Abstract
Over the last decade, culture-independent surveys of marine picoeukaryotic diversity based on 18S ribosomal DNA clone libraries have unveiled numerous sequences of novel high-rank taxa. This newfound diversity has significantly altered our understanding of marine microbial food webs and the evolution of eukaryotes. However, the current picture of marine eukaryotic biodiversity may be significantly skewed by PCR amplification biases, occurrence of rDNA genes in multiple copies within a single cell, and the capacity of DNA to persist as extracellular material. In this study we performed an analysis of the metagenomic dataset from the Global Ocean Survey (GOS) expedition, seeking eukaryotic ribosomal signatures. This PCR-free approach revealed similar phylogenetic patterns to clone library surveys, suggesting that PCR steps do not impose major biases in the exploration of environmental DNA. The different cell size fractions within the GOS dataset, however, displayed a distinct picture. High protistan diversity in the <0.8 µm size fraction, in particular sequences from radiolarians and ciliates (and their absence in the 0.8–3 µm fraction), suggest that most of the DNA in this fraction comes from extracellular material from larger cells. In addition, we compared the phylogenetic patterns from rDNA and reverse transcribed rRNA 18S clone libraries from the same sample harvested in the Mediterranean Sea. The libraries revealed major differences, with taxa such as pelagophytes or picobiliphytes only detected in the 18S rRNA library. MAST (Marine Stramenopiles) appeared as potentially prominent grazers and we observed a significant decrease in the contribution of alveolate and radiolarian sequences, which overwhelmingly dominated rDNA libraries. The rRNA approach appears to be less affected by taxon-specific rDNA copy number and likely better depicts the biogeochemical significance of marine protists.
Highlights
In the last decade, 18S rDNA clone libraries have been considered as the gold standard approach for conducting molecular surveys of marine protist diversity in the environment [1,2]
Rather we argue that rDNA copy number and extracellular DNA are major issues that introduce biases in current studies of protist diversity
Taxonomic affiliation of sequences retrieved from PCR amplified 18S rDNA clones libraries performed on the picoplankton size fraction (0.2 to 3 mm) of samples collected in the photic zone around the globe [2] was compared to that of 18S rDNA sequences found in the,3 mm size fraction of the Global Ocean Survey (GOS) metagenomic dataset (Figure 1A)
Summary
18S rDNA clone libraries have been considered as the gold standard approach for conducting molecular surveys of marine protist diversity in the environment [1,2]. These investigations, almost exclusively performed on the picoplanktonic size fraction (0.2–3 mm), have unveiled high rank taxa such as the so-called MALV (marine alveolates, [3]), MAST (marine stramenopiles, [4]), and picobiliphytes [5], many of which have become cornerstone taxa for microbial ecologists. Epifluorescence microscopy typically reveals a dominance of photosynthetic or mixotrophic cells over heterotrophic cells (ca 80% vs 20%, respectively) in the oceans [9] This suggests that 18S rDNA clone libraries may give a significantly biased view of diversity. Biases during DNA extraction and PCR amplification steps [12], primer selectivity, multiple rDNA gene copy number [13], and the existence of pseudogenes [14] or extracellular DNA [15], are relevant
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