Journal of the Torrey Botanical Society 136(4), 2009, pp. 433–444 In vitro ecology of Calopogon tuberosus var. tuberosus (Orchidaceae) seedlings from distant populations: implications for assessing ecotypic differentiation1 Philip J. Kauth2 and Michael E. Kane Plant Restoration, Conservation, and Propagation Biotechnology Program, Environmental Horticulture Department, University of Florida, 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 plants 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 433 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 North America, 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