Vestimentiferan tubeworm specimens were retrieved from two different colonies termed colony #1 and #2 on the western summit of the Palinuro volcanic complex (Mediterranean Sea, 39°32.44′N, 14°42.38′E, depth: 630 m) during the Pos412 cruise of R/V Poseidon in spring 2011. Sampling was conducted using a Mohawk-type remotely operated vehicle (ROV) supplied by Oceaneering Inc. (Aberdeen, UK) fitted with a robotic arm. Locations of the tubeworm collections are given in Table 1.

The ROV was also equipped with a fluid sampling system (Kiel in situ pumping system, KIPS, Garbe-Schönberg et al., 2006) capable of acquiring four 550 mL water samples per dive with in situ filtration. Parallel to the sampling nozzle was a temperature probe attached to a data logger. Fluids and temperatures around the colonies were sampled using KIPS and the temperature probe by maneuvering the ROV’s robotic arm into the fluid in close proximity to the living tubeworms. Live tubeworms were sampled using the ROV’s robotic arm. Immediately after tubeworm sampling, the dive was terminated, and the ROV was recovered. Upon recovery, the tubeworms obtained from colony #1 were put into sterile Petri dishes using sterile tweezers followed by dissection with a sterile scalpel. The animal was then separated from the tube; subsamples were recovered from vestimentum (host tissue free of symbionts) and trophosome (endosymbiont) tissue, and then were stored at −20°C until further molecular analysis. Samples from colony #2 were immediately stored at −20°C until further processing in the home laboratory. As the tubeworms from colony #2 exhibited several morphotypes, these were stored in separate vials.

Gulf of Mexico vestimentiferan tubeworms were sampled from hydrocarbon seep sites during several research cruises between 1997 and 2011 (Table 2; Figure 2). Lamellibrachia luymesi and Seepiophila jonesi were collected using the Johnson Sea Link submersible from two sites on the Upper Louisiana Slope from about 540 m depth. Lamellibrachia sp. 1 and Lamellibrachia sp. 2 as well as Escarpia laminata were collected from three sites on the Lower Louisiana Slope ranging in depth from 1,975 to 2,604 m. While on board the research vessel, tubeworms were dissected and vestimentum (host tissue free of symbionts) and trophosome (endosymbiont) tissue was preserved at −80°C or in 95% ethanol solution. All tissue samples were transported to the Pennsylvania State University where whole genomic DNA was obtained using a modified version of the high salt extraction protocol and ethanol precipitation as in Liao et al. (2007). Isolated DNA is currently stored at −80°C at the Pennsylvania State University.

Genomic DNA was extracted from the trophosome and vestimentum tissues of the vestimentiferan tubeworms. The tubeworms were dissected and washed three times in 0.2 μm filtered seawater prior to DNA extraction. DNA of Mediterranean tubeworm samples was isolated using the MoBio Power Biofilm Kit (Mo Bio Laboratories, Carlsbad, CA, USA) according to the protocol provided. DNA of GoM tubeworm samples was extracted following the protocol of Liao et al. (2007). Cytochrome c oxidase I (COI) genes, mitochondrial and bacterial ribosomal (16S rRNA) genes, as well as cbbM and ACL type II genes were analyzed from all individuals. Further functional genes and eukaryotic ribosomal (18S rRNA) genes were analyzed from one individual of colony #1 from the Palinuro volcanic complex.

For all gene amplifications of Mediterranean samples PCR reactions were conducted using Ready-To-Go PCR Beads (GE Healthcare, Munich, Germany) in a total volume of 25 μL. PCR from GoM samples were conducted using 1 U BioBasic TaqPolymerase (BioBasic Inc., Markham, ON, Canada) and 1× Thermopol Buffer (NEB Inc., USA) in a total volume of 50 μL. If not stated otherwise 10 pmol of each primer and 100 ng template DNA was used. For all amplifications, initial denaturation was 2 min at 94°C, final annealing was 1 min at annealing temperature, and final elongation 5 min at 72°C. For the cycles denaturation was 40 s at 94°C, annealing duration 40 s at the respective annealing temperature and elongation was 1 min at 72°C. If not stated otherwise, 35 PCR cycles were applied. Fragments of the tubeworms’ 18S rRNA and mitochondrial 16S rRNA as well as COI genes were amplified using the (i) primer pairs 5′-start (5′-GGT TGA TCC TGC CAG-3′) and 1753rev (5′-GCA GGT TCA CCT ACG G-3′) targeting the 18S rRNA gene (30 cycles, 50°C annealing temperature), (ii) primer pair 16Sar/16Sbr (Palumbi et al., 2002) targeting the mitochondrial 16S rRNA gene (30 cycles, 50°C annealing temperature), and (iii) primers LCO 1490 (5′-GGT CAA CAA ATC ATA AAG ATA TTG G-3′) and HCO 2198 [5′-TAA ACT TCA GGG TGA CCA AAA AAT CA-3′; 40 cycles, 47°C annealing temperature (Folmer et al., 1994)] using DNA extracted from the symbiont-free vestimentum tissue. Gene fragments of the endosymbiont were amplified using DNA extracted from trophosome tissue as the template. Bacterial 16S rRNA gene fragments were amplified in a 30 cycle PCR at an annealing temperature of 50°C with the general bacterial primer set 27F and 1390R (Palinuro samples; 5′-GAC GGG CRG TGT GTA CAA-3′) or 1492R (GoM samples; Lane, 1991). Amplification for fragments of dsrA and aprA genes was performed using the primer sets rdsrA240F/rdsrA403R and aps1F/aps4R, respectively (Meyer and Kuever, 2007b; Lavik et al., 2009). Fragments of soxB were amplified using the primers soxB432F/soxB1446B [10 cycles with 55°C annealing temperature and 25 cycles with 47°C annealing temperature (Petri et al., 2001)]. For the amplification of fragments of the genes coding for the large subunit of RubisCO form I and II, the primer sets cbbLF/cbbLR and cbbMF/cbbMR were used [both include two initial cycles of 2 min annealing at 37°C and 3 min elongation at 72°C, as well as additional 35 cycles of 53 and 58°C annealing temperature for cbbL and cbbM respectively (Campbell and Cary, 2004)]. A fragment of the large subunit of the putative type II ATP citrate lyase gene was amplified using the newly designed primer acl2F1 (5′-CGT CGC CAA GGA AGA GTG GTT C-3′) and acl2R1 (5′-GGC GAT GGC CTC AAA GCC GTT-3′) in a 30 cycle PCR with annealing temperatures of 45–56°C (gradient). Fragments of the hydrogen uptake hydrogenase gene hupL were amplified with the primer set HUPLX1/HUPLW2 (Csaki et al., 2001). A fragment of the norCB gene for nitric oxide reductase subunits C and B was amplified using the primer set norC21mF and norB6R (Tank, 2005) in a 35 cycle PCR using annealing temperatures of 60–50°C (10 touchdown cycles 60°C/−1°C, 25 cycles of 50°C).

Additional primer pairs used in this study include: F2/R5 and 892F/1204R for the two subunits of ATP citrate lyase (Campbell et al., 2003; Hügler et al., 2005); MxaF1003, MxaR1555, MxaR1561 for methanol dehydrogenase gene mxaF (Neufeld et al., 2007; Kalyuzhnaya et al., 2008); mmoXA/mmoXB for genes encoding the conserved alpha-subunit of the hydroxylase component of the cytoplasmatic soluble methane monooxygenase (sMMO; Auman et al., 2000); and A189F/MB661R for the particulate methane monooxygenase (pMMO) genes present in methanotrophs (Costello and Lidstrom, 1999).

All PCR products were purified via gel extraction using QIAquick gel extraction kit (QIAgen, Hilden, Germany) for Mediterranean samples, and BioBasic EZ-10 spin columns (BioBasic Inc., Markham, ON, Canada) for GoM samples respectively, and either directly sequenced by Sanger sequencing (18S rRNA gene fragments, COI, and functional gene fragments) or cloned into pCR4-TOPO vectors with the TOPO-TA cloning kit (Invitrogen, Carlsbad, CA, USA) as described by the manufacturer before sequencing (16S rRNA gene fragments). Sequencing was conducted using amplification primers and additional internal primers in the case of 16S rRNA genes (342F, 534R; Muyzer et al., 1996); 790F (Thiel et al., 2007). Amplification and sequencing of clones was conducted using vector specific primers M13 forward and M13 reverse (PCR) and T3 and T7 (sequencing), respectively. Sanger sequencing was performed using the BigDye Terminator v1.1 sequencing kit in a 3730xl DNA Analyzer (Applied Biosystems, Carlsbad, CA, USA) as specified by the manufacturer. Sequencing was conducted by the Institut für Klinische Molekularbiologie (IKMB), Universitäts-Klinikum Schleswig-Holstein (UK-SH), Kiel, Germany and the sequencing core facility at The Pennsylvania State University, University Park, PA, USA.

All sequences were edited with ChromasPro c.c1.33 and compared to the NCBI database using BLAST (Altschul et al., 1997). Functional gene nucleotide sequences were also compared with the non-redundant protein sequence database using the blastx algorithm. The endosymbiont 16S rRNA gene sequences were aligned with the ARB software (www.arb-home.de) using the ARB FastAligner utility (Ludwig et al., 2004). The sequence alignment was manually refined based on known secondary structures. Sequences of functional genes as well as mitochondrial rRNA genes were aligned using Clustal X (Thompson et al., 1997) and manually adjusted using BioEdit (Hall, 1999). Maximum Likelihood based trees and 100 bootstrap replicates were constructed using PhyML (Guindon and Gascuel, 2003). In order to verify the tree topology, further phylogenetic analyses using Neighbor-Joining and Maximum-Parsimony algorithms were conducted using MEGA5 (Tamura et al., 2011).

The morphology of endosymbiotic bacteria in trophosome tissue of the tubeworms was examined using light microscopy and scanning electron microscopy (SEM). Samples for light microscopy were prepared by removing small pieces of tissue from different parts of the trophosome and subsequent squeezing preparation in a drop of particle-free seawater and examined under 100-fold magnification using a Zeiss Axiophot Epifluorescence Microscope.

Samples for SEM were prepared by disrupting small trophosome samples in 0.2 μm filtered seawater, and then concentrated by filtration on to 0.2 μm polycarbonate membrane filters followed by dehydration through ascending concentrations of ethanol. Subsequently, the samples were critical-point-dried using a Balzers CPD 030 and CO₂ as a transition medium. The filters were sputter-coated with gold-palladium using a Balzers SCD 004 and observed with a Zeiss DSM 940 electron microscope.

After recovery of the ROV Mohawk all KIPS fluid samples were immediately transferred to the onboard ship lab and sub-sampled for subsequent analyses. Both pH and Eh of the fluids were determined immediately after sub-sampling using electrochemical techniques after calibration with certified standards. Dissolved oxygen was determined using standard Winkler titration protocols modified for small volumes. The concentration of dissolved sulfide was determined in 1 mL aliquots using a zinc acetate gelatin solution, which precipitates the dissolved sulfide as colloidal zinc sulfide. Subsequently, the color agent, N,N-dimethyl-1,4-phenylenediamine-dihydrochloride, and a catalyst, iron chloride solution, were added to form methylene blue (Cline, 1969). After 1 h, the solutions were measured photometrically at a wavelength of 660 nm using a Genesys Spectra 10 spectrophotometer. The detection limit was 1 μmol/L. Potential oxidation of dissolved hydrogen sulfide during sampling and sample recovery cannot be ruled out, but is likely minimal. Nonetheless, hydrogen sulfide concentration data given in this paper should be considered minimum values. Aliquots for the analysis of nutrients were stored in polypropylene bottles, sealed, and stored in the dark at 4°C until analysis. Aliquots for cation and trace element analysis were pressure-filtrated through 0.2 μm Nucleopore polycarbonate (PC) membrane filters using Sartorius PC filtration units and high purity nitrogen. Samples were acidified with subboiled nitric acid to pH <2 and stored in perfluoralkoxy (PFA) bottles until analysis. Multielement analysis for major ion composition (Cl, B, Si, Na, K, Ca, Mg, Fe, Mn) of the water samples was performed with a SPECTRO Ciros SOP ICP-OES spectrometer after 10-fold dilution and using Y for internal standardization. Trace elements (As, Li, W) were determined by ICP-MS (Agilent 7500 cs at University of Kiel) after 12.5-fold dilution using both In and Re for internal standardization. Certified reference materials NIST1643e, NASS-5, and IAPSO were used for validation and accuracy checks.

The organic carbon isotopic composition of tubeworm tissue (δ¹³C_ORG) was determined via continuous flow EA-IRMS using an elemental analyzer interfaced to a ThermoFinnigan Delta Plus isotope ratio mass spectrometer. Briefly, about 40–60 μg of freeze-dried worm tissue was weighed in tin capsules, combusted to CO₂, and chromatographically purified carbon dioxide was transferred to the mass spectrometer in a He gas stream. Results are reported in the standard delta notation as per mil difference to the Vienna Pee Dee Belemnite. Sample measurements were done in duplicate, and analytical performance was monitored with international reference materials (USGS 24; USGS 40) and lab standards (anthracite; brown coal) and the reproducibility was generally better than ±0.15‰.

The carbon isotopic composition of dissolved inorganic carbon (δ¹³C_DIC) from vent fluids was measured using a Thermo Finnigan Gas Bench coupled to a Thermo Finnigan Delta Plus XL. Briefly, 0.5–1.0 mL of hydrothermal fluid was injected into an exetainer that contained phosphoric acid, liberating DIC as carbon dioxide. Prior to sample injection, the exetainer was flushed with helium. CO₂ was flushed from the exetainer with a stream of helium and injected into the mass spectrometer. Results are reported in the standard delta notation as per mil difference to the Vienna Pee Dee Belemnite. Sample measurements were done in duplicate, and analytical performance was monitored with a sodium carbonate lab standard.

The sequence data have been submitted to EMBL/GenBank/DDBJ databases under accession numbers HE9744464-85, HE978212-46, and HE983327-53.

For the present study, two colonies of vestimentiferan tubeworms as well as their biogeochemical environment were sampled by means of a ROV. The tubeworms occurred within a sediment-filled depression at the western summit of the Palinuro volcanic complex in water depths of around 630 m and formed small bushes, up to 1 m² in diameter, mainly on sedimented surfaces but some patches also occurred in areas where volcanic rocks were present at the seafloor. Frequently, shimmering water was observed rising above the tubeworm colonies suggesting active fluid emanation. The first colony (colony #1) appeared to be comprised mainly of equally sized animals. The second colony (colony #2), however, consisted of animals of different sizes, indicating different ages, or variation in exposure to hydrothermal fluid at different places within the colony.

Remotely operated vehicle assisted measurements of water temperature across and within the tubeworm colonies revealed a maximum recorded temperature of 19.4°C, about 5°C above ambient seawater temperatures of 14°C (Table 1). Diffusely venting warm hydrothermal fluids reached a maximum temperature of 58.4°C within small depressions in close proximity to the tubeworms. Chloride concentrations in these fluids were significantly higher than in ambient seawater indicating influx of hydrothermal brine eventually leading to the formation of small stratified brine pools in these depressions. Chemical analyses of vent and pore fluids sampled from sediment cores collected in the same area revealed that local hydrothermal fluids were anoxic, H₂S-rich, acidic and displayed an elevated salinity. Concentrations of dissolved alkali and alkali earth elements (potassium, calcium, lithium, cesium), silica, arsenic, and tungsten (“Fluid end-member” in Table 3) were significantly higher when compared to normal bottom seawater. Fluid samples confirmed that this hydrothermal fluid was highly diluted with ambient seawater as it passed through the two tubeworm colonies leading to partly oxygenated waters (139 and 227 μmol/L dissolved O₂, Table 3) and reduced levels of sulfide. Still, dissolved sulfide concentrations of 32 and 72 μmol/L were measured in water samples from among the tubes in the tubeworm colonies. In contrast, a maximum concentration of dissolved sulfide of 5,172 μmol/L was measured for the “hottest” hydrothermal fluids (58°C) sampled at Palinuro.

Bulk organic carbon isotopes analyses of gill tissue from two Palinuro tubeworms resulted in δ¹³C values of −22.5 and −23.4‰, which are in accordance to previous measurements of Mediterranean Lamellibrachia spp. (Olu-Le Roy et al., 2004; Carlier et al., 2010) but more positive than most Lamellibrachia spp. from non-Mediterranean hydrocarbon seeps (Becker et al., 2011). The carbon isotopic composition of dissolved inorganic carbon in emanating diffuse fluids sampled at the tubeworm colonies display δ¹³C values of −0.39 and −0.68‰. The δ¹³C_DIC values of additional samples of shimmering water in the area range from −1.62 to +1.76‰.

The discovery of living vestimentiferan tubeworm colonies associated with active hydrothermal venting during a seafloor survey of the Palinuro volcanic complex (Mediterranean Sea) in July 2006 (Petersen et al., 2008; Monecke et al., 2009) came as a surprise, as until then, vent-associated tubeworms were only known from the Pacific Ocean. Living individuals of the tubeworms were sampled during a research cruise in 2011 and this communication is the first description of the worms and their endosymbionts. Phylogenetic analyses of 18S rRNA, COI and mitochondrial 16S rRNA genes showed that the tubeworms from Palinuro are Lamellibrachia anaximandri recently described from mud volcanoes of the Eastern Mediterranean (Southward et al., 2011). The highest in vivo temperatures measured among the tubes in tubeworm aggregations at the Palinuro hydrothermal vent field were 15.6–19.4°C, elevated by as much as 5.4°C from the surrounding Mediterranean Seawater (14°C) and the previously published tubeworm-bearing locations in the Eastern Mediterranean (13–14°C), extending the previously described temperature range of the species (Southward et al., 2011). L. anaximandri has also been detected in a mud volcano field in the Western Mediterranean (Hilário et al., 2011), as well as on two ship wrecks in the Eastern Mediterranean (110 km southeast of Crete, Hughes and Crawford, 2008) and the Southern Tyrrhenian Sea (Gambi et al., 2011; Figure 1). Even though this species has not been detected in the Northeastern Atlantic it has been hypothesized to occur at the West African and Lusitanian margins as well (Hilário et al., 2011).

The high diversity of habitats for the Mediterranean Lamellibrachia species is in accordance with Escarpia spp. and other Lamellibrachia spp. Originally regarded as seep species, they were subsequently found in several non-seep habitats, i.e., at sediment covered hydrothermal sites in the Pacific (Juniper et al., 1992; Fujikura et al., 2006; Miake et al., 2006; Miura and Kojima, 2006), as well as ship wrecks (Dando et al., 1992), and whale falls (Feldman et al., 1998). Considering the high diversity of so-called “seep” tubeworm habitats a high site-flexibility of these organisms becomes apparent and supports the importance of different non-seep habitats in their geographic distribution and the stepping stone hypothesis (Kimura and Weiss, 1964; Smith and Kukert, 1989; Black et al., 1994; Olu et al., 2010). Larval survival of at least three weeks and about five weeks has been demonstrated for the vestimentiferans Riftia pachyptila and Lamellibrachia luymesi respectively, suggesting potential dispersal distances on the order of 100 km for seep and vent vestimentiferans (Young et al., 1996; Marsh et al., 2001; Tyler and Young, 2003). A variety of reducing habitats, functioning as dispersal stepping-stones separated by days or weeks and connected by currents or shared water masses could facilitate the large species ranges described for many vestimentiferans, including the seep species L. barhami, which has been found in seep and low activity vent sites spanning at least 4,000–6,000 km of geographical distance (McMullin et al., 2003).

The trophosome of the L. anaximandri specimens from Palinuro analyzed in this study harbored a single gammaproteobacterial phylotype regardless of collection site or morphotype. The endosymbiont was closely related (99.7%) to the phylotype 2 found in L. anaximandri from the Amon mud volcano east of the Nile deep-sea fan (Duperron et al., 2009). The dominating endosymbiont (phylotype 1) of the seep specimen from the Amon mud volcano was not found in the tubeworms at the Palinuro hydrothermal vents.

This study is the first to characterize vestimentiferan tubeworm endosymbionts of shallow hydrothermal vents in the Mediterranean Sea. The gammaproteobacterial endosymbiont clusters with endosymbionts of other seep-associated tubeworms and are clearly distinct from the endosymbionts of vent tubeworms like Riftia and Tevnia (Fisher et al., 1997; Nelson and Fisher, 2000; McMullin et al., 2003; Vrijenhoek et al., 2007). The phylogenetic affiliation of the Palinuro L. anaximandri endosymbiont with “group 3” endosymbiont 16S rRNA gene sequences, as well as the affiliation of the dominating phylotype of L. anaximandri specimen obtained from 1,157 m depth at Amon mud volcano with “group 1,” may indicate separation by depth, as suggested for other seep vestimentiferan endosymbionts (McMullin et al., 2003). However, both Mediterranean phylotype sequences show considerable differences to the “group 1” and “group 3” cluster sequences in signature nucleotide positions (Table A1 in Appendix) and Mediterranean and GoM tubeworms do not share identical endosymbiont phylotypes. Further, in the Amon mud volcano specimen, phylotypes of “group 1” and “group 3” are present, yet in assumed different abundances (deduced from the numbers of sequences in the clone libraries; Duperron et al., 2009). Endosymbionts of different groups are also present in individuals of Lamellibrachia sp. 2 (AC601_L20) and E. laminata (AC601_L1) tubeworms from the GoM site AC601 (this study). Thus separation by depth alone cannot explain these observations and further studies are needed in order to reveal the question how endosymbionts are selected by their vestimentiferan hosts.

Based on the functional gene analyses of this study, the endosymbiont of Lamellibrachia anaximandri from Palinuro is a sulfide-oxidizing chemoautotroph. δ¹³C values measured in this study are in consistence with previous studies of L. anaximandri from Eastern Mediterranean mud volcano fields (Olu-Le Roy et al., 2004; Carlier et al., 2010), and together with delta δ¹⁵N and δ³⁴S from previous studies support a chemoautotrophic endosymbiont based nutrition for the host tubeworm (Carlier et al., 2010). Due to the presence of dsrAB and aprA genes, sulfide oxidation most likely is carried out via the APS pathway with sulfite and adenosine phosphosulfate as intermediates. As in the Riftia pachyptila endosymbiont, soxB is also present in the endosymbiont of L. anaximandri from Palinuro. In thiosulfate-utilizing bacteria, SoxB functions as sulfate thiohydrolase. However, since tubeworm endosymbiont carbon fixation is not stimulated by thiosulfate, its function in the tubeworm endosymbionts remains uncertain (Fisher et al., 1989; Markert et al., 2011).

Although high methane fluxes were noted in the habitat of L. anaximandri in mud volcano habitats (Olu-Le Roy et al., 2004), genes of methane oxidation were not successfully amplified with the primer sets used in this study. In contrast, the potential to use hydrogen as an energy source was suggested by the detection of the key gene for hydrogen oxidation, hupL in the endosymbiont from the Palinuro L. anaximandri. The hupL gene was most similar to the respective genes of the Riftia and Tevnia endosymbionts, where it is even expressed in situ (Markert et al., 2011; Petersen et al., 2011). Hydrogen concentrations have not been measured in the hydrothermal fluids from Palinuro volcano complex. However, hydrogen is present in the fluids at many hydrothermal vents, and elevated hydrogen contents are present at vent systems associated with ultramafic (mantle) rocks, or, e.g., following a volcanic eruption (Wetzel and Shock, 2000; Allen and Seyfried, 2003; Kumagai et al., 2008; Petersen et al., 2011). Such H₂-rich fluids found at vent systems associated with ultramafic rocks have recently been shown to be used as an energy source by the endosymbionts of a mussel, Bathymodiolus puteoserpentis (Petersen et al., 2011). The endosymbionts in Bathymodiolus spp. mussels are located on the external edge of the cells of gill filaments that are themselves only two cells thick. As a result, passive diffusion of the energy source (hydrogen, sulfide, and/or methane in different species) is sufficient to fuel the chemoautotrophic life style of these animals (Childress and Fisher, 1992). In contrast, the endosymbionts in tubeworms are deep in an interior tissue and must rely on the host blood to supply the electron donor for chemoautotrophy. Sulfide is transported in millimolar concentrations to the trophosome, bound to hemoglobin molecules in vestimentiferan tubeworms. Transport molecules for hydrogen or methane have not been found in these animals and thus both hydrogen and methane are unlikely to contribute significantly to the metabolism of the intact symbiosis in most environments (Childress and Fisher, 1992). However, the gammaproteobacterial endosymbiont might use hydrogen as potential energy source in its free-living stage. Thus, the potential for use of hydrogen by tubeworm endosymbionts deserves additional study.

Detection of cbbM sequences coding for a form II RubisCO in all vestimentiferan tubeworms reviewed in this study indicates that the potential to fix CO₂ via the CBB cycle is widespread in vestimentiferan tubeworms (Elsaied and Naganuma, 2001; Elsaied et al., 2002; Naganuma et al., 2005; Vrijenhoek et al., 2007; Duperron et al., 2009) and the detection of all genes of this cycle in the metagenome of the Riftia endosymbiont suggest this pathway is fully functional in vestimentiferans (see Markert et al., 2011 for further details). Enzyme activity measurements of RubisCO and phosphoribulokinase add further evidence to the usage of the CBB cycle in Riftia and Lamellibrachia endosymbionts (Felbeck, 1981; Felbeck et al., 1981). Up to now, there is no evidence for the presence of RubisCO form I (cbbL) in either the Riftia/Tevnia or any Lamellibrachia endosymbiont (this study; Elsaied and Naganuma, 2001; Elsaied et al., 2002; Naganuma et al., 2005; Duperron et al., 2009).

In addition, acl genes, coding for ATP citrate lyase, the key enzyme of the rTCA cycle were recovered from the Palinuro L. anaximandri endosymbiont using newly designed primers, suggesting the presence of the rTCA cycle as alternate carbon fixation pathway. The operation of the rTCA cycle in addition to the CBB cycle was first shown for the Riftia and Tevnia endosymbiont using a combination of metagenomic, proteomic and enzymatic approaches (Markert et al., 2007; Robidart et al., 2008; Gardebrecht et al., 2012). In the case of the Riftia/Tevnia endosymbiont, citrate cleavage is accomplished by an unusual type of ATP citrate lyase, tentatively named ACL type II (Hügler and Sievert, 2011). The recovered acl sequence from the Mediterranean Palinuro L. anaximandri endosymbiont showed high similarities to the sequence of the Riftia/Tevnia endosymbiont (Figure 8A). Subsequent analyses of seep vestimentiferan (Escarpia, Seepiophila, and Lamellibrachia) endosymbionts from different sites at the Gulf of Mexico showed the presence of type II ACL genes there as well. This implicates a wider distribution of these genes than previously thought. The presence of two different carbon fixation pathways – the CBB cycle and the rTCA cycle – in a single bacterium seems not restricted to the Riftia/Tevnia endosymbiont, but rather seems to be a common feature of vestimentiferan tubeworm endosymbionts, regardless of genus or habitat.

Despite the still rather scarce dataset of type II ACL gene sequences, these sequences appear to be monophyletic in tubeworm endosymbionts (Figure 8A). Similarly, ribosomal genes as well as aprA genes support a monophyletic origin for the tubeworm endosymbionts (Figures 6 and 7). In contrast, a monophyletic origin of the cbbM gene of tubeworm endosymbionts is not clearly supported by the phylogenetic analyses performed here (Figure 8B). This could mean that the rTCA cycle is the evolutionary older CO₂ fixation pathway in the endosymbionts, and the cbbM gene is acquired afterward, e.g., via lateral gene transfer. This evolutionary aspect clearly requires further studies.

The presence of two different carbon fixation pathways increases the metabolic versatility of the tubeworm endosymbionts. In case of the Riftia/Tevnia endosymbiont proteomic data suggest the usage of both pathways simultaneously (Markert et al., 2007, 2011; Gardebrecht et al., 2012). This is also supported by the isotopic signature of the Riftia tubeworms (Markert et al., 2007). The carbon isotopic fractionation associated with the rTCA cycle is generally smaller than the one observed for the CBB cycle (House et al., 2003). Considering the carbon isotopic composition of ca. −23‰ measured for plume tissue of two L. anaximandri tubeworms from Palinuro low temperature diffuse vent sites and a respective carbon isotopic composition of dissolved inorganic carbon (δ¹³C_DIC between −0.7 and −0.4‰), the isotopic difference would be consistent with the operation of the CBB cycle for autotrophic carbon fixation. Neither a greatly attenuated isotopic fractionation characteristic for the rTCA cycle nor an isotopic signature reflecting a substantial contribution from methane-derived carbon is discernible at the site studied here. Yet, one has to keep in mind, that the isotopic signature provides only indirect evidence and neither the actual fractionation by the enzymes involved in the rTCA cycle present in tubeworm endosymbionts, nor fractionation during uptake and transport of DIC to the endosymbionts are known. Thus future studies are needed in order to determine the conditions for the usage, as well as the regulation of the two different carbon fixation pathways in vestimentiferan tubeworm endosymbionts (Hügler and Sievert, 2011).