Microeukaryote community patterns along an O2/H2S gradient in a supersulfidic anoxic fjord (Framvaren, Norway)

Abstract

To resolve the fine-scale architecture of anoxic protistan communities, we conducted a cultivation-independent 18S rRNA survey in the superanoxic Framvaren Fjord in Norway. We generated three clone libraries along the steep O(2)/H(2)S gradient, using the multiple-primer approach. Of 1,100 clones analyzed, 753 proved to be high-quality protistan target sequences. These sequences were grouped into 92 phylotypes, which displayed high protistan diversity in the fjord (17 major eukaryotic phyla). Only a few were closely related to known taxa. Several sequences were dissimilar to all previously described sequences and occupied a basal position in the inferred phylogenies, suggesting that the sequences recovered were derived from novel, deeply divergent eukaryotes. We detected sequence clades with evolutionary importance (for example, clades in the euglenozoa) and clades that seem to be specifically adapted to anoxic environments, challenging the hypothesis that the global dispersal of protists is uniform. Moreover, with the detection of clones affiliated with jakobid flagellates, we present evidence that primitive descendants of early eukaryotes are present in this anoxic environment. To estimate sample coverage and phylotype richness, we used parametric and nonparametric statistical methods. The results show that although our data set is one of the largest published inventories, our sample missed a substantial proportion of the protistan diversity. Nevertheless, statistical and phylogenetic analyses of the three libraries revealed the fine-scale architecture of anoxic protistan communities, which may exhibit adaptation to different environmental conditions along the O(2)/H(2)S gradient.

FIG. 1.

Characteristics of the sampling site in May 2004.

FIG. 2.

Sampling saturation profile (phylotype accumulation curve). The number of phylotypes is plotted as a function of the number of clones sampled. Clone samples were randomly resampled to completion without replacement to quantify coverage of phylotype diversity. Phylotypes were defined to encompass clones that exhibited at least 98.0% sequence similarity based on a pairwise comparison of the 18S rRNA gene sequences.

FIG. 3.

Phylotypes (OTUs) shared by the three 18S rRNA clone libraries along the vertical O₂/H₂S gradient in Framvaren Fjord. The area of an oval is proportional to the size of the corresponding clone library. The numbers in the overlapping areas are the numbers of OTUs shared by the relevant libraries, and the overlap area is proportional to the amount.

FIG. 4.

Taxonomic distribution of 18S rRNA phylotypes retrieved from three protistan communities along the vertical O₂/H₂S gradient in Framvaren Fjord. Phylotypes were defined to encompass clones that exhibited at least 98.0% sequence similarity based on a pairwise comparison of the 18S rRNA gene sequences.

FIG. 5.

Minimum evolution phylogenetic tree of eukaryotic 18S rRNA gene sequences showing the positions of alveolate sequences. The tree was constructed with maximum likelihood criteria by using a GTR+I+G DNA substitution model with the variable-site gamma distribution shape parameter (G) at 0.5647, the proportion of invariable sites (I) at 0.0734, and the base frequencies and rate matrix for the substitution model suggested by Modeltest (59), based on 1,005 unambiguously aligned positions. Distance bootstrap values greater than 50% from an analysis of 1,000 bootstrap replicates are indicated at the nodes. Sequences recovered from the Framvaren Fjord are in bold. The numbers in triangles indicate the numbers of FV library sequences identified in taxonomic units. The numbers in parentheses indicate the primer sets (Table 1), and the colors indicate the three different libraries, as follows: green, FV18 (18 m); red, FV23 (23 m); and blue, FV36 (36 m).

FIG. 6.

Maximum likelihood tree of eukaryotic 18S rRNA gene sequences showing the positions of stramenopile sequences. The tree was constructed by using a GTR+I+G DNA substitution model with the variable-site gamma distribution shape parameter (G) at 0.5436, the proportion of invariable sites (I) at 0.2751, and the base frequencies and rate matrix for the substitution model suggested by Modeltest (59), based on 1,361 unambiguously aligned positions. Maximum likelihood bootstrap values greater than 50% from an analysis of 500 bootstrap replicates are indicated at the nodes. For additional details see the legend to Fig. 5.

FIG. 7.

Minimum evolution phylogenetic tree of eukaryotic 18S rRNA gene sequences showing the positions of opisthokont clones and several bikont clones of the crown radiation. The tree was constructed with maximum likelihood criteria by using a GTR+I+G DNA substitution model with the variable-site gamma distribution shape parameter (G) at 0.5877, the proportion of invariable sites (I) at 0.1415, and the base frequencies and rate matrix for the substitution model suggested by Modeltest (59), based on 1,316 unambiguously aligned positions. For additional details see the legend to Fig. 5.

FIG. 8.

Minimum evolution phylogenetic tree of eukaryotic 18S rRNA gene sequences showing the positions of early branching sequences. The tree was constructed with maximum likelihood criteria by using a GTR+I+G DNA substitution model with the variable-site gamma distribution shape parameter (G) at 0.7597, the proportion of invariable sites (I) at 0.0990, and the base frequencies and rate matrix for the substitution model suggested by Modeltest (59), based on 924 unambiguously aligned positions. For additional details see the legend to Fig. 5.