Genetic Variation and Phylogeny of Spongospora subterranea f.sp. subterranea Based on Ribosomal DNA Sequence Analysis

American Journal of Potato Research, Nov/Dec 2004 by Qu, Xinshun, Christ, Barbara J

PCR Amplification and Sequencing of S. subterranea SSU rDNA

The primer pair NS3/SsB was initially used for PCR amplification of S. subterranea SSU rDNA. The S. subterranea-specific primer SsB was designed from the ITSl sequence of S. subterranea. After PCR amplification of DNA from two S. subterranea isolates No. 1 and No. 7 (represented two ITS groups), a single fragment of approximately 1,300 bp was generated from each collection. After the NSS-SsB region was sequenced, another S. swoiemmeo-specific primer SsA was designed from this sequence data, and a single fragment of approximately 800 bp was generated following PCR amplification of DNA of the two S. subterranea isolates with the primer pair A/SsA. These fragments from each isolate were also sequenced. When the sequences of the two PCR products, c. 800 bp and 1,300 bp, from the two S. subterranea collections were aligned, it was found that the full S. subterranea SSU rDNA region consisted of 1,837 bp, and that the SSU rDNA sequences in both S. subterranea isolates were identical.

Phylogenetic Analysis of S. subterranea SSU rDNA Sequences

The complete S. subterranea SSU rDNA sequences were aligned with 61 SSU rDNA sequences from a very broad diversity of eukaryotes. The data set consisted of 2244 characters, but 651 ambiguously aligned characters were excluded from further phylogenetic analysis. Of the 1593 included characters, 313 were constant, 233 were variable uninformative, and 1047 were parsimony informative.

The heuristic search of the sequence data with the Archaea Haloferax alexandrinus as an outgroup species yielded three equally parsimonious trees with a length of 7602, consistency index = 0.348, and retention index = 0.652. One arbitrarily chosen tree is shown as a phylogram in Figure 2. The neighbour-joining analysis using Kimura's two-parameter distance produced similar topologies to those seen in the most parsimonious trees. The neighbour-joining distance and maximum parsimony analyses show the following phylogenetic patterns: S. subterranea forms a monophyletic group with other species from Plasmodiophorida with 100% bootstrap in both neighbor joining distance and maximum parsimony analysis. Spongospora subterranea f.sp. subterranea is distinct from S. subtenunea f.sp. nasturtii. The plasmodiophorid clade forms a sister group to a clade consisting of chlorarachneans, euglyphids, and sarcomonads with a high bootstrap support, 98% in neighbour-joining distance and 96% in maximum parsimony analysis, respectively. Euglyphids and sarcomonads clustered in a group as a sister taxon to chlorarachneans.

The resulting phylogenetic tree also shows strong bootstrap support for most major groups: plants form a group; animals and fungi are closely related; stramenopiles are in a group; apicomplexans, ciliates, and dinozoans group together. Mycetozoa and kinetoplastida fall within the crown group.

DISCUSSION

Spongospora subterranea is a member of the plasmodiophorids, and the taxonomy of the Plasmodiophorida has been debated for a long time. The relationships between plasmodiophorids and other eukaryote groups have been reported in several recent SSU rDNA phylogenetic analyses. Cavalier-Smith and Chao (1997) found that P. brassicae is a sister to the chlorarachneans, euglyphids and sarcomonads clade. Bulman et al. (2001) reported that several plasmodiophorid species are related to sarcomonads and chlorarachneans. However, Castlebury and Domier (1998), Ward and Adams (1998), K�hn et al. (2000), and Down et al. (2002) reported that that the plasmodiophorids are not related to any other eukaryotes and suggested that the plasmodiophorids are a distinct group. In this investigation S. subterranea formed a monophyletic group with other species of plasmodiophorids, and this group was related to chlorarachneans, euglyphids, and sarcomonads based on SSU rDNA sequence analysis. This result is in agreement with the studies of Cavalier-Smith and Chao (1997) and Bulman et al. (2001).