1. New structure-based methods for the phylogenetic analysis of ribosomal RNA sequences using the parsimony optimality criterion Joseph J. Gillespie Matthew J. Yoder* Anthony I. Cognato 1 2 3

2. 1. RAA/RSC/REC coding 2. RNA basepair coding RNA molecules have characteristic higher order structure that is conserved across all life Using structure to guide the assignment of positional nucleotide homology, we offer two new approaches to analyzing rRNA sequences:

3. RAA/RSC/REC coding* Methods for characterizing regions of RNA sequence alignments wherein positional nucleotide homology cannot be assigned with confidence Based on the premise of using information from secondary structure (i.e., compensatory base change evidence) to delimit unalignable positions *Gillespie (2004) Mol. Phylogenet. Evol. 33: 936-943

4. RAA/RSC/REC coding

5. RAA/RSC/REC coding* Region of ambiguous alignment Two or more adjacent, non-pairing positions within a sequence wherein positional homology cannot be confidently assigned due to the high occurrence of indels in other sequences RAA *Gillespie (2004) Mol. Phylogenet. Evol. 33: 936-943

6. RAA/RSC/REC coding* Region of slipped-strand compensation Region involved in base-pairing wherein positional homology cannot be defended across a multiple sequence alignment; inconsistency in pairing likely due to slipped-strand mispairing RSC *Gillespie (2004) Mol. Phylogenet. Evol. 33: 936-943

7. RAA/RSC/REC coding* Region of expansion and contraction Variable helical region flanked by conserved basepairs at the 3’ and 5’ ends, and an unpaired terminal bulge of at least three nucleotides; characteristic of RNA hairpin-stem loops REC *Gillespie (2004) Mol. Phylogenet. Evol. 33: 936-943

8. RAA/RSC/REC coding* Subdividing large ambiguously aligned regions into smaller components provides: why? 1. a means for comparing structurally similar nucleotides in fragment level alignment methods (INAASE, POY) 2. fewer character state transformations between taxa, with less potential to exceed the number of allotted states in a given phylogenetic software 4. improvements to existing global structural models for the various rRNA molecules on public databases *Gillespie (2004) Mol. Phylogenet. Evol. 33: 936-943 3. the ability to objectively assign different substitution weights to pairing (RSC, REC) and non-pairing (RAA) regions 5. a more explicit set of homologies

10. RNA basepair coding

11. A = AAA = CCA = HGA = MUA = T C = RAC = QCC = IGC = FUC = W G = NAG = ECG = LGG = PUG = Y U = DAU = GCU = KGU = SUU = V *adopted from Smith et al. (2004) Mol. Biol. Evol. 21: 419-427 non-pairing pairing code (20 states)*RNA basepair coding

12. substitution matrix non-pairing canonical non- canonical (-) RNA basepair coding

13. weighting, i.e.RNA basepair coding

14. scripts available via the Jrna script package

15. http://hymenoptera.tamu.edu/rna This project was funded by NSF-PEET DEB grants 0328922 to Robert Wharton and 0358920 to Anthony Cognato.