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

Abstract

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 highthroughput 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 stateof-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.

High‐contiguity genome assembly of the chemosynthetic gammaproteobacterial endosymbiont of the cold seep tubeworm Lamellibrachia barhami

“…This level of variation contrasts markedly with analyses based on the 16S rRNA gene, which have so far implied that tubeworm symbionts are genetically very similar, at least within vent and seep groups (McMullin et al, 2003). In contrast to prevailing beliefs, recent studies have shown that tubeworm symbionts can have varying degrees of intrahost diversity (Zimmermann et al, 2014; Reveillaud et al, 2018; Polzin, Arevalo, Nussbaumer, Polz, & Bright, 2019; Breusing, Franke, & Young, 2020) – a finding that is supported by our analyses. Despite potential inter‐strain competition, symbiont heterogeneity is predicted to be maintained if symbiont strains are functionally distinct and thereby promote adaptation of their hosts to fluctuating environmental conditions (Zimmermann et al, 2014; Perez & Juniper, 2016; Reveillaud et al, 2018; Polzin et al, 2019).…”

Contrasting influences on bacterial symbiont specificity by co‐occurring deep‐sea mussels and tubeworms

“…Other studies, using both 16S rRNA surveys, conserved, yet variable, gene markers (e.g. ITS, RuBisCo, and gyrB), and high‐coverage metagenomics, have similarly concluded that multiple phylotypes of very closely‐related symbionts can exist within the trophosome of a single vent tubeworm (Vrijenhoek et al ., 2007; Duperron et al ., 2009; Zimmermann et al ., 2014; Reveillaud et al ., 2018; Polzin et al ., 2019; Breusing et al ., 2020a). This pattern underscores the importance of using rapid barcode sequencing of more variable genes to expose underlying endosymbiont diversity and fine‐scale differences, previously hidden by traditional direct 16S rRNA sequencing approaches.…”

Shining light on a deep-sea bacterial symbiont population structure with CRISPR

“…in the northeast Pacific, belong to two vicariant populations [27–29]. At different scales, previous studies of intra-host symbiont diversity in tubeworms inhabiting hydrothermal vents or hydrocarbon seeps [30–37], have consistently found low genetic diversity at the species-level but evidence for multiple strains. Such results also highlight the fact that conventional genetic markers do not provide a high enough resolution to uncover the true strain-level diversity of the symbionts.…”