Gene loss and symbiont switching during adaptation to the deep sea in a globally distributed symbiosis

Abstract

Chemosynthetic symbioses between bacteria and invertebrates occur worldwide from coastal sediments to the deep sea. Most host groups are restricted to either shallow or deep waters. In contrast, Lucinidae, the most species-rich family of chemosymbiotic invertebrates, has both shallow- and deep-sea representatives. Multiple lucinid species have independently colonized the deep sea, which provides a unique framework for understanding the role microbial symbionts play in evolutionary transitions between shallow and deep waters. Lucinids acquire their symbionts from their surroundings during early development, which may allow them to flexibly acquire symbionts that are adapted to local environments. Via metagenomic analyses of museum and other samples collected over decades, we investigated the biodiversity and metabolic capabilities of the symbionts of 22 mostly deep-water lucinid species. We aimed to test the theory that the symbiont played a role in adaptation to life in deep-sea habitats. We identified 16 symbiont species, mostly within the previously described genus Ca. Thiodiazotropha. Most genomic functions were shared by both shallow-water and deep-sea Ca. Thiodiazotropha, though nitrogen fixation was exclusive to shallow-water species. We discovered multiple cases of symbiont switching near deep-sea hydrothermal vents and cold seeps, where distantly related hosts convergently acquired novel symbionts from a different bacterial order. Finally, analyses of selection revealed consistently stronger purifying selection on symbiont genomes in two extreme habitats - hydrothermal vents and an oxygen-minimum zone. Our findings reveal that shifts in symbiont metabolic capability and, in some cases, acquisition of a novel symbiont accompanied adaptation of lucinids to challenging deep-sea habitats.

Fig. 1. Diverse Lucinidae from a range of deep and shallow-water habitats around the world were investigated using metagenomics.

A The lucinids sampled in this study, colored by genus, originated from diverse shallow- and deep-water habitats including intertidal, cold-seep, hydrothermal vent, and oxygen-minimum zone sediments. B Sampling locations of the lucinid species sequenced in this study, colored by genus. Shape of symbols indicate sampling depth (shallower or deeper than 200 m below the sea surface).

Fig. 2. Deep-sea Lucinidae host divergent Ca. Thiodiazotropha.

A maximum likelihood phylogenetic tree was reconstructed from GTDB’s multisequence alignment using the best-fit model LG + F + I + G4. Circles indicate bootstrap support values above 95%. Symbiont MAGs originating from the present study are indicated by the asterisk. Host subfamilies are labeled according to the color scheme used in the most recent molecular phylogenetic analysis of the Lucinidae [104]. Previously known symbiont species clades and novel species clades consisting of a single host and location were collapsed for ease of interpretation. Alignment and phylogeny are available on FigShare [105]. Geographical locations of Ca. T. taylori and Ca. T. gloverae samples are detailed in Fig. S3. Australia (AU), Papua New Guinea (PG), Madagascar (MG), New Caledonia (NC), Philippines (PH), United Kingdom (UK), US (United States of America), Sweden (SE), Namibia (NB).

Fig. 3. Several deep-water lucinid species host sulfur-oxidizing bacteria from the order Thiohalomondales that are closely related to the symbionts of hydrothermal vent gastropods.

A Phylogenetic relationships of Thiohalomonadales assigned MAGs. A maximum likelihood phylogenetic tree was reconstructed from GTDB’s multisequence alignment of highly conserved bacterial marker genes using the best-fit model LG + F + I + G4. Circles indicate bootstrap support values above 95%. Novel species groups consisting of a single host and location were collapsed. The lucinid Thiohalomonadales symbionts are in bold font. Alignment and phylogeny are available on FigShare [106]. * MAGs encoding the nitrogen fixation pathway. B Relative abundance of ASVs within the gills of lucinids hosting Thiohalomonadales symbionts. C Distribution of bacteria within gills of (i) Bathyaustriella thionipta and (ii) Lucinoma myriamae detected using CARD-FISH. Probes specifically targeting Thiohalomonadales 16S rRNA (sequence in methods) - magenta, nuclei are stained using DAPI - yellow.

Fig. 4. The genomes of the L. aequizonata and B. thionipta symbionts experienced the strongest purifying selection.

Adaptive evolution in protein-coding sequences of lucinid symbiont genomes was inferred using the ratio of non-synonymous to synonymous substitutions (dN/dS ratio; omega). Only symbiont species groups with five or more high-quality MAGs were used for this analysis. The distribution of dN/dS ratios is shown with a violin plot surrounding a boxplot of the median omega value of each species. The omega values of Ca. T. “Aeq1” and “Thiohalo2” were significantly lower than all other species analyzed (***p < 0.001, two-sample Wilcoxon Signed Rank Test corrected for multiple testing).