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@ARTICLE{TreeBASE2Ref17164, author = {Sylvain G. Razafimandimbison and Elizabeth A. Kellogg and Birgitta Bremer}, title = {Recent Origin and Phylogenetic Utility of Divergent ITS Putative Pseudogenes: A Case Study from Naucleeae (Rubiaceae)}, year = {2004}, keywords = {}, doi = {10.1080/10635150490423278}, url = {}, pmid = {}, journal = {Systematic Biology}, volume = {53}, number = {2}, pages = {177--192}, abstract = {The internal transcribed spacer (ITS) of nrDNA has been widely used by systematists for reconstructing phylogenies of closely related taxa. While the occurrence of ITS putative pseudogenes is well documented for many groups of animals and plants, their potential utility in phylogenetic analyses has often been underestimated or even ignored in part because of deletions that make them difficult to align unambiguously. In addition, their long branches often can lead to spurious relationships, particularly in parsimony analyses. We have discovered unusually high levels of ITS polymorphism (up to 30%, 40%, and 14%, respectively) in three tropical tree species of the coffee family (Rubiaceae), Adinauclea fagifolia, Haldina cordifolia, and Mitragyna rubrostipulata. Both secondary structure stability and patterns of nucleotide substitutions in a highly conserved region (5.8S gene) are used for distinguishing presumed functional sequences from putative pseudogenes. We find that the combination of both criteria is the most powerful approach. The sequences from A. fagifolia appear to be a mix of functional genes and highly distinct putative pseudogenes, whereas those from H. cordifolia and M. rubrostipulata are identified as putative pseudogenes. We explore the potential utility of the identified putative pseudogenes in the phylogenetic analyses of Naucleeae sensu lato. Both Bayesian and parsimony trees identify the same monophyletic groups and show that the polymorphisms do not transcend species boundaries, implying that they do not predate the divergence of these three species. Furthermore, the resulting trees are similar to those produced in previous studies by analysis of chloroplast genes. In contrast to previous studies, therefore, divergent putative pseudogenes can be useful for phylogenetic analyses, especially, when no sequences of their functional counterparts are available. Our studies clearly show that ITS polymorphism may not necessarily mislead phylogenetic inference. Despite using many different PCR conditions (different primers, higher denaturing temperatures, and absence or presence of DMSO and BSA-TMACl), we recovered only a few functional ITS copies from A. fagifolia and none from H. cordifolia and M. rubrostipulata. We conclude that PCR selection may be occurring and/or the presumed functional alleles are located in minor loci (with few rDNA copies).} }