Figure 1.

An overview of the three ethanol treatment experiments used in this study. (A) Leaf tissues from the Vitaceae species were immediately immersed in 96% ethanol after harvest and stored in this ethanol until they were homogenized in ethanol residue and used for DNA extraction. (B) Rhizophora mangle leaf tissues were desiccated in silica gel and then soaked in ethanol for several days. After the ethanol was removed and the residue evaporated, the tissue was homogenized and used for cell lysis and DNA extraction. (C) Herbarium specimen leaf fragments were soaked in ethanol for 2 h and homogenized after the ethanol was removed and the residue evaporated. The tissue homogenate was resuspended in sorbitol buffer and centrifuged to produce a tissue pellet that was subjected to cell lysis with proteinase K digestion. The same herbarium leaf samples were also extracted using the standard CTAB procedure (Doyle and Doyle, 1987). (1) Leaf tissue is placed into a tube of 96% ethanol and homogenization beads to incubate. (2) Ethanol is removed by pipetting and ethanol residue is left in tubes (A) or is allowed to evaporate (B and C). (3) Tissue is ground into a tissue powder without (A) or with freezing in liquid nitrogen (B and C). (4) Tissue homogenate is first washed with sorbitol buffer (4a in C) or is directly vortex‐mixed with SDS lysis buffer and additives (i.e., proteinase K and β‐mercaptoethanol). 4b shows vortexed lysate with SDS lysis buffer and additives.