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