Culturing and environmental DNA sequencing uncover hidden kinetoplastid biodiversity and a major marine clade within ancestrally freshwater Neobodo designis

Abstract

Bodonid flagellates (class Kinetoplastea) are abundant, free-living protozoa in freshwater, soil and marine habitats, with undersampled global biodiversity. To investigate overall bodonid diversity, kinetoplastid-specific PCR primers were used to amplify and sequence 18S rRNA genes from DNA extracted from 16 diverse environmental samples; of 39 different kinetoplastid sequences, 35 belong to the subclass Metakinetoplastina, where most group with the genus Neobodo or the species Bodo saltans, whilst four group with the subclass Prokinetoplastina (Ichthyobodo). To study divergence between freshwater and marine members of the genus Neobodo, 26 new Neobodo designis strains were cultured and their 18S rRNA genes were sequenced. It is shown that the morphospecies N. designis is a remarkably ancient species complex with a major marine clade nested among older freshwater clades, suggesting that these lineages were constrained physiologically from moving between these environments for most of their long history. Other major bodonid clades show less-deep separation between marine and freshwater strains, but have extensive genetic diversity within all lineages and an apparently biogeographically distinct distribution of B. saltans subclades. Clade-specific 18S rRNA gene primers were used for two N. designis subclades to test their global distribution and genetic diversity. The non-overlap between environmental DNA sequences and those from cultures suggests that there are hundreds, possibly thousands, of different rRNA gene sequences of free-living bodonids globally. INTRODUCTIONKinetoplastea comprise the mainly free-living bodonid flagellates and parasitic trypanosomatids within the phylum Euglenozoa (Cavalier-Smith, 1981, 2003a. Bodonids are common, heterotrophic protozoa that occur in a wide variety of environments, including marine, freshwater and soil, from the tropics to the Arctic (Al-Qassab et al., 2002;Larsen & Patterson, 1990;Patterson & Simpson, 1996;Vørs, 1992Vørs, , 1993. The bodonids Bodo saltans, Neobodo designis and Rhynchomonas nasuta are among the 20 most commonly seen zooflagellates (Patterson & Lee, 2000); it is often suggested that such protists are cosmopolitan and may have wide ecological tolerances (Atkins et al., 1998(Atkins et al., , 2000Finlay, 2002;Patterson & Lee, 2000;Patterson & Simpson, 1996). This frequent assumption of broad physiological tolerance and easy interchange between marine and freshwater environments was recently clearly contradicted for members of the genus Goniomonas, heterotrophic cryptomonads with deeply divergent marine and freshwater clades (von der Heyden et al., 2004a). In bodonids, recent evidence for N. designis suggested marked divergence between freshwater and marine strains, and all bodonid morphospecies, especially B. saltans, exhibited extensive genetic diversity (von der Heyden et al., 2004b). Despite considerable interest in free-living bodonids (Callahan et al., 2002; Doležel et al., 2000;Simpson et al., 2002), their true biodiversity has probably been grossly ...

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“…The proposed method is particularly well suited to detect minute genotypic differences between individuals of the same morphospecies. Genotypic variation within nominal protist taxa is of special interest with respect to the debate on microbial distribution and biogeographies (3,22,23,33). We applied our method to check for differences in the dominant genotypes in protist taxa affiliated with the same morphospecies but originating from different habitats or seasons.…”

“…A high molecular variation has also been demonstrated for other nominal nanoflagellate taxa (e.g., for Spumella sp. [K. Pfandl et al, submitted for publication], Paraphysomonas vestita [2], Neobodo designis [33], and Rhynchomonas nasuta [22]). A conclusive judgment would require in-depth investigation of the respective morphospecies, which was, however, not in the scope of this study.…”