experiment (Fig. 2A). However, shoot bio- 3.64b 0.33 2.99b 0.81 2.63b 1.04 1.58c 1.07 2.34ab 1.33 1.55b 0.96 0.37b 1.22 mass allocation was significantly higher on var. 6 6 6 6 6 6 6 south Florida seedlings than all other popula- tions. South Florida seedlings allocated more biomass to shoots compared to roots and corms over the 20 wk period. Shoot and root biomass allocation of Michigan seedlings decreased simultaneously. After week 10, 3.54a 43.9 3.41a 30.9 2.82a 31.0 1.90a 19.1 2.32a 21.7 1.70a 5.4 0.85a 1.3 shoot and root biomass allocation in South Calopogon tuberosus 6 6 6 6 6 6 6 Carolina and north central Florida seedlings followed the same trend. Root biomass allocation was significantly higher on south Florida seedlings compared to all other populations, while root biomass was lowest

Root Length (mm) on Michigan seedlings (Fig. 2B). 3.99b 65.5 2.59b 58.5 2.39b 52.4 1.07b 42.4 1.26a 29.1 1.48a 23.3 0.60a 10.0 0.05. Correlation analysis revealed a strong neg- 6 6 6 6 6 6 6 5

among population comparisons by week; uppercase ative correlation between corm biomass allo- a

5 cation and growing season length so that as percent corm biomass allocation increased the length of growing season decreased. Growing season was considered the number of days SE) root length and root number on 3.43c 41.1 4.89c 39.7 1.91c 38.6 2.13c 33.6 2.21b 27.1 1.30b 19.2 0.93a 10.7 AA A ACA ABA A A BC A A AA A BBCA A A BC B AB A BC A ACCABACDA AABC BDCA A DABCD B CDDB D C DABA ACDD D DDB E C AD DEDABB between the first spring and last fall frost. 6 6 6 6 6 6 6 6 Pearson’s correlation coefficients (all P values Michigan South Carolina North Central Florida South Florida Michigan South Carolina North Central Florida South Florida , 0.0001) were as follows: 20.73 (all weeks), 20.67 (week 8); 20.81 (week 10); 20.87 (week 12); 20.93 (week 14); 20.96 (week 16); 20.91 (week 18); 20.95 (week 20). Regression analysis also revealed a negative trend for all Table 3. Mean ( according to Tukey’s HSD test at Week 20 7.3 Week 18 17.3 Week 16 13.1 Week 14 12.9 Week 12 16.8 Week 10 9.6 Week 8 10.0 with the same letter (lowercase weeks (Fig. 3). With the exception of week 8

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Table 4. ANOVA results showing main effects and interactions contributing to variation in percent shoot, root, and corm biomass allocation of Calopogon tuberosus seedlings over a 20 week period.

Shoot Root Corm Source of variation df FPdf FPdf FP Population 3 642.6 , 0.0001 3 166.8 , 0.0001 3 2154.7 , 0.0001 Week 6 236.1 , 0.0001 6 31.7 , 0.0001 6 527.9 , 0.0001 Population 3 week 18 6.72 , 0.0001 18 35.3 , 0.0001 18 54.4 , 0.0001

and 10, regression models accounted for much were combined the r2 was 0.54, but the model of the data variance with strong r2 values over was significant. 0.75 (Fig. 3). Due to the lack of corm formation in week 8 and 10 data, r2 values Discussion. This study represents the appli- were not as strong (Fig. 3). When weekly data cation of in vitro ecology to assess the extent of ecotypic differentiation of a latitudinally widespread orchid species. Although informa- tion connecting the timing of biomass alloca- tion to ecotypic development is scarce (Galla- gher 1983, Gallagher and Howarth 1987, Gross et al. 1991, Seliksar et al. 2002, Yoshie 2007), timing of corm formation is an important factor in the ecotypic development of Calopogon tuberosus. Few published articles exist that utilize in vitro techniques to correlate ecotypic life history traits with in vitro growth strategies of orchids (Dijk and Eck 1995, Kauth et al. 2008). The present results also indicate the potential use of in vitro common garden studies to detect unique growth strat- egies. In particular biomass allocation in C. tuberosus ecotypes is influenced by growing season length. Biomass allocation dynamics and storage organ function have been previously described in situ for single orchid populations (Whigham 1984, Snow and Whigham 1989, Zimmerman and Whigham 1992, Tissue et al. 1995, Øien and Pederson 2003, 2005). However, biomass allocation in orchids has not been explored with respect to ecotypic differentiation. In the present study, Calopogon tuberosus biomass allocation to corms ranged from 7% to 97%, depending on seed source. Whigham (1984) reported nearly 80% of biomass in a single Tipularia discolor population was allocated to underground storage organs. Zimmerman and Whigham (1992) reported that 61% and 66% of the total non-structural carbohydrates were allocated to the youngest corms in vegetative and dormant plants, respectively. In a detailed FIG. 2. Shoot, root, and corm biomass alloca- analysis of biomass allocation in T. discolor, tion of Calopogon tuberosus seedlings over 20 weeks 66% of the total biomass was allocated to in vitro culture. Each data point represents the mean of three replications 6 1 standard error. Data points corms during fruit maturation and 80% during with the same letter are not significantly different leaf senescence (Tissue et al. 1995). These data according to Tukey’s HSD test at a 5 0.05. are comparable to C. tuberosus since more

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FIG. 3. Correlation of growing season length and percent corm biomass allocation represented as mg of dry weight per total dry weight. Each value point represents the mean response of three replications with nine seedlings each. Corm biomass percentages were arcsine transformed prior to regression analysis. Regression analysis was performed for each week as well as pooled data combing all weekly data. Note that N 5 99 in week 10 due to contamination of one South Carolina replication. biomass was allocated to corms just prior to to ensure survival during unfavorable growing and during leaf senescence. Although carbo- conditions. In Tipularia discolor corms are vital hydrate analysis of C. tuberosus was not to support growth and reproduction (Zimmer- investigated, reallocation of carbohydrates man and Whigham 1992, Tissue et al. 1995), from leaves to corms might explain increased and serve as sinks for nutrient reserves (Whig- corm biomass allocation in C. tuberosus as was ham 1984). Corms may also aid in long term similarly reported for Dactylorhiza lapponica survival by protecting the shoot meristem tubers (Øien and Pederson 2005). during periods of stress (Whigham 1984). Regardless of orchid species, storage organs Greater and faster biomass allocation to such as corms represent ecological adaptations underground organs in northern Calopogon

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tuberosus ecotypes followed a similar trend to availability in the form of ground water ecotypes of Spartina alterniflora (Gallagher (Nelson 1986, Cohen and Kost 2008), while 1983, Gallagher and Howarth 1987, Gross et populations in Florida experience distinct dry al. 1991) and Sagittaria latifolia (Kane et al. seasons (Davis 1943). Ecotypes in areas prone 2000, Kane et al. 2003). The faster biomass to flooding allocated more biomass and allocation to corms in C. tuberosus is likely a carbohydrates to corms and tubers indicating selection pressure favored by the shorter a vegetative growth strategy (Li et al. 2001, growing season at northern latitudes as Sun et al. 2001, Pen˜as-Fronteras et al. 2009). reported with ecotypes of S. alterniflora Higher biomass to underground storage organs (Seliksar et al. 2002), Plantago asiatica (Sa- may be a response to prolonged flooding when wada et al. 1994), grass species (Potvin 1986, plants would need a readily available source of Liancourt and Tielbo¨rger 2009), and Erio- carbohydrates (Pen˜as-Fronteras et al. 2009). phorum vaginatum (Fetcher and Shaver 1990). Growth differences may be related to repro- Northern ecotypes of Calopogon tuberosus ductive strategy as well. Florida populations in may allocate larger carbohydrate reserves in the present study produce more flowers and storage organs to survive winter conditions, seed capsules then the plants in Michigan and and subsequently reallocate those carbohy- South Carolina, which may lead to higher seed drates to rapid growth the following spring production (Pen˜as-Fronteras et al. 2009). (Seliksar et al. 2002). Greater corm biomass in Higher seed production may be necessary in northern C. tuberosus ecotypes could be order to colonize areas of earlier succession influenced by faster reallocation of carbohy- such as prairies and non-wooded areas in south drates from shoots to corms leading to faster Florida (Abrahamson 1975, 1979). shoot senescence compared to southern eco- Differences in root number, length, and types (Mooney and Billings 1960). Further biomass of Calopogon tuberosus ecotypes may investigation may also determine whether be related to soil nutrient and water availabil- northern ecotypes are more tolerant to freez- ity. Biomass allocation to roots was greater in ing temperatures due to higher corm carbohy- several annual plant species and Populus drate reserves. davidiana ecotypes under low nutrient and A short life cycle from initial shoot produc- water stressed soils (McConnaughay and tion to shoot senescence as well as low Coleman 1999, Zhang et al. 2005). Massachu- temperature tolerance is an adaptation to setts ecotypes of Spartina alterniflora were northern environments where the growing found to have shorter roots due to the shallow, season is short (Potvin 1986). Even under the organic soils compared to the deeper sand- same environmental conditions in vitro, north- based soils in Georgia (Seliksar et al. 2002). ern Calopogon tuberosus ecotypes expressed a Longer or deeper roots on southern C. shorter growth cycle and faster corm biomass tuberosus ecotypes may be an adaptation to allocation. Since seeds were collected directly water-stressed environments where the upper from wild populations, pre-conditioned envi- soil layers have poor water availability ronmental carry-over effects may have ex- (Kondo et al. 2003). plained this adaptation. A long-term genetic Shoot biomass as well as shoot length on adaptation to shorter growing seasons may Calopogon tuberosus was highest in Florida also explain the differences in growth (Shaver populations that experience higher growing et al. 1986), and plants from northern latitudes temperatures. The larger shoots on Florida C. may always express the shorter life cycle and tuberosus seedlings may be a selection pressure greater corm biomass allocation regardless of to maximize photosynthesis to outcompete environmental conditions. The adaptation vegetation during a longer growing season may also be a consequence of primary (Gallagher and Howarth 1987). A higher productivity where plants from northern shoot biomass may be a requirement to reach latitudes are not able to take advantage of reproductive size to set seed before adverse increased temperatures or constant growing environmental conditions are experienced conditions (Fetcher and Shaver 1990). (Rice et al. 1992). Faster shoot growth in Greater biomass to corms may represent a Michigan seedlings may be due to earlier successful survival strategy. The populations carbohydrate allocation. used in the present study from Michigan and Common garden studies are useful tools to South Carolina have long periods of water detect local adaptation influenced by genetics,

Journal of the Torrey Botanical Society tbot-136-04-02.3d 30/11/09 14:04:15 441 Cust # 09-RA-013R1 442 JOURNAL OF THE TORREY BOTANICAL SOCIETY [VOL.136 but often diminish the impact of environmen- terrestrial orchid habitats. Mycol. Res. 107: tal conditions in situ (Nuismer and Gandon 1210–1220. CLAUSEN, J., W. M. KECK, AND W. M. HIESEY. 1941. 2008). Transplant and reciprocal transplant Regional differentiation in plant species. Am. studies better indicate environmental effects Nat. 75: 231–250. on local adaptation (Nuismer and Gandon CLIMATOLOGICAL DATA ANNUAL SUMMARY. 2007. 2008). Conditions in vitro can be controlled to Retrieved February 11, 2009 from the National Climactic Data Center. ,http://www.ncdc.noaa. represent in situ conditions by controlling gov/oa/ncdn.html. environmental conditions experienced across COHEN,J.G.AND M. A. KOST. 2008. Natural a species’ distribution such as photoperiod, community abstract for northern fen, 18 p. temperature, and humidity. 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