title: Secondary structure analyses of the nuclear rRNA internal transcribed spacers and assessment of its phylogenetic utility across the Brassicaceae (mustards).
abstract: The internal transcribed spacers of the nuclear ribosomal RNA gene cluster, termed ITS1 and ITS2, are the most frequently used nuclear markers for phylogenetic analyses across many eukaryotic groups including most plant families. The reasons for the popularity of these markers include: 1.) Ease of amplification due to high copy number of the gene clusters, 2.) Available cost-effective methods and highly conserved primers, 3.) Rapidly evolving markers (i.e. variable between closely related species), and 4.) The assumption (and/or treatment) that these sequences are non-functional, neutrally evolving phylogenetic markers. Here, our analyses of ITS1 and ITS2 for 50 species suggest that both sequences are instead under selective constraints to preserve proper secondary structure, likely to maintain complete self-splicing functions, and thus are not neutrally-evolving phylogenetic markers. Our results indicate the majority of sequence sites are co-evolving with other positions to form proper secondary structure, which has implications for phylogenetic inference. We also found that the lowest energy state and total number of possible alternate secondary structures are highly significantly different between ITS regions and random sequences with an identical overall length and Guanine-Cytosine (GC) content. Lastly, we review recent evidence highlighting some additional problematic issues with using these regions as the sole markers for phylogenetic studies, and thus strongly recommend additional markers and cost-effective approaches for future studies to estimate phylogenetic relationships.
authors.affiliation: Division of Biological Sciences, University of Missouri, Columbia, Missouri, United States of America; Department of Plant and Microbial Biology, University of California Berkeley, Berkeley, California, United States of America.
authors.forename: Patrick P
authors.initials: PP
authors.lastname: Edger
authors.affiliation: Division of Biological Sciences, University of Missouri, Columbia, Missouri, United States of America.
authors.forename: Michelle
authors.initials: M
authors.lastname: Tang
authors.affiliation: Division of Biological Sciences, University of Missouri, Columbia, Missouri, United States of America.
authors.forename: Kevin A
authors.initials: KA
authors.lastname: Bird
authors.affiliation: Division of Biological Sciences, University of Missouri, Columbia, Missouri, United States of America.
authors.forename: Dustin R
authors.initials: DR
authors.lastname: Mayfield
authors.affiliation: Informatics Institute, University of Missouri, Columbia, Missouri, United States of America; Division of Animal Sciences, University of Missouri, Columbia, Missouri, United States of America.
authors.forename: Gavin
authors.initials: G
authors.lastname: Conant
authors.affiliation: Department of Biology, University of Osnabrück, Osnabrück, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Mummenhoff
authors.affiliation: Department of Biodiversity and Plant Systematics, Heidelberg University, Heidelberg, Germany.
authors.forename: Marcus A
authors.initials: MA
authors.lastname: Koch
authors.affiliation: Division of Biological Sciences, University of Missouri, Columbia, Missouri, United States of America; Informatics Institute, University of Missouri, Columbia, Missouri, United States of America.
authors.forename: J Chris
authors.initials: JC
authors.lastname: Pires
mesh_terms: D019607:Brassicaceae / Q000235:genetics*; D021903:DNA, Ribosomal Spacer / Q000737:chemistry*; D020459:Genes, rRNA; D009690:Nucleic Acid Conformation; D010802:Phylogeny
chemical_list: D021903:DNA, Ribosomal Spacer