DNA extractions and preparation for PCR. Field collected material will be thoroughly washed, rinsed, and inspected for epiphytes. DNA extractions will be done following our modification of the CTAB protocol listed in Hillis et al. (1996). The PIs lab has been able to successfully extract, PCR, and sequence DNA from hundreds of bryophyes, including herbarium specimens up to 10 years old.

Two different molecules will be used for the two different relationship questions at issue in this study. For the higher-level question (the proper circumscription of the Calymperaceae and relationships with putativly close outgroups), we will sequence the chloroplast rbcL gene using the standard bryophyte PCR primers we have developed. Based on our experience with sequencing the rbcL gene in many mosses, including a special survey of the haplolepideous mosses, we are very confident that this gene will provide useful characters at the level of relationships.

For the lower-level question (the relationships of populations and species within a small clade such as Mitthyridium, we will use the nuclear-encoded ITS region of the rDNA repeat. Our lab has successfully sequenced ITS using the standard PCR primers derived from angiosperm studies (Baldwin, 1992) for two genera of Pottiaceae (Tortula and Eucladium), and in both cases found significant variation among populations within species. We were thus able to reconstruct phylogenies within and between groups of closely related species.

Alignment of rbcL sequence data is straightforward by hand due to the presence of codon structure. ITS alignments can be more difficult if highly variable, but in comparisons among close relatives there should be no problem based on our past experience. All data will be independently checked against the original gels or sequence run files and the sequence data will be confirmed using both forward and reverse primers, in order to minimize error.

We will also use PAUP to analyse the molecular data sets, both separately and together with the morphological data (the best-supported trees are usually obtained in such "total evidence" analyses, which are appropriate as long as there is no evidence for separate histories for a particular gene versus other genes or morphology (such as might occur with hybridization and lineage sorting (Huelsenbeck et al, 1996).

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