Diverse molecular signatures for ribosomally 'active' Perkinsea in marine sediments

Abstract

Background: Perkinsea are a parasitic lineage within the eukaryotic superphylum Alveolata. Recent studies making use of environmental small sub-unit ribosomal RNA gene (SSU rDNA) sequencing methodologies have detected a significant diversity and abundance of Perkinsea-like phylotypes in freshwater environments. In contrast only a few Perkinsea environmental sequences have been retrieved from marine samples and only two groups of Perkinsea have been cultured and morphologically described and these are parasites of marine molluscs or marine protists. These two marine groups form separate and distantly related phylogenetic clusters, composed of closely related lineages on SSU rDNA trees. Here, we test the hypothesis that Perkinsea are a hitherto under-sampled group in marine environments. Using 454 diversity 'tag' sequencing we investigate the diversity and distribution of these protists in marine sediments and water column samples taken from the Deep Chlorophyll Maximum (DCM) and sub-surface using both DNA and RNA as the source template and sampling four European offshore locations.

Results: We detected the presence of 265 sequences branching with known Perkinsea, the majority of them recovered from marine sediments. Moreover, 27% of these sequences were sampled from RNA derived cDNA libraries. Phylogenetic analyses classify a large proportion of these sequences into 38 cluster groups (including 30 novel marine cluster groups), which share less than 97% sequence similarity suggesting this diversity encompasses a range of biologically and ecologically distinct organisms.

Conclusions: These results demonstrate that the Perkinsea lineage is considerably more diverse than previously detected in marine environments. This wide diversity of Perkinsea-like protists is largely retrieved in marine sediment with a significant proportion detected in RNA derived libraries suggesting this diversity represents ribosomally 'active' and intact cells. Given the phylogenetic range of hosts infected by known Perkinsea parasites, these data suggest that Perkinsea either play a significant but hitherto unrecognized role as parasites in marine sediments and/or members of this group are present in the marine sediment possibly as part of the 'seed bank' microbial community.

Figure 1

BioMarKs sampling locations and recovery of Perkinsea-like sequences. A: Map of BioMarKs sampling locations across Europe with GPS position of the sampling sites. Number of 454 sequences recovered from each sampling site, B: Perkinsea sequences recovered from source template (i.e. DNA or cDNA), C: Sampling provenance of Perkinsea sequences across sampling location (water column sub-surface, water column DCM or sediment).

Figure 2

Bayesian phylogenetic tree of Perkinsea diversity using the V4 region of SSU rDNA gene (part 1) and provenance of the Perkinsea BioMarKs sequences. Subsection of the phylogenetic tree is shown (see Figure 3 for the rest of the phylogeny). Bayesian posterior probability, Maximum Likelihood bootstrap (1,000 replicates), and LogDet distance bootstrap (1,000 replicates) values are added at each node using the following convention: support values are summarised by black circles on the branch when support are equal to or higher than 0.9/80%/80% and ringed circles when bootstrap values are between 0.6/60%/60% and 0.9/80%/80%. When the bootstrap value is below 60%, a “+” is added if the topology of the tree is recovered in the ML and LogDet analyses. A “-” is shown when these tree topologies are not consistent. Nine sequences of Dinoflagellata and five sequences of Marine Alveolata group II were used as an outgroup. Branches shortened by ½ are labelled with a double slashed line. The black and grey branches on the tree indicate marine and freshwater lineages respectively. Distribution and provenance of sequences across RNA and DNA derived libraries are illustrated down the right columns as shaded triangles if they represent a cluster group. Number in brackets refers to multiple identical sequence reads from the same sample. Circles are used to represent the provenance of a single environment unique sequence cluster. The colour of triangles designates the number of sequences recovered from each location (surface, DCM and sediment) or rDNA/rRNA for each cluster group. White represent between 0 and 5 sequences, Grey between 6–10 and black higher than 10. For correspondence between the 17 freshwater cluster groups identified by Bråte et al. (2010) and the 5 freshwater cluster groups identified in our analyses see Additional file 4: Table S3.

Figure 3

Bayesian phylogenetic tree of Perkinsea diversity using the V4 region of SSU rDNA gene (part 2) and provenance of the Perkinsea BioMarKs sequences. Remaining part of Bayesian phylogenetic tree of Perkinsea diversity using the V4 region of SSU rDNA gene. This phylogeny is illustrated using the same conventions as Figure 2.