Molecular Studies of Parasitic Plants Using Ribosomal rna
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| The 16S rDNA phylogenetic tree shown in Fig. 7 indicates (by means of branch lengths) the highly diverged sequence of Cytinus. The sequences used in this analysis were subjected to formal relative rate tests (Wu and Li, 1985) as described in Nickrent and Starr (1994). The purpose of the test is to determine whether the rates of substitution are the same or different between two lineages - here taxon 1 and 2 (see Fig. 8). A hypothetical common ancestor of taxa 1 and 2 is designated X. The actual number of substitutions (transitions and transversions) on the branch from X to 1 (K1) cannot be determined directly but can be calculated assuming that the time since divergence from X to taxon 1 and 2 is the same. The null hypothesis is that the number of substitutions from taxon 1 to taxon 3 (K13) is equal to that from taxon 2 to 3 (K23), or K13 – K23 = 0. A significant deviation of this value from 0 indicates that the two lineages are evolving at different rates. Generally, when K13 – K23 / standard error > 3, the probability of rejecting the null hypothesis is less than 1%. Taxon 3 (the reference) must be an unambiguous outgroup to taxa 1 and 2 for this test to be meaningful. For this reason, Pinus was chosen as the reference since the topology shown in Fig. 7 does not place Cytinus with the dicots.
The results of the relative rate tests shown in Fig. 8 clearly indicate the gross rate asymmetry found in the branch leading to Cytinus. In the study by Wolfe et al. (1992), most substitutions were seen along the Epifagus and Conopholis branch, hence the authors concluded that the rate of substitution was higher in these holoparasites. The formal relative rate tests reported here do not show these two plants to exhibit any greater rate asymmetry than in comparisons of other plants (such as Vicia). CONCLUSIONS This paper has focused upon the utility of nuclear and plastid ribosomal RNA in molecular phylogenetic and molecular evolutionary studies of parasitic angiosperms. The use of nuclear 18S rDNA in examining the phylogeny of the component families and genera of Santalales has been demonstrated. This molecular phylogeny clearly indicates the progressive evolution from root parasitic (and nonparasitic) Olacaceae to the most advanced clade, the aerially parasitic Viscaceae. Mistletoes are represented by at least four, independently evolved groups (Viscaceae, Loranthaceae, Misodendraceae, and Santalaceae/Eremolepidaceae). Data from nuclear-encoded rDNA and plastid-encoded rbcL indicate that Antidaphne and Eubrachion (Eremolepidaceae) are best classified within Santalaceae. The difficulty in placing the holoparasite families Balanophoraceae, Hydnoraceae, and Rafflesiaceae within the overall classification of angiosperms can be attributed to their extremely derived vegetative and floral features combined with very high rates of molecular evolution. Preliminary evidence from 18S rDNA analyses place Hydnoraceae with the paleoherbs (Magnoliidae). Nuclear 18S rDNA sequence analysis is in agreement with recent classifications that recognize Orobanchaceae as a component of Scrophulariaceae. 18S rDNA data are concordant with analyses of rps2 in that both recover a monophyletic group composed of rhinanthoid Scrophulariaceae and Orobanchaceae, thus indicating the unique origin of parasitism in the family. Genera traditionally considered transitional between Scrophulariaceae and Orobanchaceae (Lathraea, Harveya and Hyobanche) emerged as components of the Orobanche clade. The utility of 18S rDNA sequences in addressing subfamilial phylogenetic relationships has been demonstrated but not fully realized owing to incomplete sampling. Holoparasitic plants (such as Epifagus and Conopholis) have undergone extreme reorganization of the plastid genome, mainly manifested by the loss of photosynthetic genes. Sequences from plastid rDNA from representatives of Balanophoraceae, Hydnoraceae, and Rafflesiaceae have been obtained thereby suggesting that these plants have also retained a plastid genome. These plants have the most divergent 16S rRNAs ever documented, yet structural studies provide evidence that these molecules are still functional. Extreme rate heterogeneity, as compared with other vascular plants, was demonstrated for 16S rDNA in Cytinus ruber using relative rate tests. This amount of change at highly conserved ribosomal loci provides unprecedented opportunities to study the molecular evolution of the plastid and nuclear genomes. For example, the study of these "fast rate" rRNAs can provide general insight into the structure and function of all rRNA molecules by providing compensatory mutations in regions previously thought to be invariant. Unlike Orobanche or Striga, which are the topic of many papers at this symposium, many of the plants discussed here are rare and endangered. For this reason, they are worthy of conservation efforts and should be viewed as valuable model organisms that can increase our understanding of the mode and tempo of evolutionary change at the molecular level. ACKNOWLEDGMENTS The laboratory assistance of Joseph Agan, Jamie Beam, Mark Berning, Brent Beilschmidt, Erica Grimm, Jennifer Patrick, and Ellen Starr is gratefully acknowledged. Special thanks go to Alison Colwell who provided several unpublished 18S sequences of Scrophulariaceae. Sincere thanks to the colleagues who collected samples of the myriad parasite species reported herein. We could not have achieved a worldwide perspective on the evolution of these plants without the assistance of the following people: Carol Augspurger, Wilhelm Barthlott, Donaldo E. 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The presence of hemiparasitism is indicated by grey borders surrounding Santalales and Polygalales and holoparasitism by means of black borders. Both trophic conditions occur in Cuscutaceae and Scrophulariales. Concepts derived from results of molecular analyses (this paper) are incorporated, such as affinity of Hydnoraceae with Magnoliidae and inclusion of Eremolepidaceae in Santalaceae. 
Figure 2. Comparison of plant nuclear and plastid ribosomal DNA cistrons. A. Nuclear rDNA cistron showing small-subunit (18S), large-subunit (26S), and 5.8S rDNAs. Also shown are the intergenic spacer (IGS), external transcribed spacer (ETS), nontranscribed spacer (NTS) and the internal transcribed spacers (ITS-1, -2). Variable domains are shaded (V1-V9 on 18S, D1-D12 on 26S). B. Plastid rDNA cistron as found in tobacco and most other higher plants. The spacer between the small-subunit (16S) and large-subunit (23S) rDNAs contains two tRNA genes (trnIGAU and trnAUGC), each containing introns. 
Figure 3. The strict consensus tree derived from a heuristic search with 62 18S rDNA sequences from representatives of Santalales. A total of 144 trees of length 1538 steps were found and are summarized by this consensus phylogram where branch lengths are indicated. The consistency index (minus uniformative sites) is 0.331 and the retention index is 0.610. All Olacaceae are found at the base of the tree with the exception of Schoepfia () which is sister to Misodendraceae within the Loranthaceae. Eremolepidaceae, represented by Antidaphne and Eubrachion (*) are components of Santalaceae (see text). 
Figure 4. The single tree of length 784 steps found via a branch and bound search of 18S rDNA sequences of Balanophoraceae (and Cynomorium). The consistency index (minus uniformative sites) is 0.685 and the retention index is 0.677. Number of steps are indicated above and bootstrap values (from 100 replications) are indicated below the branches. 
Figure 5. The strict consensus 18S rDNA tree derived from a branch and bound search with 18 sequences from representatives of Orobanchaceae and Scrophulariaceae. Four trees of length 246 steps were found and are summarized by this consensus phylogram. Number of steps are indicated above the branches. The consistency index (minus uniformative sites) is 0.553 and the retention index is 0.643. The evolution of parasitism marks a monophyletic group composed of rhinanthoid Scrophulariaceae and Orobanchaceae. 
Figure 6. Secondary structural model for plastid-encoded 16S rRNA from Cytinus ruber (Cytinaceae or Rafflesiaceae). Bases in reverse font indicate mutations relative to the sequence of Nicotiana (totalling 130). Bases in lower case occur at the primer sites, hence were not determined. 
Figure 7. The single tree of length 808 steps found via a branch and bound search of a matrix containing16S rDNA sequences from plant plastids and cynobacterial outgroups (Anacystis, Microcystis, and Anabaena). The consistency index (minus uniformative sites) is 0.634 and the retention index is 0.655. Number of steps are indicated above and bootstrap values (from 100 replications) are indicated below the branches. Clades recovered in less than 50% of the trees are indicated by an asterisk (*). The loss of the inverted repeat is indicated by IR. 
Figure 8. Histogram showing results of relative rate tests using plastid 16S rDNA sequences from 12 angiosperms and Pinus (as the reference taxon 3). The three-taxon tree uses K1, the number of nucleotide substitutions per site for taxon 1, K2 for taxon 2 and K3 for taxon 3. The difference in nucleotide substitutions per site (K13 – K23) is multiplied by 1000 for graphical purposes. Standard error values are included above the bar for each taxon. 
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