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Supplement
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Material and methods
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Sample collection and preparation
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Symbiotic bivalves were collected at deep-sea hydrothermal vents (Table S1) using the ROV
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MARUM Quest (Marum, Germany) and DSV Alvin (Woods Hole Oceanographic Institution,
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USA). We collected B. puteoserpentis from the Logatchev hydrothermal vent field on the Mid
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Atlantic Ridge at 14°45.2’N, 44°58.7’W in February 2004 during research cruise M 60/3 with
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RV Meteor and B. azoricus from the Menez Gwen hydrothermal vent field on the MAR at
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37°50.7’N, 31°31.2’W in September 2010 during RV Meteor cruise M 82/3. Host species
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identification was based on their collection site: Numerous previous studies have shown that
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Logatchev is colonized by B. puteoserpentis mussels while Menez Gwen is colonized by B.
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azoricus and that these two species do not overlap at these sites (Comtet and Desbruyeres,
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1998; Faure et al., 2009; O'Mullan et al., 2001; Won et al., 2003). Whole juvenile specimens
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(5-29 mm shell length) of both Bathymodiolus species as well as symbiont containing gill
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tissues of adult B. azoricus mussels (55-100 mm shell length) (Table S1) were preserved for
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fluorescence in situ hybridization (FISH) analyses as previously described (Wendeberg et al.,
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2012).
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Fluorescence in situ hybridization (FISH) of rRNA
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Samples were prepared for FISH as described in detail in Wendeberg et al. (2012). Briefly,
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the fixed juvenile specimens of B. puteoserpentis and B. azoricus were embedded whole with
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their shells in Steedman’s wax (Steedman, 1957) and their shells dissolved subsequently
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through acid washing. Embedded specimens were sectioned and mounted onto slides for
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FISH analyses. Hybridizations on tissue sections were also performed as described by
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Wendeberg et al. (2012). Both investigated Bathymodiolus species harbor identical sulfur-
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oxidizing and methane-oxidizing symbionts based on 16S rRNA sequences (Duperron et al.,
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2006). Therefore we used probe BNMAR_193_thio (5’-CGA AGG TCC TCC ACT TTA-3’)
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targeting the sulfur oxidizer and probe BNMAR_845_meth (5’-GCT CCG CCA CTA AGC
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CTA-3’) targeting the methane oxidizer for both Bathymodiolus species (Duperron et al.,
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2006). Tissue sections were analyzed using an Axioskop II epifluorescence microscope
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(Zeiss).
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References
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Comtet T, Desbruyeres D (1998) Population structure and recruitment in mytilid bivalves
from the Lucky Strike and Menez Gwen hydrothermal vent fields (37 degrees 17 ' N and 37
degrees 50 ' N on the Mid-Atlantic Ridge). Marine Ecology-Progress Series 163: 165-177.
Duperron S, Bergin C, Zielinski F, Blazejak A, Pernthaler A, McKiness ZP et al. (2006) A
dual symbiosis shared by two mussel species, Bathymodiolus azoricus and Bathymodiolus
puteoserpentis (Bivalvia: Mytilidae), from hydrothermal vents along the northern MidAtlantic Ridge. Environ Microbiol 8: 1441-1447.
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hydrothermal vent mussels. PLoS One 4: e6485.
O'Mullan GD, Maas PAY, Lutz RA, Vrijenhoek RC (2001) A hybrid zone between
hydrothermal vent mussels (Bivalvia : Mytilidae) from the Mid-Atlantic Ridge. Molecular
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Steedman HF (1957) Polyester wax; a new ribboning embedding medium for histology.
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