Ecological Archives E086-140-A4

Phyllis D. Coley, John Lokvam, Kathleen Rudolph, Keryn Bromberg, Tara E. Sackett, Leslie Wright, Tania Brenes-Arguedas, Dan Dvorett, Seth Ring, Alex Ponge, Caroline Baptiste, R. Toby Pennington, and Thomas A. Kursar. 2005. Divergent defensive strategies of young leaves in two species of Inga. Ecology 86:2633–2643.

Appendix D. PCR and sequencing of trnD-T and atpB-rbcL.

Methods

PCR was peformed on a MJ Research PTC-200 Thermal Cycler, and sequencing used an ABI Prism TM DNA sequencer. PCR reactions were 50 µL using reagents from Bioline (UK) and containing: 5 µL 10x NH4 buffer, 5 µL of 2mM dNTPs, 2.5 µL of 50mM MgCl2, 1.5 µL of each 10 µL primer, 1.25 units of Biotaq polymerase and 2 µL total genomic DNA. PCR Amplification for trnD-T used 1 cycle at 94°C for 4 min; then 30 cycles of: 45 sec at 94°C, 45 sec at 54.5°C, 2 min. at 72°C; followed by 1 cycle at 72°C for 10 min. PCR Amplification for atpB-rbcL used 1 cycle at 94°C for 2 min.; then 30 cycles of: 45 sec at 94°C, 1 min 15 sec at 49°C, 1 min 15 sec at 72°C; followed by 1 cycle at 72°C for 10 min. Prior to sequencing, PCR products were purified using the QIAquick R PCR purification kit (Qiagen Ltd., UK). Sequencing used the PCR primers, and for trnD-T, an internal primer trnD-Ti (5’-CCACGATYCAACTTCCTTTCG-3’) designed by A. Ponge specifically for this study. Sequencing reactions were 20 µL, containing: 4 µL Thermo Sequenase II (Amersham Pharmacia, UK), 0.5 µL primer (10mM), 13.5 µL dH 2O and 2 µL purified PCR product. Sequencing PCR conditions were: 25 cycles of 96°C for 10 sec, 50°C for 5 sec and 60°C for 4 min. Products were purified following manufacturer’s recommendations via ethanol precipitation prior to sequencing.

 
   FIG. D1. A single most-parsimonious tree from the trnL-F analysis. The trnL-F data matrix had 98 variable characters of which 17 were potentially informative. Heuristic search produced 5000 trees of 109 steps with CI 0.69 (excluding autapomorphies) and RI = 0.85. Numbers above the branches are numbers of nucleotide substitutions. Numbers after species names are accession numbers (see Richardson et al. 2001). The node marked by a bold line is resolved in the strict consensus tree, demonstrating that all most-parsimonious hypotheses of relationships indicate I. goldmanii and I. umbellifera (highlighted by arrows) to be distantly related.

 

LITERATURE CITED

Richardson, J. A., R. T. Pennington, T. D. Pennington and P. Hollingsworth. 2001. Recent and rapid diversification of a species-rich genus of Neotropical trees. Science 293: 2232-45.



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