In general, nuclear rDNA, particularly the ITS region, has proved to be valuable in phylogeny reconstruction and in the study of reticulate evolution and the origin of polyploids. Some examples of its use in investigating these different aspects of plant evolution are discussed below.
Phylogenetic reconstruction
Available data indicate that ITS sequences are phylogenetically useful at various intrafamilial levels in angiosperms (depending on the lineage), but are unlikely to retain sufficient evolutionary signal or alignability for the examination of relationships among species in different plant families (Baldwin et al., 1995). However, this generalisation is valid only to the extent that family rank implies an ancient origin, which is not true in all cases. Furthermore, low levels of ITS variation in some ancient plant groups raise the possibility that the ITS region may prove useful for the appraisal of relationships between old families that have experienced exceptionally low rates of spacer evolution. The arbitrariness of taxonomic rank is a major limitation to any general statement about taxonomic limits on the phylogenetic utility of ITS sequences (Baldwin et al., 1995; Hamby & Zimmer, 1992).
Baldwin et al. (1995) have cited several plant families (e.g. Asteraceae, Fabaceae, Rosaceae, Saxifragaceae, Viscaceae and Polemoniaceae) where ITS sequences were
utilised effectively for examining relationships within genera and among closely related genera. Also, within species, ITS sequences have been used successfully for investigating relationships among allopatric or disjunct populations. For example, up to 4.3% ITS sequence divergence was found between individuals from conspecific, allopatric populations in Calycadenia (Baldwin, 1993). Moreover, it became evident that in this genus the ITS region had evolved primarily by point mutations, based on the moderately high levels of sequence divergence between and within species, and even among subspecies.
The small number of nucleotide positions available for phylogenetic analysis in both ITS spacers is often compensated for by the high levels of variation found in ITS1 and ITS2. In several studies, ITS sequences are reported to be much more variable than the total cpDNA from the same set of DNA accessions (Baldwin et al., 1995), an instance that has been found, for example, in the families Astragalus (Wojciechowski
et al., 1993), Madiinae (Baldwin, 1992), Rudbeckckiinae (Urbatsch & Baldwin, 1993) and Viburnum (Donoghue & Systma, 1993), among others.
Reticulate evolution and the origin of polyploids
Since nrDNA is inherited biparentally, it is useful for studying hybridisation, introgression and reticulate evolution in plants. Unlike cpDNA, nrDNA data can provide direct evidence of reticulate evolution if concerted evolution fails to act across repeat units contributed by different parental species (Baldwin et al., 1995; Chase et al., 1993). For instance, such lack of sequence homogenisation may occur if: (1) the hybridisation event was recent; (2) nrDNA repeats are at different loci in the parental taxa, and interlocus gene conversion is inoperative in their hybrid; or, (3) the hybrid is asexual. The parentage of suspected early generation hybrids may be resolved simply by screening for presence or absence of restriction sites diagnostic for ITS sequences of each of the putative parental species. In such cases, additivity for the parental restriction patterns provides excellent evidence of hybridity. However, resolution of ancient hybridisation is likely to require more detailed analysis of ITS variation, e.g. by sequencing of ITS clones (Baldwin et al., 1995).
If concerted evolution fails to homogenise ITS paralogues (i.e. those at different chromosomal loci) through a series of speciation events, the possibility of unknowingly sampling sequences with different evolutionary histories is a real danger to phylogenetic analysis (Sanderson & Doyle, 1992). On the other hand, if such paralogues are retained in most or all members of a species lineage, thorough sampling of these sequences can offer independent estimates of organismal phylogeny, and even a means of rooting a portion of the tree in the absence of outgroup data (Iwabe et al., 1989; Baldwin et al., 1995). In other words, non- homogenised paralogues represent positive phylogenetic opportunities along with some potential danger.
One example of the use of ITS in a study of genetic diversity and reticulate evolution comes from the work of Soltis et al. (1991) in the genus Heuchera (Saxiffagaceae). Here, cpDNA restriction site variation had suggested that both northern and southern populations of Tellima grandiflora in the USA were distantly related. In contrast, ITS data strongly indicated that both groups of populations were conspecific, as was also indicated by their morphology and allozyme data (Soltis & Kuzoff, 1995). Consequently, it was postulated that introgressive hybridisation between T. grandiflora and a species of Mitella had led to chloroplast capture of the Mitella
plastome by some populations of T. grandiflora, thus causing the high level of cpDNA divergence found within this species (Soltis etal., 1991).
Ribosomal genes have been used in several investigations dealing with the origins of polyploid taxa, particularly in cereals (Appels et al., 1980; Saghai-Maroof et al.,
1984). One study examined spacer length variation within populations and among species of Triticum (Appels & Dvorak, 1982). These authors analysed a 130 bp repeat unit found within the spacer region; two out of 11 of the 130 bp variants were sequenced, and the lowered thermal stabilities of heterologous versus homologous hybrids were employed for estimating sequence differences among the other 130 bp variants. Subsequently, different cultivars of hexaploid T. aestivum and tetraploid T. dicoccoides were assayed for sequence differences in the spacer region, and it was estimated that differences from the 'standard' cultivar Chinese Spring ranged from 0.6
to 2.2% at the nucleotide sequence level. These findings, along with cytological evidence, suggested that factors other than simple hybridisation have been involved in the origin of hexaploid wheat. It was suggested that structural changes and deletion of some nrDNA genes in the diploid and tetraploid genomes must have occurred prior to domestication of the polyploid wheats, that is, over 10000 years ago.
To summarise, ITS characters have aided the understanding of plant evolution by providing: (1) corroboration of unexpected findings and the resolution of conflict between data sets (e.g. morphological, cpDNA-based vs. ITS sequences); (2) improved resolution of species relationships (e.g. aiding clarification of taxonomic, biogeographic and cytological data); (3) direct resolution of reticulate evolution; and, (4) evidence of the parentage of polyploids. For a more detailed discussion of these various aspects, the reader is referred to Baldwin et al. (1995) and references therein. In a more broader context, nuclear ribosomal RNA genes have provided much of the molecular data for phylogenetic reconstructions among several branches in the Tree of Life (Hillis & Dixon, 1991; Mindell & Honeycutt, 1990; Maddison & Maddison,
1996).
The increasing number of nrDNA-based studies (e.g. the reviews by Hamby & Zimmer, 1992; Avise, 1994; and, Baldwin et al., 1995) attests to their value and potential, combined with the relative simplicity of automated sequencing methods. Therefore, there seems to be little question that sequencing of the highly conserved regions encoding nrDNA is a powerful tool in many fields of biological study.
4.1.6. Objectives and aims of sequencing the ITS region of Pachyrhizus