18S rDNA Entire Region (˜ 2000 bp): The 18S sequence has proven to be useful for addressing higher level relationships in insects (divergence times of ~300MYA) (Pashley, MCPheron et al., 1993; Campbell, Steffen-Campbell et al., 1995; Whiting, Carpenter et al., 1997). Complete 18S sequences have been obtained from 40 taxa. In this study 18S will be used primarily to properly determine ordinal relationships of basal pterygotes and higher level relationships within Ephemeroptera. Some regions of 18S are hypervariable and may also provide resolution at deeper nodes. 18s will also play an important role in the polarization character states.
28S rDNA Entire Region (˜ 2300 bp): Portions of 28S r DNA have been used in insect phylogenetics, however it tends to show higher levels of variation at the ordinal level than (Whiting, Carpenter et al., 1997; Yeates and Weigmann, 1999). Complete 28S sequences have been obtained from 25 taxa. As with 18S, portions of this gene are hypervariable and pose obstacles for alignment, but may also provide resolution at the lower phylogenetic levels.
Histone 3 (H3; 376 bp): Histone H3 is part of the histone family of genes and forms the basic structural unit of the nucleosome (Maxson, Cohn et al., 1983; Adams, Knowler et al., 1992). H3 is organized in tandem repeats and is highly expressed, but has been shown to undergo concerted evolution between copies (Hood et al., 1975; Zernick et al., 1980). H3 has been successfully used in insect phylogenetics (Colgan, McLauchlan et al., 1998), and congruence between the H3 topology and other molecular markers supports the use of the gene in this study. Sequence has been obtained for 39 taxa. H3 should provide further support for more terminal relationships.
Elongation Factor 1-µ(EF-1µ; ˜ 1200 bp): EF-1µ codes for a key protein used in the translational elongation process and has a well conserved amino acid sequence making alignment straightforward. This gene has paralogous copies in some insects (Danforth and Ji, 1998) and so one must be careful to use ortholgous copies. It has been used to resolve higher level arthropod relationships (Regier and Shultz, 1997) and higher level insect relationships (Yang, Wiegmann et al., 2000). Preliminary PCR and sequencing have shown that this is a viable marker that can be used for this study.
Cytochrome Oxidase II (COII; ˜ 600 bp): COII is a common mitochodrial gene used in insect phylogenetics. The conservation of the codon reading frame makes the alignment straightforward. As with EF-1µ, COII has amplified well and appears to give good levels of divergence.