Molecular systematics, GISH and the origin of hybrid taxa in Nicotiana (Solanaceae)

Abstract

Phylogenetic relationships in the genus Nicotiana were investigated using parsimony analyses of the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (nrDNA). In addition, origins of some amphidiploid taxa in Nicotiana were investigated using the techniques of genomic in situ hybridization (GISH), and the results of both sets of analyses were used to evaluate previous hypotheses about the origins of these taxa. Phylogenetic analyses of the ITS nrDNA data were performed on the entire genus (66 of 77 naturally occurring species, plus three artificial hybrids), comprising both diploid and polyploid taxa, and on the diploid taxa only (35 species) to examine the effects of amphidiploids on estimates of relationships. All taxa, regardless of ploidy, produced clean, single copies of the ITS region, even though some taxa are hybrids. Results are compared with a published plastid (matK) phylogeny using fewer, but many of the same, taxa. The patterns of relationships in Nicotiana, as seen in both analyses, are largely congruent with each other and previous evolutionary ideas based on morphology and cytology, but some important differences are apparent. None of the currently recognized subgenera of Nicotiana is monophyletic and, although most of the currently recognized sections are coherent, others are clearly polyphyletic. Relying solely upon ITS nrDNA analysis to reveal phylogenetic patterns in a complex genus such as Nicotiana is insufficient, and it is clear that conventional analysis of single data sets, such as ITS, is likely to be misleading in at least some respects about evolutionary history. ITS sequences of natural and well-documented amphidiploids are similar or identical to one of their two parents-usually, but not always, the maternal parent-and are not in any sense themselves 'hybrid'. Knowing how ITS evolves in artificial amphidiploids gives insight into what ITS analysis might reveal about naturally occurring amphidiploids of unknown origin, and it is in this perspective that analysis of ITS sequences is highly informative.

Fig. 1. Phyletic diagram of the genus Nicotiana (from Goodspeed, 1954). A, Nicotiana subgenera Tabacum, Rustica and Petunioides; B, N. subgenus Petunioides, expanded.

Fig. 2. One of the most parsimonious, all‐taxon trees showing cladistic relationships in Nicotiana. Shaded bars indicate Goodspeed’s (1954) taxonomic categories. Branch lengths (ACCTRAN optimization) are indicated above the branches and bootstrap percentages below (any clade with a hyphen has BP <50). An arrowhead indicates nodes collapsing in the strict consensus of all most‐parsimonious trees. A, All taxa excluding N. section Suaveolentes; B, N. section Suaveolentes.

Fig. 2. One of the most parsimonious, all‐taxon trees showing cladistic relationships in Nicotiana. Shaded bars indicate Goodspeed’s (1954) taxonomic categories. Branch lengths (ACCTRAN optimization) are indicated above the branches and bootstrap percentages below (any clade with a hyphen has BP <50). An arrowhead indicates nodes collapsing in the strict consensus of all most‐parsimonious trees. A, All taxa excluding N. section Suaveolentes; B, N. section Suaveolentes.

Fig. 3. One of the most parsimonious diploid‐only trees showing relationships in Nicotiana. Shaded bars indicate Goodspeed’s (1954) taxonomic categories, subgeneric membership as in Fig. 2. Branch lengths (ACCTRAN optimization) are indicated above the branches and bootstrap percentages below (any clade with a hyphen has BP <50). An arrowhead indicates nodes collapsing in the strict consensus of all most‐parsimonious trees.

Fig. 4. Genomic in situ hybridization to metaphases of Nicotiana species. In all parts, yellow fluorescence indicates hybridization to the probe, whereas unlabelled chromatin fluoresces red with propidium iodide counterstain. In each of the three pairs of GISH experiments, putatively complementary sets of chromosomes (12) are labelled by each of the parental diploids suggested by Goodspeed (1954). A, Nicotiana rustica probed with N. paniculata DNA; B, N. rustica probed with N. undulata DNA; C, N. arentsii probed with N. undulata DNA; D, N. arentsii probed with N. wigandioides DNA; E, N. quadrivalvis probed with N. attenuata DNA; F, N. clevelandii probed with N. attenuata DNA.

Fig. 5. Comparison of results (strict consensus tree) of ITS with those produced by the matK data (Aoki and Ito, 2000) for the same set of taxa analysed by the latter. Side bars indicate the sections of Goodspeed (1954); arrows show positions of taxa that differ in placements between the plastid and ITS trees.