Intra-host symbiont diversity in eastern Pacific cold seep tubeworms identified by the 16S-V6 region, but undetected by the 16S-V4 region

Vestimentiferan tubeworms are key taxa in deep-sea chemosynthetic habitats worldwide. As adults they obtain their nutrition through their sulfide-oxidizing bacterial endosymbionts, which are acquired from the environment. Although horizontal transmission should favor infections by various symbiotic microbes, the current paradigm holds that every tubeworm harbors only one endosymbiotic 16S rRNA phylotype. Although previous studies based on traditional Sanger sequencing have questioned these findings, population level high-throughput analyses of the symbiont 16S diversity are still missing. To get further insights into the symbiont genetic variation and uncover hitherto hidden diversity we applied state-of-the-art 16S-V4 amplicon sequencing to populations of the co-occurring tubeworm species Lamellibrachia barhami and Escarpia spicata that were collected during E/V Nautilus and R/V Western Flyer cruises to cold seeps in the eastern Pacific Ocean. In agreement with earlier work our sequence data indicated that L. barhami and E. spicata share one monomorphic symbiont phylotype. However, complementary CARD-FISH analyses targeting the 16S-V6 region implied the existence of an additional phylotype in L. barhami. Our results suggest that the V4 region might not be sufficiently variable to investigate diversity in the intra-host symbiont population at least in the analyzed sample set. This is an important finding given that this region has become the standard molecular marker for high-throughput microbiome analyses. Further metagenomic research will be necessary to solve these issues and to uncover symbiont diversity that is hidden below the 16S rRNA level.

10.1371/journal.pone.0227053

1932-6203

Breusing, Corinna

91adf7f8-3220-4e4b-b450-b0ac3d34d56a

Franke, Maximilian

26631ff8-2848-43e2-a32f-08b7cf25a579

Young, Curtis Robert

c15e536a-84a3-4e28-a8b1-ccea9a37b3cb

Montoya, Jose M.

7cd589ff-7cbc-4d26-b22d-2c160f447b45

15 January 2020

Breusing, Corinna

91adf7f8-3220-4e4b-b450-b0ac3d34d56a

Franke, Maximilian

26631ff8-2848-43e2-a32f-08b7cf25a579

Young, Curtis Robert

c15e536a-84a3-4e28-a8b1-ccea9a37b3cb

Montoya, Jose M.

7cd589ff-7cbc-4d26-b22d-2c160f447b45

Breusing, Corinna, Franke, Maximilian and Young, Curtis Robert , Montoya, Jose M. (ed.) (2020) Intra-host symbiont diversity in eastern Pacific cold seep tubeworms identified by the 16S-V6 region, but undetected by the 16S-V4 region. PLoS ONE, 15 (1), [e0227053]. (doi:10.1371/journal.pone.0227053).

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Accepted/In Press date: 11 December 2019

Published date: 15 January 2020

Additional Information: Funding Information: This work was funded through grants of the David and Lucile Packard Foundation (to MBARI) and the German Research Foundation (grant number BR 5488/1-1 to C.B.). C.R.Y was supported by UK Natural Environment Research Council funding Calibrating eDNA Tools for Biodiversity Monitoring in the Ocean (grant number NE/N006496/1) and National Capability funding to the National Oceanography Centre, as part of the Climate Linked Atlantic Section Science (CLASS) programme (grant number NE/R015953/1). The Ocean Exploration Trust?s Nautilus Exploration Program, Cruise NA090, was funded through NOAA-OER (grant number NA15OAR0110220). The work by M.F. was funded by the Max Planck Society and the DFG Cluster of Excellence?The Ocean in the Earth System? at MARUM (University of Bremen). We thank the ship crews and ROV pilots for their able assistance in sampling tubeworm specimen for this study, Bob Vrijenhoek for providing samples from his collection as well as SeqMatic (44846 Osgood Rd, Fremont, CA 94539) for sequencing our 16S amplicon libraries. This research used samples provided by the Ocean Exploration Trust?s Nautilus Exploration Program, Cruise NA090. The Symbiosis Group at the Max Planck Institute for Marine Microbiology Bremen is gratefully acknowledged for their excellent advice and lab support on the CARD-FISH analyses. Especially, we thank Silke Wetzel, Miriam Sadowski and Martina Meyer for helping with the lab procedures. We also want to thank Roxanne Beinart, Peter Girguis and Jessica Mitchell for providing the PFA fixed material. Finally, we want to acknowledge an anonymous reviewer for thoughtful comments that helped to improve this manuscript. Publisher Copyright: © 2020 Breusing et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.