Complex communities of small protists and unexpected occurrence of typical marine lineages in shallow freshwater systems

Abstract

Although inland water bodies are more heterogeneous and sensitive to environmental variation than oceans, the diversity of small protists in these ecosystems is much less well known. Some molecular surveys of lakes exist, but little information is available from smaller, shallower and often ephemeral freshwater systems, despite their global distribution and ecological importance. We carried out a comparative study based on massive pyrosequencing of amplified 18S rRNA gene fragments of protists in the 0.2-5 μm size range in one brook and four shallow ponds located in the Natural Regional Park of the Chevreuse Valley, France. Our study revealed a wide diversity of small protists, with 812 stringently defined operational taxonomic units (OTUs) belonging to the recognized eukaryotic supergroups (SAR--Stramenopiles, Alveolata, Rhizaria--Archaeplastida, Excavata, Amoebozoa, Opisthokonta) and to groups of unresolved phylogenetic position (Cryptophyta, Haptophyta, Centrohelida, Katablepharida, Telonemida, Apusozoa). Some OTUs represented deep-branching lineages (Cryptomycota, Aphelida, Colpodellida, Tremulida, clade-10 Cercozoa, HAP-1 Haptophyta). We identified several lineages previously thought to be marine including, in addition to MAST-2 and MAST-12, already detected in freshwater, MAST-3 and possibly MAST-6. Protist community structures were different in the five ecosystems. These differences did not correlate with geographical distances, but seemed to be influenced by environmental parameters.

Fig. 1. Principal Component Analysis (PCA) plot of the measured physicochemical parameters.

Sampled ecosystems appear in blue and physico-chemical parameters in black. MG, Mare Gabard; EV, Etang des Vallees; LC, La Claye; RSA, RuSainte Anne; SR, Saint Robert. TDS, Total Dissolved Solutes; DOC, Dissolved Organic Carbon; OrthoP, orthophosphate.

Fig. 2. Histogram showing the relative proportion of 18S rRNA gene amplicon reads assigned to high rank taxa in the five shallow ecosystems studied.

Replicate samples are labeled as ‘b’ or ‘c’. In all cases, the distribution corresponds to protists in the 0.2-5 μ size range, except in the Etang des Vallees, where the 5-30 μ size fraction was additionally analyzed.

Fig. 3. Correspondence Analysis (CoA) plot showing protist community composition similarities and differences among the five ecosystems studied.

MG25 and MG25b, Mare Gabard; LC25 and LC25b, La Claye; SR25 and SR25b, Saint Robert; RSA25 and RSA25b, Ru Saint Anne; EV33 and EV33c, Etang des Vallees (0.2-5 μm size samples). EV34 and 34b, Etang des Vallees (5-30 μm size samples).

Fig. 4. Rank abundance curve for the total 768 OTUs detected collectively in the 0.2-5 μm size fraction of the five shallow freshwater systems studied.

Sequence data from all samples were pooled to define OTUs with high stringency. The relative abundance of protist OTUs representing more than 0.5% of the total number of reads are shown in the inset. The identity number of the respective OTUs and their taxonomic affiliation are shown below and above the histogram bars.

Fig. 5. Distribution of protist OTUs in the five shallow freshwater systems studied.

Sequence data from replicates were pooled. A, five-set Venn diagram showing the number of specific and shared OTUs in the different freshwater systems (0.2-5 μm fraction size). B, Venn diagram showing OTUs shared by the two fraction sizes analyzed in Etang des Vallees. C, clustering analysis of the five ecosystems based on the presence of shared OTUs, as shown by the heatmap. Only 0.2-5 μm size fractions are considered. Heatmaps show all OTUs and OTUs shared by 2, 3 or 4 ecosystems. No OTU is shared by all the 5 ecosystems. Each row represents an ecosystem and each vertical bar an OTU. Black: OTU present, Grey: OTU absent. The colored sidebar indicates the taxonomic affiliation of the OTU represented by the bar below. Color codes representing different phylogenetic affiliation are indicated in the box. CCTHK: Cryptophyta, Centroheliozoa, Telonemia, Haptophyta and Katablepharida. For presentation reasons, the width of OTU bars is not the same in all the heatmaps. 29 RSA25d, Ru Sainte Anne; LC25d, La Claye; SR25d, Saint Robert; MG25d, Mare Gabard; EV33d, EV34d, Etang des Vallées.

Fig. 6. Approximate Maximum Likelihood (ML) phylogenetic tree of partial 18S rRNA gene stramenopile sequences showing the presence of several MAST clades in shallow freshwater systems.

Non-MAST stramenopile lineages have been collapsed. A total of 392 unambiguously aligned positions were used to reconstruct the tree. Two alveolate sequences were used to root the tree. Representative sequences ofOTUs from this work are shown in bold green. The name of samples where MAST OTUs were detected and the respective proportion of reads are shown within brackets. The scale bar represents the number of estimated substitutions per position for a unit branch length. RSA25/RSA25b, Ru Saint Anne; MG25/MG25b, Mare Gabard; EV33/ EV33b (0.2-5 μm) and EV34/EV34b (5-30 μm), Etang des Vallées.

Fig. 7. Canonical Correspondence Analysis (CCA) plot.

Only 0.2-5 μm size fractions are considered. Each dot represents an OTU. The colorsindicate the taxonomic affiliation. Black triangles indicate samples. Duplicate samples appear superimposed. CCTHK: Cryptophyta, Centroheliozoa, Telonemia, Haptophyta and Katablepharida. TDS: Total Dissolved Solutes, DOC: Dissolved Organic Carbon, OrthoP: orthophosphate.