Summary: Smith et al. Patterns of diversification in Yucca Electronic Supplementary Material
Electronic Supplementary Materials
Materials and Methods
DNA extraction procedures were as described by Pellmyr et al. (2007). PCR reactions
were performed in 30 µl reaction volumes, with 0.3 µL of 10 µM primers and 0.6 µL of 10 mM
dNTP mixture, 3 µL of 10X reaction buffer, and 1.5 units of Fisher brand Taq polymerase.
MgCl2 and template volumes varied depending on the primers used for amplification (Table
S1). PCR reactions were performed in an MJ Research PTC-100 thermal cycler. For all gene
regions except the rps16 intron, sequences were amplified using standard PCR conditions (see
Table S1 for primer-specific annealing temperatures). The rps16 intron was amplified using a
touchdown procedure, with a starting annealing temperature of 56°C and a final annealing
temperature of 42°C.
PCR reactions were analyzed by electrophoresis in 2% agarose gels stained with
ethidium bromide and visualized using a UV light trans-illuminator. Successful PCR reactions
were purified using QIAquick PCR Purification Kits (© 2002, Qiagen Corporation).
Sequencing of purified PCR products followed Althoff et al (2006).
Raw sequence data were edited in Sequencher v. 4.1.2 (GeneCodes Corporation, Ann
Arbor, MI). Multiple sequence alignment was completed in Clustal W using a gap opening
penalty = 15 and a gap extension penalty = 6.66.
Phylogenetic analysis- Aligned sequences were concatenated in Mesquite v. 1.11 (Maddison &