MOLECULAR PHYLOGENY AND PHENOTYPIC VARIATION IN THE HETEROTROPHIC GREEN ALGAL GENUS PROTOTHECA (TREBOUXIOPHYCEAE, CHLOROPHYTA)¹

Abstract

Species of the heterotrophic green microalgal genus Prototheca and related taxa were phylogenetically analyzed based on the nuclear small subunit (SSU) and the 5 0 end of the large subunit (LSU) rRNA gene (rDNA) sequences. We propose restricting the genus Prototheca to the four species: P. moriformis Krüger, P. stagnora (Cooke) Pore, P. ulmea Pore, and P. zopfii Krüger. The main diagnostic feature of these taxa is the absence of growth on trehalose.Of these, it was suggested that P. moriformis should be merged into P. zopfii; P. moriformis and three varieties of P. zopfii constituted a paraphyletic assemblage with estimated short evolutionary distances. The trehalose-assimilating strains (Prototheca wickerhamii Tubaki et Soneda strains and Auxenochlorella protothecoides (Krüger) Kalina et Punčochářová SAG 211-7a), together with an invertebrate pathogen Helicosporidium sp., diverged before the radiation of the four species of Prototheca in the SSU rDNA and composite (SSU rDNA plus LSU rDNA) analyses. Comparison between the results from physiological data in this work (fermentative pattern) and those described earlier (growth requirements) lead us to propose a hypothesis that the phenotypic variation, which did not represent diagnostic characters for species delimitation, may reflect the history of genetic diversification within the genus Prototheca as inferred from rDNA sequence characters.

Direct evidence for redundant segmental replacement between multiple 18S rRNA genes in a single Prototheca strain

“…Most of the base insertions were found in the evolutionarily variable regions V1-V9 (Wuyts et al, 2000). None of them accounted for group I (Dávila-Aponte et al, 1991; Jackson et al, 2002;Ueno et al, 2005) or spliceosomal introns (Cubero et al, 2000), which was confirmed by PCR of first-strand cDNAs synthesized from purified 18S rRNA. Clones A and B had almost identical sequences with respect to helices H29 (V5) and E45-1 (Marin et al, 2003) (V8), with only one nucleotide substitution in each of the two helices (Figs 1a and 2b), but were markedly different from each other in a large helix E23 (V4:~300 bp) and H37 (V6:~100 bp).…”

“…To remove precursor rRNAs, 18S rRNAs were purified from total RNA by gel excision without UV shadowing. The absence of group I introns in 18S rRNA has been shown experimentally (Ueno et al, 2005).…”

“…Both libraries were constructed as previously described (Ueno et al, 2005). The cDNA pools were derived from total RNA extracted from an early stationary phase culture of the alga grown in Sabouraud liquid medium (Difco).…”

“…P. wickerhamii was defined on the basis of its ability to utilize trehalose as a sole source of carbon for growth (Pore, 1998). Although a sister relationship between ATCC 16529 and SAG 263-11 was not resolved in the phylogenies based on 18S and 26S rRNA gene sequences, both strains were placed within one clade containing all species of the non-photosynthetic genus Prototheca and a photosynthetic analogous strain A. protothecoides SAG 211-7a (Ueno et al, 2005). P. wickerhamii SAG 263-11 has a single 18S rRNA gene sequence (GenBank accession number X74003).…”

“…In order to examine the nature of these small insertions in H37, RT-PCR analysis was performed. Preparation and reverse transcription of 18S rRNAs were performed as previously described (Ueno et al, 2005). A 270-280 bp region containing helix H37 was amplified by PCR with a set of primers shown in Supplementary Fig.…”