Fig 1.
Composite map of the sampling sites used in this study, as well as the type locality of O. webbi. 1: Nyegga/Storegga slide, 2: Lofoten canyon site, 3: type locality of O. webbi, off Kvaløya, Tromsø, 4: Håkon Mosby mud volcano (type locality of O. haakonmosbiensis, 5: pingo site, 6: crater site, 7: Laptev Sea site. Bathymetry was obtained from IBCAO [33].
Fig 2.
A: overview of the pingo site (location 5 in Fig 1). Individual pingos are visible in the bathymetry as raised bumps on the seafloor. B: Gas hydrate pingo 3 (GHP3) with sampling locations marked with black dots. C: overview of the crater site (location 6 in Fig 1). D: the Yin Yang pingo-crater complex from the crater site, with sampling locations marked.
Fig 3.
Maximum likelihood (ML) tree obtained with PhyML on a 473-bp alignment of a fragment of the mitochondrial COI gene for frenulate Siboglinidae, with a SPR-tree-searching approach. The moniliferan siboglinid Sclerolinum spp. was used as the outgroup. For clades with specimens from different geographic origins, the area of capture is indicated. The accession numbers are provided for each sequence in parentheses after the sample number. Branch support: left = approximate likelihood ratio test ALRT; right = bootstrap (%) with the Kimura-2-Parameter distance method. ‘-‘ indicates incongruence of topologies. Only nodes with aLRT values greater than 0.7 are indicated.
Table 1.
Probe details.
Table 2.
Morphological features of O. haakonmosbiensis, O. webbi and the pingo and crater worms.
Fig 4.
Haplotype network and barcode gap detection.
A: Haplotype network obtained with a TCS methods in PopART for COI sequences (n = 32) in the Oligobrachia spp. clade (see Fig 3). Tick marks indicate mutational steps. B: Automatic Barcode Gap Discovery (ABGD) pairwise distance distribution histogram showing two groups of sequences (prior maximal distance P = 0.00176).
Fig 5.
Morphology of the pingo-crater worms and O. haakonmosbiensis.
A: Live profile view of one of the pingo and crater worms (individual #1078–1 from the pingo site). The different body parts are shown (most of the trophosome and the opisthosome are missing). The head is anterior to the frenulum. The forepart is anterior to the diaphragm. The pre-annular trunk is anteriorly limited by the diaphragm, and posteriorly by the two girdles. The post-annular trunk starts with a narrowing behind the girdles and posteriorly contains the black trophosome. B: Close up view of the head and forepart of O. haakonmosbiensis (Storegga) showing the bridle keels of the frenulum. C: Close up view of the head and forepart of a pingo-crater worm (individual # 1125–7 from the crater site) showing the bridle keels of the frenulum.
Fig 6.
Enlarged papillae of pingo and crater worms.
Scanning electron micrographs of the feature that could possibly be unique to the pingo and crater worms: enlarged papillae before the anterior girdle. Arrows indicate the papillae. A line of multi-toothed chaetae is visible just anterior to the each girdle. AG: anterior girdle, PG: posterior girdle.
Fig 7.
Phylogenetic affiliation of the bacterial symbionts in sampled worms from the pingo and crater sites based on 16SrRNA gene sequences (in bold). A Maximum Likelihood method using the General Time Reversible model with Gamma-distributed rates with invariants was used (1393 positions used). Scale bar represents estimated 5% base substitution. Percentages at nodes correspond to boostrap support values (100 replicates, >50% shown).
Fig 8.
Phylogenetic affiliation of APS-encoding genes sequences (aprA) from symbionts of Oligobrachia specimens 1054–6 and 1078–13 (in bold), inferred using a Maximum Likelihood method and a GTR model (5 categories and invariants, 352 nt positions). Scale bar represents estimated 20% nucleotide substitution. Percentages at nodes correspond to bootstrap support values (100 replicates).
Fig 9.
FISH image of pingo worm trophosome cross section.
Fluorescence in situ hybridization of bacterial symbionts on a cross section of a pingo worm (sample 1078–13): Epifluorescence images showing host nuclei, stained with DAPI (blue), and bacterial symbionts in red, hybridized with LaSp probes from Duperron et al., 2009. A: entire cross section at the level of the trophosome. B: close up of top right part of cross section shown in A. Note the high densities of bacterial symbionts.
Fig 10.
FISH image of crater worm longitudinal section.
Epifluorescence image of a longitudinal section of the trophosome of a crater worm (sample 1125–6). Host nuclei appear blue due to DAPI staining. Bacterial cells (yellow) were hybridized with the GAM42 probe. From this point of view, the trophosome appears to contain multiple folds and the bacteria appear to follow these folds.
Fig 11.
TEM images of pingo and crater worms.
Electron micrographs of cross sections of the symbiont bearing trophosome tissue of the pingo and crater worms. Round or rod shaped bacteria were seen, depending on the plane across which the cuts were made, typical morphology of sulfur oxidizing symbionts. A: Entire bacteriocyte densely packed with bacteria. Note the alignment all along a single plane such that they appear circular (cut through cross sections). B: Close up view of the symbionts in cross section. Bacterial cells occur singly, or in pairs or groups of three within individual vacuoles. C. Bacteriocytes where many of the symbionts have been cut longitudinally, showing the rod shaped morphology.