NOTE / NOTE 5.8S motifs for the identification of pseudogenic ITS regions Doerte Harpke and Angela Peterson Abstract: The internal transcribed spacer (ITS) region (ITS1, 5.8S rDNA, ITS2) represents one of the most popular mo- lecular markers in phylogenetics. The number of investigations revealing high degrees of intra-individual polymorphism connected with the presence of pseudogenic ITS regions is on the increase. Studies including pseudogenic ITS regions can lead to erroneous phylogenetic trees and false taxonomic conclusions. For their recognition, we focus on the 5.8S rDNA as the functional part of this region, which is also affected by degeneration processes. We outline three conserved Viridi- plantae 5.8S motifs: GAATTGCAGAAwyC, TTTGAAyGCA, CGATGAAGAACGyAGC, which can be simply checked in sequence alignments. The latter 5.8S motif we also recognised in the large subunit RNA (LSU) of Escherichia coli. The utility of different methods for pseudogene detection based on easily recognisable 5.8S sequence motifs by comparison with 5.8S secondary structure reconstructions and statistical tests are discussed and illustrated with three previously pub- lished angiosperm data sets. Key words: 5.8S rDNA, motif, pseudogene, internal transcribed spacer region. Re ´sume ´: La re ´gion (ITS) de l’espaceur interne transcrite (ITS1, 5.8S rADN, ITS2) repre ´sente un des marqueurs des plus populaires en phyloge ´nie. On observe une augmentation du nombre de recherches montrant une pre ´sence importante ac- crue de polymorphisme intra individuel, relie ´e avec la pre ´sence de re ´gions ITS pseudoge `nes. Les e ´tudes incluant des re ´- gions ITS pseudoge `nes peuvent conduire a ` des arbres phyloge ´ne ´tiques errone ´s et a ` de fausses conclusions taxonomiques. Afin de les reconnaı ˆtre, les auteurs se concentrent sur le 5.8S rADN comme partie fonctionnelle de cette re ´gion, laquelle est e ´galement affecte ´e par le processus de de ´ge ´ne ´rescence. Ils soulignent trois motifs 5.8S Viridiplantae : GAATTGCA- GAAwyC, TTTGAAyGCA, CGATGAAGAACGyAGC, qu’on peut simplement ve ´rifier en alignements de se ´quences. Ils reconnaissent e ´galement le dernier motif 5.8S dans la grande sous-unite ´ ARN (LSU) chez l’Escheria coli. On discute l’utilite ´ de diffe ´rentes me ´thodes pour de ´tecter les pseudoge `nes, base ´es sur des motifs de se ´quences facilement reconnaissa- bles du 5.8S, comparativement aux reconstructions secondaires et aux tests statistiques; trois ensembles de donne ´es prove- nant d’angiospermes et pre ´alablement publie ´es, servent a ` illustrer ce propos. Mots-cle ´s : 5.8 rADN, motif, pseudoge `ne, re ´gion de l‘espaceur interne transcrite. [Traduit par la Re ´daction] Introduction Molecular data, in combination with phylogenetic analysis, have become the most widely applied tools for systematic analysis. Sequences of the biparental inherited internal transcribed spacer region (ITS1, 5.8S rDNA, ITS2) of nuclear ribosomal DNA (nrDNA) are frequently used to estimate phylogenetic relationships (e.g., A ` lvarez and Wendel 2003). Phylogenetic trees based on ITS regions provide an insight into classification, hybridization, polyploidy, specia- tion, and species diversity (Baldwin et al. 1995). The ITS region belongs to a multigene family with hundreds to thousands of copies in a single genome, and is therefore subjected to concerted evolution, i.e., the homogenization of the tandem-repeated copies through processes such as unequal crossing over and gene conversion (Li 1997). The homogenization of this region allows for direct sequencing of PCR products for many eukaryotes, and is generally connected with a single dominant sequence. Incompletely concerted or nonconcerted evolution caused, for example, by hybridization, disadvantageous loci, or poly- ploidy results in intra-individual sequence polymorphism (e.g., Zhang and Sang 1999; Wissemann 2003; Peterson et al. 2004). Generally, intra-individual nrDNA polymorphism connected with the presence of pseudogenic ITS regions has been considered to be an exception, but the number of investigations detecting pseudogenes in different eukaryotic Received 5 September 2007. Published on the NRC Research Press Web site at botany.nrc.ca on 29 February 2008. D. Harpke and A. Peterson. 1 Biozentrum, Martin-Luther-University of Halle-Wittenberg, Weinbergweg 22, 06120 Halle/Saale, Germany. 1 Corresponding author (e-mail: angela.peterson@biozentrum.uni-halle.de). 300 Botany 86: 300–305 (2008) doi:10.1139/B07-134 # 2008 NRC Canada