Integrating metagenomic and amplicon databases to resolve the phylogenetic and ecological diversity of the Chlamydiae

Abstract

In the era of metagenomics and amplicon sequencing, comprehensive analyses of available sequence data remain a challenge. Here we describe an approach exploiting metagenomic and amplicon data sets from public databases to elucidate phylogenetic diversity of defined microbial taxa. We investigated the phylum Chlamydiae whose known members are obligate intracellular bacteria that represent important pathogens of humans and animals, as well as symbionts of protists. Despite their medical relevance, our knowledge about chlamydial diversity is still scarce. Most of the nine known families are represented by only a few isolates, while previous clone library-based surveys suggested the existence of yet uncharacterized members of this phylum. Here we identified more than 22,000 high quality, non-redundant chlamydial 16S rRNA gene sequences in diverse databases, as well as 1900 putative chlamydial protein-encoding genes. Even when applying the most conservative approach, clustering of chlamydial 16S rRNA gene sequences into operational taxonomic units revealed an unexpectedly high species, genus and family-level diversity within the Chlamydiae, including 181 putative families. These in silico findings were verified experimentally in one Antarctic sample, which contained a high diversity of novel Chlamydiae. In our analysis, the Rhabdochlamydiaceae, whose known members infect arthropods, represents the most diverse and species-rich chlamydial family, followed by the protist-associated Parachlamydiaceae, and a putative new family (PCF8) with unknown host specificity. Available information on the origin of metagenomic samples indicated that marine environments contain the majority of the newly discovered chlamydial lineages, highlighting this environment as an important chlamydial reservoir.

Figure 1

Flow chart illustrating the main steps in the analysis of metagenomic and amplicon sequence data for inferring diversity and ecology of defined microbial taxa. In this study, this approach was used for investigating the phylum Chlamydiae. A detailed description of each step is provided in supplementary information.

Figure 2

Ecological and taxonomic classification of putative chlamydial proteins in metagenomic sequence data. Proteins were classified based on their respective closest neighbor in maximum likelihood trees. Environmental origins grouped in four general categories are color coded. ‘All' refers to all putative chlamydial proteins; ‘specific' refers to the subgroup of proteins with exclusively known chlamydial homologs, ‘virulence' includes all metagenomic proteins with homology to known chlamydial virulence-associated proteins. The number of proteins in each group is indicated in parenthesis. Most of the detected putative chlamydial metagenomic proteins originated from marine environments and are most similar to Simkaniaceae or Parachlamydiaceae homologs.

Figure 3

Relationships of described and predicted families in the phylum Chlamydiae based on near full-length 16S rRNA gene sequences (>1100 nt). The phylogenetic tree was calculated using Bayesian inference (MrBayes; (Huelsenbeck and Ronquist, 2001)). Branches with a posterior probability lower than 0.50 were collapsed. Those with posterior probability values between 0.50 and 0.70 are indicated with red color. The monophyly of all chlamydial families is well supported (>0.90); family level OTUs obtained by sequence similarity-based clustering with ESPRIT (Sun et al., 2009) and including only yet undescribed sequences are labeled as PCF. Details for the sequences included in tree calculation and clustering are available as Supplementary Table S3. Bar, 0.1 expected substitutions per site.

Figure 4

Diversity and ecology of chlamydial families based on NMDS of OTU distances. Filled circles represent family-level OTUs, with the size corresponding to the number of sequences included. The distance between circles indicates the relative distance between OTUs. Colors represent the environment from which the sequences that form the OTUs originated from. OTUs formed by a single sequence only (singletons) were not included in the plot. The majority of family-level OTUs contain only marine-derived sequences (dark blue circles) indicating a high diversity of marine Chlamydiae (see also Supplementary Figure S2). Three prominent OTUs comprise the majority of sequences, the Rhabdochlamydiaceae, followed by the Parachlamydiaceae and PCF8.