Recent Origin and Phylogenetic Utility of Divergent ITS Putative Pseudogenes: A Case Study from Naucleeae (Rubiaceae)

Abstract

The internal transcribed spacer (ITS) of nuclear ribosomal DNA has been widely used by systematists for reconstructing phylogenies of closely related taxa. Although the occurrence of ITS putative pseudogenes is well documented for many groups of animals and plants, the potential utility of these pseudogenes in phylogenetic analyses has often been underestimated or even ignored in part because of deletions that make unambiguous alignment difficult. In addition, 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) were used for distinguishing presumed functional sequences from putative pseudogenes. The combination of both criteria was 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 were identified as putative pseudogenes. We explored the potential utility of the identified putative pseudogenes in the phylogenetic analyses of Naucleeae sensu lato. Both Bayesian and parsimony trees identified the same monophyletic groups and indicated that the polymorphisms do not transcend species boundaries, implying that they do not predate the divergence of these three species. The resulting trees are similar to those produced by previous analyses of chloroplast genes. In contrast to results of 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, which suggests that PCR selection is occurring and/or the presumed functional alleles are located at minor loci (with few ribosomal DNA copies).

Testing the utility of matK and ITS DNA regions for discrimination of Allium species

“…However, several species could not be discriminated from each other by either ITS or matK regions. On the other hand, the ITS region requires cloning before sequencing because of the allelic polymorphisms (presence of insertion/deletion, single nucleotide polymorphism), pseudogenes, and paralogous copies of the ITS region in a plant species (Buckler et al, 1997;Denduangboripant and Cronk, 2000;Kita and Ito, 2000;Bailey et al, 2003;Li et al, 2004;Razafimandimbison et al, 2004;Ruggiero and Procaccini, 2004;King and Roalson, 2008). In our study, the nucleotide sequences of ITS ranged from 720 bp in A. pskemense (W6 12754) to 748 bp in A. cernuum (W6 20304).…”

DNA barcodes for ecology, evolution, and conservation

“…In addition, the concern that DNA barcodes will give poor results or faulty identifications because of the complications of ancestral polymorphisms, hybridization, and/or introgression certainly applies to both plants and animals [10,11]. These complications can be particularly acute in some groups of plant in which hybridization is widespread and pseudogenes in the nuclear genome are common [12]. More concerted work is needed on taxa with extensive hybridization to verify whether DNA barcodes can successfully provide accurate identifications across all species.…”

The Colchic region as refuge for relict tree lineages: cryptic speciation in field maples

“…The potential of ITS to reconstruct complex processes of intrageneric evolution, partly including information from pseudogenous variants (Razafimandimbison et al, 2004;Grimm and Denk, 2008), has been demonstrated in many cases. Studies with a comparable sampling and geographic cover are available for Acer section Acer , Fagus (Denk et al, 2002, Quercus (Denk and Grimm, 2010), and Zelkova Christe et al, 2014).…”