Novel eukaryotes from the permanently anoxic Cariaco Basin (Caribbean Sea)

Abstract

Present knowledge of microbial diversity is decidedly incomplete (S. J. Giovannoni and M. S. Rappé, p. 47-84, in D. Kirchman, ed., Microbial Ecology of the Oceans, 2000; E. Stackebrandt and T. M. Embley, p. 57-75, in R. R. Colwell and D. J. Grimes, ed., Nonculturable Microorganisms in the Environment, 2000). Protistan phylogenies are particularly deficient and undoubtedly exclude clades of principal ecological and evolutionary importance (S. L. Baldauf, Science 300:1703-1706, 2003). The rRNA approach has been extraordinarily successful in expanding the global prokaryotic record (S. J. Giovannoni and M. S. Rappé, p. 47-84, in D. Kirchman, ed., Microbial Ecology of the Oceans, 2000; E. Stackebrandt and T. M. Embley, p. 57-75, in R. R. Colwell and D. J. Grimes, ed., Nonculturable Microorganisms in the Environment, 2000) but has rarely been used in protistan discovery. Here we report the first application of the 18S rRNA approach to a permanently anoxic environment, the Cariaco Basin off the Venezuelan coast. On the basis of rRNA sequences, we uncovered a substantial number of novel protistan lineages. These included new clades of the highest taxonomic level unrelated to any known eukaryote as well as deep branches within established protistan groups. Three novel lineages branch at the base of the eukaryotic evolutionary tree preceding, contemporary with, or immediately following the earliest eukaryotic branches. These newly discovered protists may retain traits reminiscent of an early eukaryotic ancestor(s).

FIG. 1.

Vertical distributions of microorganisms (A), bacterial production (B), and oxygen, hydrogen sulfide, and samples for phylogenetic analysis (C) at Station CARIACO on 8 May 2002. Chemoautotrophic production was determined by assimilation of ¹⁴CO₂ into particles in the dark, and heterotrophic production was estimated from [³H]leucine incorporation into protein.

FIG. 2.

Minimum-evolution phylogenetic tree of eukaryotic small-subunit rRNA showing the positions of clones E220, H25, H50, and H52. The tree was constructed under maximum-likelihood criteria by using a general-time-reversible DNA substitution model with the variable-site gamma distribution shape parameter (G) at 0.9831, based on 657 aligned positions. Distance bootstrap values over 50% are given at the respective nodes. The size of each shaded triangle corresponds to the total number of sequences in the respective group used in the analysis. The first and second numbers in parentheses correspond to the numbers of GenBank sequences and Cariaco sequences, respectively, included in the group.

FIG. 3.

Minimum-evolution phylogenetic tree of stramenopiles. The tree topology was obtained under a Kimura-two-parameter DNA substitution model with the variable-site gamma distribution shape parameter (G) at 0.4830 and the among-site rate variation proportion of invariable sites (I) at 0.2945 based on 648 aligned positions. Distance bootstrap values over 50% are given at respective nodes. The size of each shaded triangle corresponds to the total number of sequences in the respective group used in the analysis. The first and second numbers in parentheses correspond to the numbers of GenBank sequences and Cariaco sequences, respectively, included in the group.

FIG. 4.

Minimum-evolution phylogenetic tree of alveolates. The tree topology was obtained under a Tamura-Nei DNA substitution model with equal base frequencies and variable-site gamma distribution shape parameter (G) at 0.5042, based on 529 aligned positions. Distance bootstrap values over 50% are given at respective nodes. The size of each shaded triangle corresponds to the total number of sequences in the respective group used in the analysis. The first and second numbers in parentheses correspond to the numbers of GenBank sequences and Cariaco sequences, respectively, included in the group.

FIG. 5.

Minimum-evolutionary-distance phylogenetic tree of ciliated protozoans. The tree was constructed under maximum-likelihood criteria by using a general-time-reversible DNA substitution model with the variable-site gamma distribution shape parameter (G) being 0.4671, based on 506 aligned positions. Distance bootstrap values over 50% are given at respective nodes. Class-level classification is done according to the information in reference and is well supported by the rRNA data. Candidate class CAR_H is a sister group of classical ciliate classes.