Novel eukaryotic lineages inferred from small-subunit rRNA analyses of oxygen-depleted marine environments

Abstract

Microeukaryotes in oxygen-depleted environments are among the most diverse, as well as the least studied, organisms. We conducted a cultivation-independent, small-subunit (SSU) rRNA-based survey of microeukaryotes in suboxic waters and anoxic sediments in the great Sippewisset salt marsh, Cape Cod, Mass. We generated two clone libraries and analyzed approximately 300 clones, which contained a large diversity of microeukaryotic SSU rRNA signatures. Only a few of these signatures were closely related (sequence similarity of >97%) to the sequences reported earlier. The bulk of our sequences represented deep novel branches within green algae, fungi, cercozoa, stramenopiles, alveolates, euglenozoa and unclassified flagellates. In addition, a significant number of detected rRNA sequences exhibited no affiliation to known organisms and sequences and thus represent novel lineages of the highest taxonomical order, most of them branching off the base of the global phylogenetic tree. This suggests that oxygen-depleted environments harbor diverse communities of novel organisms, which may provide an interesting window into the early evolution of eukaryotes.

FIG. 1.

Phylogenetic tree for nearly complete SSU rRNA sequences of environmental clones from oxygen-depleted water in the Great Sippewisset salt marsh, Cape Cod, Mass., and the most closely related cultured organisms and environmental 18S rRNA signatures. Environmental clones are indicated by boldface type, and each clone is designated by the library designation (CCW) followed by a number. The tree topology was obtained by a heuristic search with stepwise addition of taxa (TBR branch swapping) under a Tamura Nei minimum evolutionary distance model, with gapped and ambiguously aligned positions excluded. Distance bootstrap values over 50% from an analysis of 1,000 bootstrap replicates are given at respective nodes. Sequences are followed by GenBank accession numbers.

FIG. 2.

Phylogenetic trees for nearly complete SSU rRNA sequences of environmental clones in the Great Sippewisset salt marsh, Cape Cod, Mass., and the most closely related cultured organisms and environmental 18S rRNA signatures. Environmental clones are indicated by boldface type, and each clone is designated by the library designation (CCI for water-sediment interface; CCA for subsurface sediment [10 cm]) followed by a number. The tree topologies were obtained by a heuristic search with stepwise addition of taxa (TBR branch swapping) under a Tamura Nei minimum evolutionary distance model (trees a, b, and c) and maximum-likelihood (tree d) trees, with gapped and ambiguously aligned positions excluded. Distance bootstrap values over 50% from an analysis of 1,000 bootstrap replicates are given at the respective nodes. Sequences are followed by GenBank accession numbers.

FIG. 2.

Phylogenetic trees for nearly complete SSU rRNA sequences of environmental clones in the Great Sippewisset salt marsh, Cape Cod, Mass., and the most closely related cultured organisms and environmental 18S rRNA signatures. Environmental clones are indicated by boldface type, and each clone is designated by the library designation (CCI for water-sediment interface; CCA for subsurface sediment [10 cm]) followed by a number. The tree topologies were obtained by a heuristic search with stepwise addition of taxa (TBR branch swapping) under a Tamura Nei minimum evolutionary distance model (trees a, b, and c) and maximum-likelihood (tree d) trees, with gapped and ambiguously aligned positions excluded. Distance bootstrap values over 50% from an analysis of 1,000 bootstrap replicates are given at the respective nodes. Sequences are followed by GenBank accession numbers.

FIG. 2.

Phylogenetic trees for nearly complete SSU rRNA sequences of environmental clones in the Great Sippewisset salt marsh, Cape Cod, Mass., and the most closely related cultured organisms and environmental 18S rRNA signatures. Environmental clones are indicated by boldface type, and each clone is designated by the library designation (CCI for water-sediment interface; CCA for subsurface sediment [10 cm]) followed by a number. The tree topologies were obtained by a heuristic search with stepwise addition of taxa (TBR branch swapping) under a Tamura Nei minimum evolutionary distance model (trees a, b, and c) and maximum-likelihood (tree d) trees, with gapped and ambiguously aligned positions excluded. Distance bootstrap values over 50% from an analysis of 1,000 bootstrap replicates are given at the respective nodes. Sequences are followed by GenBank accession numbers.

FIG. 2.

Phylogenetic trees for nearly complete SSU rRNA sequences of environmental clones in the Great Sippewisset salt marsh, Cape Cod, Mass., and the most closely related cultured organisms and environmental 18S rRNA signatures. Environmental clones are indicated by boldface type, and each clone is designated by the library designation (CCI for water-sediment interface; CCA for subsurface sediment [10 cm]) followed by a number. The tree topologies were obtained by a heuristic search with stepwise addition of taxa (TBR branch swapping) under a Tamura Nei minimum evolutionary distance model (trees a, b, and c) and maximum-likelihood (tree d) trees, with gapped and ambiguously aligned positions excluded. Distance bootstrap values over 50% from an analysis of 1,000 bootstrap replicates are given at the respective nodes. Sequences are followed by GenBank accession numbers.