Protistan microbial observatory in the Cariaco Basin, Caribbean. I. Pyrosequencing vs Sanger insights into species richness

Abstract

Microbial diversity and distribution are topics of intensive research. In two companion papers in this issue, we describe the results of the Cariaco Microbial Observatory (Caribbean Sea, Venezuela). The Basin contains the largest body of marine anoxic water, and presents an opportunity to study protistan communities across biogeochemical gradients. In the first paper, we survey 18S ribosomal RNA (rRNA) gene sequence diversity using both Sanger- and pyrosequencing-based approaches, employing multiple PCR primers, and state-of-the-art statistical analyses to estimate microbial richness missed by the survey. Sampling the Basin at three stations, in two seasons, and at four depths with distinct biogeochemical regimes, we obtained the largest, and arguably the least biased collection of over 6000 nearly full-length protistan rRNA gene sequences from a given oceanographic regime to date, and over 80,000 pyrosequencing tags. These represent all major and many minor protistan taxa, at frequencies globally similar between the two sequence collections. This large data set provided, via the recently developed parametric modeling, the first statistically sound prediction of the total size of protistan richness in a large and varied environment, such as the Cariaco Basin: over 36,000 species, defined as almost full-length 18S rRNA gene sequence clusters sharing over 99% sequence homology. This richness is a small fraction of the grand total of known protists (over 100,000-500,000 species), suggesting a degree of protistan endemism.

Figure 1

Map of study site. Station A represents the site of the US–Venezuelan Cariaco biogeochemical time series program Station A in the eastern sub-basin. Station B is a shallower station south of the Tortuga channel, the source of incoming Caribbean waters. Station C is centered in the less productive western sub-basin.

Figure 2

Vertical profiles of oxygen, hydrogen sulfide and bacterial and flagellate abundances at the time of sampling for protistan DNA in (A) January and (B) May 2005; modified from Taylor et al. (2001) and Scranton et al. (2006).

Figure 3

Phylum- and kingdom-level assignments of the 18S rRNA gene sequence collections obtained by conventional cloning/Sanger sequencing and 454 pyrosequencing approaches.

Figure 4

Subphylum and class level assignments of the alveolate 18S rRNA gene sequence collections obtained by conventional cloning/Sanger sequencing and 454 pyrosequencing approaches.

Figure 5

Order assignments of the Dinophyceae 18S rRNA gene sequence collections obtained by conventional cloning/Sanger sequencing and 454 pyrosequencing approaches.

Figure 6

The fit of the three-mixed exponential distribution (solid line) to the empirical data points (filled circles, OTUs clustered at 99% 18S rRNA gene sequence identity).