SUPPORTING INFORMATION
Asian origin and upslope migration of Hawaiian Artemisia (Compositae–
Anthemideae)
Christopher R. Hobbs and Bruce G. Baldwin
Journal of Biogeography
DNA extraction, amplification and sequencing
DNA was extracted from leaf material using the Qiagen DNEasy Plant Mini-Kit, and
for two accessions was obtained from the Hawaiian Plant DNA Library (Randell &
Morden, 1999). Primers used for PCR amplification were Ast-1 (Markos & Baldwin,
2001) and 18S-ETS (Baldwin & Markos, 1998) for ETS, and ITS-I and ITS4 (White et
al., 1990; Urbatsch et al., 2000) for the ITS region. CpDNA primers e and f (Taberlet
et al., 1991) were used to amplify the trnL–trnF intergenic spacer region, and psbA
(Sang et al., 1997) and trnH-GUG (Tate & Simpson, 2003) were used to amplify the
psbA–trnH intergenic spacer region.
PCR was performed on an MJ Research PTC-200 thermal cycler. For ITS
amplification, the initial temperature was 95 °C for 2 min, then 40 cycles of 96 °C for
10 s, 48 °C for 30 s, and 72 °C for 20 s with a 4-second extension per cycle. Final
extension was at 72 °C for 7 min. For ETS, the initial hold at 96 °C was for 1 min,
followed by the same cycling conditions used for amplifying the ITS region except for
an annealing temperature of 60 °C. The chloroplast gene regions were amplified with
an initial temperature of 94 °C for 3 min, then 29 cycles of 94 °C for 1 min, 55 °C for
1 min, 72 °C for 1 min, followed by 72 °C for 5 min. Exo-SAP (Affymetrix, Santa
Clara, CA) was used to clean the products for sequencing, and Sanger sequencing
was performed at the University of California, Berkeley DNA Sequencing Facility. The
unedited sequences were processed with CHROMAS PRO (Technelysium, South
Brisbane, Australia) and GENEIOUS 5.5 (Drummond et al., 2010) to check base
identities on each strand.
REFERENCES
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spacer (ETS) of 18S–26S rDNA: congruence of ETS and ITS trees of
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M., Heled, J., Kearse, M., Markowitz, S., Moir, R., Stones-Havas, S., Sturrock,
S., Thierer, T. & Wilson, A. (2010) Geneious v5.5. Available from
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