Many nanoflagellate morphospecies comprise an enormous variation of genotypes, probably indicating cryptic species. One of the best-investigated morphospecies with respect to molecular and ecophysiological variation are flagellates of the Spumella morphotype. Here, we have phylogenetically analysed three protein-coding genes (actin, alpha-tubulin, beta-tubulin), internal transcribed spacers (ITS1, ITS2) and the 5.8S rDNA of 17 Spumella-like strains isolated from soil, freshwater and marine samples in order to (i) test the validity of the current Spumella-like phylogenetic classification system based exclusively on small subunit (SSU) rDNA, (ii) elucidate the phylogenetic associations of SSU rDNA-unresolved strains and (iii) evaluate the validity of the assignment of ecophysiological adaptations to previously identified SSU rDNA sequence clades. All single-gene analyses show different patterns of support, are incongruent and identify a number of conflicting nodes. Likewise, a concatenation of all protein genes fails to recover specific SSU rDNA clades. However, a combined analysis of all genes confidently resolved the conflicts of the single genes and the protein-gene concatenations and resulted in a tree topology that is identical to the SSU rDNA analysis, but with enhanced phylogenetic resolution and decisively greater support. We conclude that, depending on the genes concatenated, a 'supergene' analysis minimizes artefactual effects of single genes and may be superior in its performance in phylogenetically analysing cryptic species. We confirm the validity of the SSU rDNA Spumella-like phyloclades and support the suggestion that these clades indeed seem to reflect certain ecophysiological adaptations.
INTRODUCTIONIn protistology, species descriptions often pragmatically conform to the morphological species concept: microbial eukaryotes have traditionally been identified on the basis of their morphological diversity (Finlay et al., 1996). In general, such a concept seems to work well for a number of larger protists. However, numerous smaller species (nanoand picoeukaryotes) are morphologically indistinguishable (Coleman, 2002;Hackstein, 1997;Nanney, 2004), as evolution and speciation in microbial eukaryotes are not necessarily accompanied by a perceptible morphological change (Machelon et al., 1984). As a consequence, distinct microbial eukaryote species are often classified as a single species (5cryptic species). The prevalence of such cryptic species impairs biodiversity estimates and analyses of spatial distribution patterns and ecological functions (Bickford et al., 2006;Coleman, 2002;Hackstein, 1997).A group of organisms that falls into this category are colourless chrysophytes often referred to as 'Spumella-like' flagellates or Spumella spp. (Auer & Arndt, 2001;Cleven & Weisse, 2001;Weitere & Arndt, 2003). These organisms account for a substantial proportion of the heterotrophic nanoflagellate (HNF) biomass, especially in freshwater systems (Domaizon et al., 2003;Felip et al., 1999;Zhao et al., 2003), and...