Divergent paralogues of ribosomal DNA in eucalypts (Myrtaceae)

Abstract

The presence of divergent paralogues of nuclear ribosomal DNA, from the 18S–5.8S–26S cistron, is reported in members of Eucalyptus subg. Eucalyptus. These paralogues, which include non-functional pseudogenes, probably diverged prior to the differentiation of species groups in subg. Eucalyptus. When compared with presumably functional sequences, the pseudogenes show greater sequence variation between species, particularly in the 5.8S gene. They are also characterised by reduced GC content, associated with a reduced number of CpG and CpNpG methylation sites, and an increase in the inferred number of methylation-induced substitutions. Some pseudogenes also lack motifs that are usually conserved in plants, both in ITS1 and the 5.8S gene. Two main lineages of pseudogenes are identified, one isolated from a group of western Australian species, one from a group of eastern Australian species. It is not clear whether these two lineages of pseudogenes are orthologous, or represent independent divergences from functional sequence types. The presence of divergent rDNA paralogues highlights the need for caution when interpreting eucalypt phylogenies based on ITS sequences.

Introduction

The internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA) are used extensively in phylogenetic studies of flowering plants, especially for analyses of relationships within genera, or among closely related genera. Plant genomes include multiple copies (paralogues) of these rDNA regions, in the order of thousands of copies per cell, arranged in one to several arrays of tandem repeats. Relative homogeneity of sequences across multiple copies of rDNA is maintained through concerted evolution, which involves such processes as gene conversion and unequal crossing over (Arnheim, 1983), and which recent studies have shown to be extremely efficient and rapid in some taxa (Kovarik et al., 2005). Divergence of rDNA copies will, however, occur where concerted evolution is slow, or does not act on some copies, e.g., if copies are dispersed to a different part of the genome (Childs et al., 1981). In recent years, divergent rDNA paralogues within single genomes have been reported from an increasing range of plant and fungal groups (e.g., Buckler et al., 1997, Lieckfeldt and Seifert, 2000, Hartmann et al., 2001, Mayol and Roselló, 2001, Muir et al., 2001, Bailey et al., 2003, Razafimandimbison et al., 2004, Won and Renner, 2005, Álvarez and Wendel, 2003). The presence of such paralogues can, if undetected, confound attempts at phylogenetic reconstruction (Sanderson and Doyle, 1992).

Prominent among divergent rDNA paralogues are non-functional pseudogenes. These rDNA copies, freed from functional constraints, are generally characterised by increased substitution rates in conserved regions and an increase in the number of methylation-induced substitutions, which lead, in turn, to reduced GC content and reduced stability of secondary structure (Buckler et al., 1997, Bailey et al., 2003). Methylation-induced substitutions occur because cytosines, methylated to 5-methylcytosine, frequently mutate to thymine by deamination (Vairapandi and Duker, 1994, Ng and Bird, 1999). DNA methylation in plants and, therefore, these kinds of mutations, occur chiefly at CpG and CpNpG sites (where N is any nucleotide; Gardiner-Garden et al., 1992, Bender, 2003). In contrast to pseudogenes, functional copies of rDNA maintain large numbers of CpG and CpNpG sites, presumably because of functional constraints.

In this paper, we report the occurrence of divergent paralogues of rDNA in Eucalyptus L’Hér. (Myrtaceae), the dominant tree genus over much of the Australian continent. These paralogues, which are probably non-functional pseudogenes, were discovered while trialling different primer combinations for direct sequencing of rDNA for a phylogenetic study of the monocalypt eucalypts, Eucalyptus subg. Eucalyptus, a group of 110 species (sensu Brooker, 2000) distributed in eastern and south-western Australia (Fig. 1). Properties of the divergent paralogues are described. Their phylogenetic histories are considered by comparison with previously published sequences of the eucalypt group (Steane et al., 1999, Steane et al., 2002, Udovicic and Ladiges, 2000), which, in the broad sense, includes seven genera (Allosyncarpia S.T.Blake, Angophora Cav., Arillastrum Pancher ex Baill., Corymbia K.D. Hill and L.A.S. Johnson, Eucalyptopsis C.T. White, Eucalyptus, Stockwellia D.J. Carr, S.G.M. Carr and B. Hyland) from Australia, Timor, New Guinea, New Britain, Sulawesi, Ceram, Mindanao and New Caledonia (Ladiges et al., 2003).