Ecological Archives E096-045-A3

Grace F. Chen and Douglas W. Schemske. 2015. Ecological differentiation and local adaptation in two sister species of Neotropical Costus (Costaceae). Ecology 96:440449.

Appendix C. Detailed description of methods for cloning juvenile Costus plants.

Plants used to produce the clones were derived from hand pollination of plants established from naturally-pollinated seed in Central Panama and grown to flowering in the greenhouse at Michigan State University (MSU). Clonal replicates derived from 16 C. allenii and 36 C. villosissimus were made in the MSU greenhouse and then transported to Panama. A healthy stem was cut into several small pieces (9 to 12 cm long), each including at least one node. Most leaf tissue was removed from the cutting before being planted to equalize size among clones. A small section (approximately 5 cm²) of healthy, green tissue on the newest leaf was retained to allow growth during cultivation. Cuttings were placed individually in test tubes (Aquatube #53, Syndicate Sales®, IN) with distilled water and grown in an incubator set for 12 hour light (6 am to 6 pm), at temperatures of 30°C daytime and 24°C nighttime, reflecting conditions of Central Panama. Cuttings were checked twice a week to remove damaged tissue and to change water. After 2 months of growth in the incubator, cuttings were sealed with parafilm and air-shipped to Panama. After being released from a seven-day quarantine, cuttings were planted into 1-L plastic grow bags containing potting soil (manufactured by Do it Center®, Panama), and grown in a greenhouse in Gamboa for three to eight months. To obtain enough healthy clones for all transplant sites, this cloning/shipping process was repeated three times in 2006 and 2007. Importation permits were obtained from Autoridad Nacional del Ambiente (SIM/P-3-06, No. SIM/P-3-07), and from Ministerio de Desarrollo Agropecuario Direccion ( #560569, #560575, #561805, #580448, #580459, #580448, #607295, #607296, #607297).

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