Non-concerted ITS evolution in Mammillaria (Cactaceae)

Abstract

Molecular studies of 21 species of the large Cactaceae genus Mammillaria representing a variety of intrageneric taxonomic levels revealed a high degree of intra-individual polymorphism of the internal transcribed spacer region (ITS1, 5.8S rDNA, ITS2). Only a few of these ITS copies belong to apparently functional genes, whereas most are probably non-functional (pseudogenes). As a multiple gene family, the ITS region is subjected to concerted evolution. However, the high degree of intra-individual polymorphism of up to 36% in ITS1 and up to 35% in ITS2 suggests a non-concerted evolution of these loci in Mammillaria. Conserved angiosperm motifs of ITS1 and ITS2 were compared between genomic and cDNA ITS clones of Mammillaria. Some of these motifs (e.g., ITS1 motif 1, ‘TGGT’ within ITS2) in combination with the determination of GC-content, length comparisons of the spacers and ITS2 secondary structure (helices II and III) are helpful in the identification of pseudogene rDNA regions.

Introduction

Dating back to around 30 million years, the Cactaceae represents a relatively young (Hershkovitz and Zimmer, 1997) but species-rich family, including approximately 100 genera and 1500 species (Barthlott and Hunt, 1993). Within this family, Mammillaria is a species-rich genus of about 180 species (Pilbeam, 1999), mainly found in Mexico and extending northwards into the south-western United States and southwards into Central America and Venezuela (Pilbeam, 1999). On a higher systematic level, the phylogenetic relationships within this family have been well studied (Hershkovitz and Zimmer, 1997, Nyffeler, 2002, Edwards et al., 2005). Molecular studies and null hypothesis testing (Butterworth and Wallace, 2004) revealed that the genus Mammillaria is paraphyletic.

An initial project was launched in which molecular markers were carried out to provide a closer view of the phylogenetic relationships within the species of Mammillaria. Our results of trnL–trnF sequence data showed only a low level of variation for this genus (Harpke et al., 2006). As a consequence, we tested further common molecular markers, e.g., the internal transcribed spacer (ITS) region (ITS1, 5.8S rDNA, ITS2) of the nuclear ribosomal DNA (Baldwin, 1992, Baldwin et al., 1995). The ITS region belongs to a multi-gene family and is therefore subjected to concerted evolution, i.e., the homogenisation of the tandem-repeated copies through processes such as unequal crossing over and gene conversion (Li, 1997). Two Mammillaria species (M. camptotricha and M. centralifera) were selected to check the utility of the ITS region for further phylogenetic studies. Interestingly, the amplification of the ITS region revealed two bands differing in sequence length. The direct sequencing of the upper bands of the two individuals revealed an overlap of different sequences by multiple peaks per position in the electropherogram.

Generally, intra-individual nrDNA polymorphism has been considered to be an exception (Mayol and Rosselò, 2001). Nevertheless, some studies have identified this type of polymorphism in plants (e.g., Buckler et al., 1997, Baker et al., 2000, Hartmann et al., 2001, Mayol and Rosselò, 2001, Andreasen and Baldwin, 2003, Ruggiero and Procaccini, 2004). Intra-individual nrITS polymorphism was described in several plant families (reviewed by Wissemann, 2003). Some investigations showed that individuals with polymorphic ITS copies often contain potentially non-functional nrDNA copies (pseudogenes) in addition to functional copies (reviewed by Bailey et al., 2003). 5.8S rRNA pseudogenes have been reported, e.g., for Halophila stipulacea (Hydrocharitaceae) (Ruggiero and Procaccini, 2004), the cactus Lophocereus (Hartmann et al., 2001) and for species of several angiosperm families: Malvaceae, Poaceae, Solanaceae and Winteraceae (Buckler et al., 1997). Pseudogenes are DNA sequences derived from functional genes, but have become non-functional through mutations. They are expected to evolve at a high rate (Li, 1997). Non-functional sequences are often characterized by cytosine mutations at methylation sites, because they are highly mutable (Li, 1997). Thus, they become AT-rich (Li, 1997). The identification of 5.8S rRNA pseudogenes is possible through the examination of secondary structures, substitution rates and methylation-related substitutions on methylation sites and the length (Buckler et al., 1997; reviewed by Bailey et al., 2003).

The internal transcribed spacers 1 (ITS1) and 2 (ITS2) do appear to function in the maturation of nuclear ribosomal RNAs, as specific deletions or point mutations in ITS1 can inhibit the production of mature large and small subunit rRNAs, and deletions or point mutations in ITS2 prevent or reduce the processing of large subunit rRNAs (Liang and Fournier, 1997). Both regions are often used to estimate phylogenetic relationships (Buckler et al., 1997; reviewed in Hershkovitz et al., 1999, Peterson et al., 2004).

Among angiosperms, sequences near the 5′ and 3′ end of ITS1 are highly variable and cannot be unambiguously aligned, but do form similar stem-loop structures (Liu and Schardl, 1994). However, a 25-base pair motif near the centre of ITS1 is highly conserved and can be aligned across numerous plant families, even between monocots and dicots (Liu and Schardl, 1994). Within ITS2, six conserved motifs were discovered by Hershkovitz and Zimmer (1996), playing an important role in the stem-loop formation of the secondary structure of ITS2. Liu and Schardl (1994) recognized two motifs, both of which are located within the motifs 3 and 4 of Hershkovitz and Zimmer (1996). A common ITS2 secondary structure of angiosperms and green algae was published by Mai and Coleman (1997). Four stem-loop regions (helices I–IV) were recognized. According to the position of the stem-loop regions of both spacers among angiosperms, it has been proposed that these act as a scaffold for the processing of the coding regions (Liu and Schardl, 1994).

This study should reveal the reasons for the observed phenomenon of non-concerted evolution (Li, 1997; reviewed in Wissemann, 2003) and whether this is common throughout the entire genus Mammillaria. The idea was to study more species representing different subgenera and series of the large subgenus Mammillaria and to characterize the ITS region. Cloning of genomic and cDNA was conducted in order to obtain sequences for individual copies of the ITS region. In Mammillaria, putative pseudogenes were identified by an extensive analysis of 5.8S rDNA (Harpke, 2005). According to our 5.8S rDNA investigation (Harpke, 2005; Harpke and Peterson, unpublished) the secondary structure construction together with the relative rate test were the most valuable methods for the detection of pseudogenes in Mammillaria. The 5.8S rDNA provides an excellent indicator of the functionality of ITS copies (Hershkovitz et al., 1999). As a consequence, in this study all ITS regions containing putative non-functional 5.8S rRNA genes are considered as pseudogenes. In order to find out more about the role of the internal transcribed spacers in maturation processing of nrDNA and whether these can be used to identify nrDNA pseudogenes, conserved angiosperm motifs were compared between genes and pseudogenes. Additionally, GC-contents and the length variation of both spacers were determined, the secondary structures of ITS2 constructed and minimum free energies of all genomic and cDNA sequences calculated. Furthermore, the utility of the ITS region for phylogenetic studies in Mammillaria is discussed.