Following the traces of symbiont bearing molluscs during earth history 77: 83-97, 6 figs. 2014 Anne Dreier1,2 * & Michael Hoppert1,2 1Institute of Microbiology and Genetics, Georg-August University Göttingen, Grisebachstr.8, 37077 Göttingen, Germany; Email: anne.dreier@geo.uni-goettingen.de 2Courant Centre Geobiology, Geoscience Centre, Georg-August University Göttingen, Goldschmidtstr. 3, 37077 Göttingen, Germany * corresponding author Ivan E. Wallin (1883–1969) was among the first scientists who noticed the evolutionary impact of symbiotic events. He proposed that endosymbiosis was the principal source for speciation (Wallin 1927). Mitochondria and chloroplasts as symbiotic descendants of bacteria in a eukaryotic cell are the well-known and most important endosymbiotic key play- ers, enabling and shaping the evolution of eukaryotes. In addition, a multitude of other symbioses between prokaryotes and Eukarya have been described so far. As an example, symbiosis between molluscs and sulphur- or methane- oxidising bacteria, is a widespread lifestyle in marine habitats (and perhaps yet undetected for other environments). These symbiotic associations occur worldwide at oxic–anoxic interfaces such as at the boundary layer of reducing sedi- ments, in cold seeps, in hydrothermal vents or in mangrove peat. The symbiosis between marine molluscs and chemo- synthetic bacteria increase the metabolic capabilities and therefore the possibilities to occupy ecological niches of both host and symbiotic prokaryote. Nowadays, due to molecular analyses and in situ hybridisation techniques, detection of symbioses in recent living organisms is not that difficult. But finding a path back to the point in Earth’s history were symbiotic events took place is a tricky challenge. Not long ago only analyses of morphological features of shells and fa- cies criteria were available for assessment of the lifestyle and the diet of extinct bivalves. Close phylogenetic relation- ships to recent symbiont bearing genera in a similar habitat make it likely that the extinct genera exhibited a similar life- style, but these indirect criteria are not sufficient to uncover ancient symbiosis in molluscs. In this review several ap- proaches of “molecular palaeontology” are discussed, which allow for a direct determination of a symbiotic or non- symbiotic lifestyle in recent and fossil molluscs. Received: 06 January 2013 Subject Areas: Palaeontology, Zoology, Molecular Palaeontology Accepted: 01 August 2013 Keywords: Mollusca, Bacteria, Eukarya, Recent, fossil, symbiotic lifestyle, phylogeny Introduction “It is concluded that the evolutionary potential of symbio- sis is great and that symbiosis serves as a supplementary speciation mechanism capable of producing directed evo- lutionary changes” (Taylor 1979). This conclusion may be particularly true for bivalves: bacteria and marine mol- luscs, often form mutualistic partnerships which markedly influence the physiology, ecology and evolution of both. Autotrophic bacteria assimilate inorganic carbon as primary carbon source. The bacterium is chemoauto- trophic when reducing power and energy needed for as- 84 Anne Dreier & Michael Hoppert similation of carbon dioxide derives from reduced inor- ganic compounds. The other important energy source is sunlight for photoautotrophic organisms. Chemoauto- trophic or methanotrophic bacteria are found in a wide range of reducing habitats providing these coveted energy sources, in particular H2, H2S or CH4. Most prominent sites are cold seeps and hydrothermal vents, but dysoxic conditions are frequent in marine environments, such as in seagrass beds, mangrove sediments or wood and whale falls. These habitats of free living autotrophic or metha- notrophic bacteria are usually also inhabited by molluscs hosting symbiotic chemoautotrophs or methanotrophs (Lonsdale 1977; Corliss et al. 1979; Jannasch & Wirsen 1979; Van Dover 2000; Treude et al. 2009; Kiel & Tyler 2010). Though also bacteria of other metabolic types are symbionts of marine invertebrates, most of them are sul- phur-oxidiser or methanotrophs belonging to the Gam- maproteobacteria. According to phylogenetic analyses these symbioses have been established multiple times in earth history and evolved independently (Dubilier et al. 2008). Several marine molluscs, especially some species of Cephalopoda, Gastropoda and Bivalvia, are known to cul- tivate symbiotic microbes. The basic feature of this rela- tionship is that the symbionts need reduced substrates and electron acceptors for their metabolism, which do not oc- cur in the same microenvironments (Zhang & Millero 1993). The molluscs are able to bridge the oxic–anoxic boundaries using behavioral, morphological or metabolic adaptations and supply substrates (e.g., reduced sulphur compounds) and electron acceptors (oxygen in most cas- es) to the microbes. In turn, most if not all organic carbon and also nitrogen compounds are provided by the symbi- ont (Cavanaugh et al. 2006; Dubilier et al. 2008). Photosymbiosis is most successful in oligotrophic wa- ter under nutrient-limited conditions (Hallock & Schlager 1986; Hallock 1987; Schlager 2003). Eukaryotic algae of the genus Symbiodinium (zooxanthellae) are the most preva- lent symbionts of molluscs. The zooxanthellae satisfy a major part of the host's energy demand (Trench et al. 1981; Klumpp et al. 1992; Hawkins & Klumpp 1995). In turn, zooxanthellae cover their nitrogen and phosphate demands mainly through their host’s excretion products. Multiple studies provided insight into symbiont-host in- teraction, their metabolic features and how symbiotic partners are adapted to each other. Various approaches like 16S ribosomal DNA sequence analysis, fluorescence in situ hybridisation, transmission electron microscopy, stable isotope and fatty acid analysis were applied so far (Kharlamenko et al. 1995; McKenzie et al. 2000; Colaco et al. 2007). However, studies aiming at reconstruction of evolution of symbiotic molluscs were based on compari- son of shell morphologies or were conducted in specific palaeoenvironments like vents and seeps (Fig. 1). In order to get a better understanding of the evolu- tionary steps and to give an estimate for the time point when a representative of a mollusc taxon starts its cooper- ation with microbes and shifts its diet to chemo- or pho- totrophic we need to detect a direct symbiotic fingerprint of the investigated fossil. These fingerprints or biosigna- tures must be stable in geological timescales. Fig. 1: Late Miocene seep at Montepetra (Italy) with a mass occurrence of lucinid clams (Heterodonta: Lucinoida). Endosymbiotic molluscs Recent situation Within the clades of recent molluscs, endosymbiosis with sulphur- or methane-oxidising (chemosynthetic) bacteria occur in seven bivalve families: Solemyidae, Nucinellidae, Montacutidae, Mytilidae, Thyasiridae, Lucinidae (being the most diverse familiy), Vesicomyidae (Taylor & Glover 2009, 2010; Taviani 2011, and references therein; Taylor et al. 2011; Oliver et al. 2013). In addition, the Teredinidae are known to harbour endosymbiotic cellulose-digesting symbionts (Distel et al. 2002). Symbiont bearing molluscs during earth history 85 The symbiosis seems to be obligate in all species of Lucin- idae, Vesicomyidae and Solemyidae, while some species of Thyasiridae and Mytilidae are asymbiotic. Their life styles are highly diverse, ranging from epifaunal to deep infaunal (Taylor & Glover 2010). Lucinids occur also in the deep sea at cold seeps (Callender & Powell 1997), hydrothermal vents (Glover et al. 2004) and wood or whale falls (Dubi- lier et al. 2008). Though they appear to be rare on such sites, Kiel & Tyler (2010) stated that this might be a sam- pling artifact. Nevertheless, the more common molluscs of deep-sea habits are all three families of chemosynthetic gastropods, bathymodiolian mussels, vesicomyid clams and solemyids (Dubilier et al. 2008; Kiel & Tyler 2010). A symbiotic relationship to chemosynthetic bacteria is also known from three gastropod families (Provannidae, Lepetodrilinae and Peltospiridae) and from one family of the Aplacophora (Simorthiellidae, Dubilier et al. 2008). These organisms are all inhabitants of deep sea seep and vent sites. Deep sea hydrothermal vents with their rich and con- stant supply of reduced inorganic compounds are perfect niches for a chemosynthetic lifestyle, which leads to mass development of chemosymbiotic molluscs in such envi- ronments. They are less abundant in the photic zones. The primary production in shallow water is driven by phototrophy and is usually dominated by heterotrophic communities; however, in some cases chemosymbionts could also dominate in shallow water (Dando & South- ward 1986; Little et al. 2002; Tarasov et al. 2005). Though, the role of chemosynthetic molluscs in shallow water sys- tems like coral reef sediments, seagrass meadows or man- grove sediments is not that unimportant. The evolutionary radiation of Lucinidae, for example, seems to be linked to the emergence of seagrasses in the late Cretaceous (Heide et al. 2012; and references therein). Lucinids are very im- portant for the stability of seagrass systems, because they detoxify the surrounding sediment from sulfide and lead to oxygenation, with a not negligible effect on seagrass (Heide et al. 2012). The highest diversity of recent Lucini- dae was described for tropical reefal habitats (Glover & Taylor 2007); also some solemyids (Krueger et al. 1996) and thyasirids (Dubilier et al. 2008) occur in this environ- ment as well as some photosynthetic Cardiidea and Tridacnidea. Some bivalve species within the Trapeziidae and Cardiidea (Fig. 2) maintain symbiotic associations with Symbiodinium (Yonge 1936; Kawaguti 1950, 1968, 1983; Purchon 1955; Stasek 1961; Hartman & Pratt 1976; Jacobs & Jones 1989; Jones & Jacobs 1992; Ohno et al. 1995; Persselin 1998; Vermeij 2013). These bivalves exhibit specific characteris- tics of soft body but also microstructural and macroscopic adaptations in shell morphology. Tridacnidea have very large and thick shells, others exhibit semitransparent shells; all adaptations should improve the exposure of the mantle to sunlight (ref. above). Fig. 2: Two phototrophic bivalves in their natural habitats. (A) Fragum un- edo (Heterodonta: Veneroida: Cardiidae) – mud flat of North Stradbroke Island, Queensland, Australia, and (B) Tridacna maxima (Heterodonta: Veneroida: Cardi- idae) – coral reef of One Tree Island, Queensland, Australia. Symbiont-bearing invertebrates in earth’s history Ancient shelled molluscs have a rich and well-documented fossil record; they are confirmed since the early Cambrian (Goedert & Squires 1990; Peel 1991; Gubanov et al. 2004; Vinther & Nielsen 2005; Skovsted et al. 2007; Kiel & Ty- ler 2010). The common method to detect ancient symbio- ses in some fossil bivalves/molluscs is based on structural features of their shells, e.g., the imprints of the elongated anterior adductor muscle and pallial blood vessels in the shells. In addition, palaeohabitat occupation patterns give important hints for symbiotic life styles (Taylor & Glover 2000; Amano et al. 2007). Indirect tools to date back sym- biotic molluscs evolution (estimated molecular age) are molecular clocks (Baco et al. 1999; Shank et al. 1999; Dis- tel et al. 2000; Kano et al. 2002). Upcoming direct molecu- lar tools are biogeochemical analyses of biosignatures which are described in detail below. 86 Anne Dreier & Michael Hoppert The longest fossil record and hence the oldest supposed symbiotic bivalves are the Lucinidae and Solemyidae. The existence of fossil Solemyidae dates back to the Ordovi- cian (Kiel & Tyler 2010), Lucinidae first appear during the Silurian (Taylor & Glover 2006). It was suggested that symbiotic relationship of both groups are ancient (Taylor & Glover 2000, 2006; Taylor et al. 2008). Thyasirids are possibly much younger; they were first described from the Early Cretaceous and were found at seeps and wood falls, which indicates possible symbiotic lifestyle (Kiel et al. 2008a, Kiel & Dando 2009). Vesicomyids are also associ- ated with seep deposits from the beginning of their ap- pearance in the Middle Eocene; from the Late Eocene onwards they could be found in large numbers at vents and seeps (Kiel & Tyler 2010 and references therein). Bathymodiolins also appear in the Middle to Late Eocene (Goedert & Squires 1990; Squires & Goedert 1991; Tavi- ani 1994; Kiel & Goedert 2006a; Kiel & Little 2006). All of these fossil molluscs had recent relatives, thus an an- cient symbiotic lifestyle of their ancestors living in similar environments was deduced (e.g., Goedert & Squires 1990; Taviani 1994; Goedert & Campbell 1995; Peckmann et al. 1999, 2002, 2004; Goedert et al. 2003; Gill et al. 2005; Majima et al. 2005; Campbell 2006; Kiel & Little 2006; Kiel & Peckmann 2007). Inoceramidae disappeared at the end of Cretaceous (Dhondt 1983) and were first known from the Permian (Cramton 1988). Some authors also speculated about chemosynthetic or even photosynthetic lifestyles of some inoceramid species (MacLeod & Hoppe 1992). The Cardiidea have a fossil record dating back to the Late Triassic (Keen 1980; Morton 2000; Coan et al. 2000; Schneider & Carter 2001). Recent members of Cardiidea with photosynthetic lifstyle like Fragum have a fossil rec- ord that dates back to Miocene/Holocene (Keen 1980), Tridacnids proliferate since the Eocene (Romanek et al. 1987). Futhermore, for the Neogen bivalve Mercenaria “tridacnoides” (Jones et al. 1988) for rudists and some other fossil bivalves a photosymbiotic lifestyle was postulated (Kauffman 1969; Philip 1972; Vermeij 2013). It was speculated about symbiotic relationships in some extinct non-bivalve species like brachiopods which were associated with chemosynthesis-dominated envi- ronments in their fossil record (Sandy 2010) and even photosymbiosis was postulated for some fossil rostrocon- chia and brachiopods (Cowen 1970, 1982; Vermeij 2013). Fortey (2000) reported that olenid trilobites (Late Cambri- an/Ordovician) lived under oxygen-poor and sulphur-rich conditions at the sea floor. Reduced oral structures and extended pleural areas were interpreted as an indication for a symbiotic relationship with sulphur bacteria. Also fossil members of gastropods inhabiting chemo- synthetic ecosystems, e.g., Provannids date back to the Late Cretaceous (Kiel & Tyler 2010). Though fossil deep-sea chemotrophic molluscs are rela- tively well-documented, not much attention is given to the non-seep related shallow water chemo- or phototrophic molluscs. At seep and vent sites the epifaunal molluscs densely colonise the habitat. Detecting fossil endosymbio- sis in shallow water molluscs by using biogeochemical techniques has an advantage that possibly different het- erotrophic molluscs co-occur in the same substrate. Com- paring different species in the same habitat gives a better indication of which might have had symbiotic associations with chemosynthetic bacteria or maybe phototrophic di- noflagellates. In the evolution of bathymodiolid bivalves, it was expected that the ancestors of this modern deep-sea mussels live in shallow water reducing sediments. Thus it is possible that “the first contact” between free-living chemosynthetic bacteria and heterotrophic bathymodiol- ids did not start in the deep-sea but in shallower marine environments (Duperron 2010). It will be really interesting to support these hypotheses by analysis of biosignatures. In shallower water habitats the probability to find fossils of definitive non-symbiotic molluscs among the putative symbiotic ancestor of bathymodiolids is much greater than at fossil seep and vent deposits. Here, it is possible to compare biosignatures of shell-fossils from different spe- cies of the same location, to evaluate their lifestyles (see below). Molecular markers in tissue of chemo- symbiotic vs. heterotrophic bivalves Prokaryotes are inhabitants of this planet long before the raise of eukaryotes and metazoans and consequently “in- vented” most of the biochemical key processes. They are the only organisms capable of primary energy production like chemosynthesis and photosynthesis; fixation of mo- lecular nitrogen is unique to prokaryotes. Thus, all other living organisms are able to perform primary production only with support of their ancient or current endosymbi- otic associations with prokaryotes. In any case, metazoans whose major diet is based on their autotrophic symbionts are closer to the bottom of the food chain than metazoans without relationship to such microbes. Some of the sym- biotic bacteria in molluscs are located within specialised gill cells, so-called bacteriocytes. In other cases the bacte- ria are attached extracellularly at the gill tissue (Dubilier et al. 2008; Duperron 2008; Southward 2008). Fluids, rich in oxygen and sulfide or methane, are drawn into the gill and are absorbed by the bacteriocytes. Furthermore, it was reported that Calyptogena use their foot to dig for sulfide in the sediment and then use specif- ic transport proteins which transfer sulfide to symbionts in the gill tissue (Zal et al. 2000). Symbiont bearing molluscs during earth history 87 The majority of phototrophic symbionts, the zooxanthel- lae, are located within mantle tissue, sometimes within the gill filaments of the host bivalve (Yonge 1981), so that the symbionts are exposed to sunlight. How may the lifestyle of symbiont bearing molluscs lead to identifiable features or even “patterns”, identifiable in the fossil record? In case that the major nutrients (carbon, nitrogen and sulphur) of molluscs derive from their prokaryotic symbi- onts, the host biomass is based on molecules built by the prokaryotic metabolism. In contrast, heterotrophic mol- luscs filter out or graze off particulate organic matter from their surrounding environment. Thus, search for finger- prints specific for either heterotrophic or chemo- and phototrophic molluscs must consider autotrophic and/or nitrogen metabolism of the symbionts. Carbon fixation Apart from the cellulose-degraders in Teredinidae and methanotrophs, all prokaryotic symbionts fix inorganic carbon autotrophically. The common pathway for CO2 fixation in chemo- as well as in phototrophic symbionts is the Calvin-Benson cycle (Herry & Le Pennec 1989; Duperron & Fiala-Médioni 2007; Dreier et al. 2012). The key enzyme of this pathway is ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). RubisCO catalyses the fixation of 12CO2 slightly faster than fixation of 13CO2 (Pu- rich & Allison 2000). This selection leads to an enrich- ment of 12C in the biomass relative to 13C (negative δ13C value). The host obtains the organic carbon from its sym- bionts (Fiala-Médioni & Felbeck 1990; Childress & Fisher 1992), hence δ13C ratios of host tissue reflect the carbon source. Mentionable is the fact that 13C depletion by chemoautotrophic bacteria using the Calvin-Benson cycle for CO2 fixation is higher than in photosynthetic algal or- ganisms, because of different specificities of RubisCO form I and II enzymes (Ruby et al. 1987; Blumenberg 2010; see Table 1). Moreover, these distinct forms of Ru- bisCO have been also described for chemoautotrophs (Robinson & Cavanaugh 1995). They show that form I RubisCO is expressed by the symbionts of Solemya velum and Bathymodiolus thermophilus, exhibiting relatively low δ13C ratios, whereas form II RubisCO is expressed in the tubeworms Riftia pachyptila and Tevnia jerichonana with high- er δ13C ratios (Childress & Fisher 1992). However, the δ13C ratios are also influenced by the ratios of source car- bon (CO2) and by translocation of carbon during uptake and transport from symbiont to host (Scott et al. 2004). Symbionts that oxidise methane are related to type I methanotrophs within the Gammaproteobacteria (Pe- tersen & Dubilier 2009). Methane serves as electron donor as well as carbon source. Biogenic methane exhibits highly δ13C depleted signatures (Sugimoto & Wada 1995; Zyakun 1996). Type I methanotrophs use the ribulose monophos- phate pathway for carbon fixation (Leak et al. 1985) and preferentially consume 12CH4 which leads to a further de- pletion in δ13C values (Coleman et al. 1981; Grossman et al. 2002). In summary, δ13C values of tissue from molluscs which harbor, chemoautotrophs and/or methanotrophs (“primary producers”) are all significantly depleted relative to molluscs at higher trophic levels. This depletion pattern should also be expected for tissue of phototrophic mol- luscs Nitrogen assimilation The main nitrogen sources of bacterial biomass and hence host tissue in chemoautotrophic symbioses are ammonia (NH4+) and nitrate (Johnson et al. 1988; Conway et al. 1992; Lilley et al. 1993; Lee & Childress 1994; Lee et al. 1999). Ammonia and nitrate are used by bacteria for bio- synthesis of amino acids and other nitrogen compounds (Payne 1973; Reitzer & Magasanik 1987). Molluscs receive their amino acids from their diet (e.g., Neff 1972), which is in case of chemosymbiosis mainly based on biomole- cules from the symbionts. Isotopic fractionation of nitrogen may occur during uptake and incorporation of nitrogen by bacterial symbi- onts (Hoch et al. 1992; Yoneyama et al. 1993; Dreier et al. 2012). Methane-oxidising bacteria, for instance, prefer as- similation of 14NH3 (Lee & Childress 1994). Independent of the pathway of nitrogen assimilation, it is known that δ15N ratio increases by about 3.4 ‰ per trophic level (Mi- nagawa & Wada 1984; Peterson & Fry 1987). Accordingly, primary producers must show lower δ15N in tissue than their consumers (Conway et al. 1989; Conway et al. 1992; Lee & Childress 1994; Colaco et al. 2002; Dreier et al. 2012). Sulphur oxidation Thiotrophic endosymbiosis is most common among mol- luscs (see above). Their energy source is sulfide, which originates from abiogenic reduction of sulfate or from mi- crobial sulfate reduction (Kaplan et al. 1963; Aharon & Fu 2000; Joye et al. 2004). Sulfide in sediments mostly derives from microbial sulfate reduction; both biogenically and abiogenically generated sulfide is depleted in δ34S (Kaplan et al. 1963; Kiyosu & Krouse 1993; Aharon & Fu 2000; Joye et al. 2004). The pathway of sulphur oxidation does not lead to a significant fractionation of sulphur isotopes. The depleted sulfide from sediment is possibly not just used as an energy source but is also assimilated by sulfide- oxidising symbionts and incorporated in their biomass (Dreier et al. 2012). In contrast, the sulphur compounds of non-thiotrophic molluscs derive from sea-water sul- fates with δ34S ratios being markedly different from that of sulfides in sediments (Kaplan et al. 1963; Trust & Fry 1992; Michener & Schell 1994). 88 Anne Dreier & Michael Hoppert Table 1: Some δ13C values of different chemoautotrophic symbioses and corresponding isotopic discrimination of different carbon fixing pathways (compiled after Roeske & O’Leary 1984; Brooks et al. 1987; Conway et al. 1989; Fisher 1990; Kennicutt et al. 1992; Guy et al. 1993; Goericke et al. 1994; Robinson & Cavanaugh 1995; Cavanaugh & Robinson 1996; Van Dover et al. 2003; Scott et al. 2004; Van Dover 2007). Thus δ34S ratios of biomass from thiotrophic molluscs are higher depleted than 34S ratios of non-thiotrophic mol- luscs (Mizota & Yamanaka 2003; O’Donnell et al. 2003; Mae et al. 2007; Dreier et al. 2012). Fig. 3: Chitin staining of a cross section of Tridacna maxima decalcified shell. (A) Recent, One Tree Island, Queensland, Australia; (B) Pleistocene, north of Da- hab, Sinai, Egypt. Cross sections were stained with Calcofluor White. In summary, the isotopic compositions of the biological elements carbon, sulphur and nitrogen in biomolecules from host tissue are excellent biosignatures, providing in- formation about an animal’s diet and trophic level (Mich- ener & Schell 1994; Casey & Post 2011). However, in or- der to determine diet of fossil molluscs, preserved bio- molecules are needed. Here one may take benefit from the mineralised mollusc shells, which are perfect long term conservation wrappings for organic matter. Different biosignatures and stability over geological timescales Various techniques are used to detect symbiotic prokary- otes in mollusc tissue, such as 16S ribosomal DNA se- quence analysis, fluorescent in situ hybridisation and transmission electron microscopy. After death, soft tissue is degraded and only shells are left for incorporation into the fossil record. Mollusc shells are mainly composed of calcium carbonate in aragonite and calcite conformation; these crystals are formed be- tween organic matrix layers. Frémy (1855) was the first who described conchiolin, the acid insoluble organic ma- trix in shells. Later, high proportions of acidic amino acids Asx (Asp+Asn) were found in soluble shell organics. X- ray/electron diffraction revealed matrix-crystal spatial re- lations protein structure (β-sheet), and the presence of chitin (Weiner & Traub 1980; Weiner et al. 1983). Symbiont bearing molluscs during earth history 89 Recent studies about shell proteins imply that the organic shell matrix is composed of a macromolecular framework consisting of a chitin-silk fibronin gel with acidic proteins (e.g., Marin & Luquet 2007; Evens 2008; Marin et al. 2008). To demonstrate the presence of chitin in shells, staining with the fluorescence dye Calcofluor White may be performed, which binds to cellulose and chitin. Obvi- ously, also the remaining organic shell matrix of fossil shells could be stained. This could be a hint for the persis- tence of these biomolecules in the shell matrix. Fig. 3 shows a stained cross section of Tridacna maxima shells (recent and fossil) embedded in LR white resin. The cross section was decalcified with 0.5 M EDTA over night and then stained with Calcofluor White. To exclude unspecific binding to embedding resin, an untreated (unfixed, not embedded) piece of Tridacna maxima shell was decalcified and stained with Calcofluor White (Fig. 4). It is obvious that the Calcoflour-stained material in the fossil shell of Tridacna maxima (Fig. 4C) is different from the filamentous structure in the modern shell (Figs. 4A, 4C). In addition, a cross section of a fossil (Upper Creta- ceous) Inoceramus sp. shell was stained with Calcofluor White. Treatment of cross section was identical to that of Tridacna but without decalcification. Fig. 5 shows that maybe fossil chitin was stained between the calcium car- bonate crystals of the Inoceramus sp. shell. The preserved biomolecules of the shell will provide information about the mollusc's diet. In endosymbiont- bearing molluscs, carbon, nitrogen and sulphur are taken up by the symbionts, get an isotopic fingerprint and are then incorporated in mollusc biopolymers (see above). Since the remains of the organic shell matrix are preserved after death, stable isotope analysis of the matrix serves as valuable screening tool for detecting symbiotic association in living as well as in fossil molluscs. In many studies δ13C, δ15N and sometimes δ34S in soft tissue were determined in order to analyze dietary intake (Kennicutt et al. 1992; Dando & Spiro 1993; Conway et al. 1994; Dando et al. 1994; Fischer 1995; Colaco et al. 2002; Lorrain et al. 2002; Dattagupta et al. 2004; Carlier et al. 2007, 2009). However, only few studies describe these isotopic fingerprints with respect to the organic matrix of recent and fossil shells (O’Donnell et al. 2003; Mae et al. 2007; Dreier et al. 2012). Only the study by Dreier et al. provides δ34S values of the organic matrix of empty shells from recent bivalves and subfossil (Late Pleistocene) shells. It was shown that sulphur isotopes are not useful markers to detect ancient the thiotrophic lifestyle, because after death of the molluscs δ34S values in the organic ma- trix will decrease. It was assumed that the reason could be the instability of sulphur-containing amino acids (Jones & Vallentyne 1960) or sulfides derived from proteolysis and from bacterial sulfate reduction during soft tissue degrada- tion. New results confirm the latter hypothesis: the non- symbiotic bivalve Venerupis aurea, which was used in the study of Dreier et al. (2012), was degraded in original sed- iments under laboratory conditions in an aquarium. After half a year the shells were analyzed and δ34S as well as C/N ratios were measured. The C/N ratio is an expres- sion for the grade of alteration and decay of the organic shell matrix (Ambrose 1994). The C/N ratio of the artifi- cial degraded shells of Venerupis slightly increased from 3.15 (fresh shell) to 3.27 (degraded half a year), the δ34S ratio dropped slightly from 7.8 ‰ to 7.5 ‰. Longer rest- ing time in the sediment is needed to futher decrease the δ34S ratio further (Dreier et al. 2012). Fig. 4: Chitin staining of decalcified piece of Tridacna maxima shell. (A– B) Recent, One Tree Island, Queensland, Australia, filament like structures are visible; (C) Pleistocene, north of Dahab, Sinai, Egypt, no filaments could be detect- ed. Cross sections were stained with Calcofluor White. Fig. 5: Chitin staining of a section of fossil (Upper Cretaceous of the quarry Dammann South, Söhlde, Germany) Inoceramus sp. (Heterodonta: Veneroida: Cardiidae) shells. (A) Longitudinal section of shell stained with Calcofluor White. (B) Cross section of the same shell. 90 Anne Dreier & Michael Hoppert The enrichment of sulfides during degradation of soft tis- sue may be the cause of the framboidal pyrite formation (Fig. 5; Berner 1984; Wilkin 1995) and for the decreasing δ34S ratio of shells after death. Lipids are another prominent group of biochemical markers, which are analyzed to identify symbiosis in mol- luscs. Fatty acids as main building blocks of lipids have a characteristic distribution pattern. Short-chained monoun- saturated fatty acids (MUFA) are of mainly prokaryotic origin (Bishop 1976) whereas the major component of eukaryotic lipids consist of long-chained polyunsaturated fatty acids (PUFA; Shaw 1974). Their specificity and structural diversity make them to important trophic bi- omarkers in marine ecology (Gehron & White 1982; Parkes & Taylor 1983; Guckert et al. 1985; Sargent et al. 1987; Wakeham & Canuel 1988; Findlay et al. 1990; Sar- gent et al. 1990; Bradshaw et al. 1991; Hopkins et al. 1993; Rajendran et al. 1993). Lipids were also used to characterise symbiotic associ- ations between prokaryotes and marine invertebrates (Berg et al. 1985; Conway & Capuzzo 1990, 1991; Ben- Mlih et al. 1992; Zhukova et al. 1992; Cobabe & Pratt 1995; Fullarton et al. 1995). In molluscs the lipid content depends on dietary lipid intake (Moreno et al. 1980; Piretti et al. 1987), thus lipid content of molluscs with auto- trophic symbionts will reflect a diet based on the symbi- onts. It is known from bivalve shells that they contain li- pids like fatty acids, cholesterol, phytandienes, ketones and sometimes n-alkanes. Lipids are geologically stable which make them well-suited for paleontological ap- proaches. In addition, lipids have low solubility in water at low temperatures; hence in early diagenesis the level of contamination from surrounding pore fluids and the mi- gration of lipids out of the shell is low. As mentioned above, the carbon of symbiont-derived compounds is de- pleted in δ13C, furthermore it is known that lipid carbon was found to be depleted by 3 ‰ relative to their dietary carbon (DeNiro & Epstein 1977; Crenshaw 1980). Con- sequently, δ13C ratios of most molluscs shell-lipids may reflect if they are symbiont-bearing or not. Cobabe & Pratt (1995), Conway & Capuzzo (1991) and Dreier et al. (2012) found some fatty acids of chemotropic bivalves to be more depleted in δ13C relative to heterotrophic bivalve. Lipids from fossil shells of two bivalve species about 1.4 million years old show a fatty acid distribution very similar to modern shells (with differences in their relative abun- dance; Cobabe & Pratt 1995). However, δ13C values of fossil shell lipids have been not reported so far. Future perspectives In the light of recent climatic and global changes it will be more and more important to reconstruct environmental conditions of the past. Especially the marine environment represents an important climatic driving force and chang- ing conditions could be recognised by a change in the benthic ecosystem. Today the stability of many ecosys- tems is in danger, also because of the breakdown of sym- biotic interactions, just considering e.g. bleaching events in coral reefs (Carpenter et al. 2008). Chemosymbiotic species are major players at oxic- anoxic interfaces of the sediment or at seep and vent sites, for example at sites of methane-hydrate breakdown. The influence and importance of chemosymbiotic species at places with high eutrophication, leading to anoxic events, is not well-understood, though one may expect that eu- trophication also leads to mass development of chemo- symbionts (Hesselbo et al. 2000). If we even could identify the point where the lifestyle of a species switches from heterotrophic to symbiotic, we will be also able to find factors driving emergence of co- operative microbial-host associations, which will foster our understanding of this evolutionary driving force. Mol- luscs are very suitable model organisms, because they have a well-documented fossil history and provided mineralised tissue. In some cases original organic matrix is preserved in fossil shells. By analyzing the isotopic composition of the remaining original organic matrix and of separately ex- tracted lipids of fossil molluscs shells, it is possible to dis- tinguish between “primary consumers” (chemo- and pho- totrophic) and molluscs from higher trophic levels. In order to get trustworthy data it is recommendable to analyze at least two species with different diets from the same habitat or location, otherwise the reliability of isotopic dates are questionable (Dreier et al. 2012). For instance, Dreier et al. (2012) found δ13C and δ15N values for the heterotrophic bivalve Venerupis (δ13C of -24.1 ‰ and δ15N of +4.2 ‰) in the same range as for chemo- trophic bivalves from other sites. But in contrast, com- pared to the values of the chemotrophic bivalves from the same site, the large differences between the isotopic signa- tures allowed to distinguish between the two lifestyles. With this respect it is also important to keep in mind that some diets of endosymbiotic molluscs are not completely based on their symbionts. Some of the molluscs still use filter-feeding as an additional option (Duplessis et al. 2004). To date no isotopic data are available for chemo- compared with phototrophic molluscs inhabiting the same site, so is not known if there is a resolution limit between the two different primary producer's lifestyles. Generally it should be possible to confirm either auto- trophy or heterotrophy by comparing carbon and nitrogen isotopies of the organic matrices from different candidate shell specimens from the same location. This method is not limited to molluscs – all invertebrates with mineralised tissue and embedded organic matrix could be analyzed, for example also shells of brachiopods and perhaps even organic matrices of tubeworm tubes. Symbiont bearing molluscs during earth history 91 Fig. 6: Scanning electron micrographs of a Venerupis shell (Heterodonta: Veneroida: Veneridae) after 6 months incubation in sediment. (A) general view of the shell; (B) higher magnification, with framboidal pyrite [circle]; (C) higher magnification of left picture, with bacteria attached to the shell surface [arrow]. Acknowledgements The fossil Tridacna maxima shell (GZG.INV.76418) was donated from Geoscience Center, Museum, Collections & Geopark. AD is supported by the Studienstiftung des deutschen Volkes. We are grateful to a anonymous referee for appropriate and con- structive suggestions and corrections. Additional thanks are due to Mike Reich and Frank Wiese, their comments greatly im- proved the final version of our manuscript. References Aharon, P. & Fu, B. S. (2000): Microbial sulfate reduction rates and sulfur and oxygen isotope fractionations at oil and gas seeps in deepwater Gulf of Mexico. Geochimica et Cosmochimica Acta 64: 233-246. http://dx.doi.org/10.1016/S0016- 7037(99)00292-6 Amano, K.; Jenkins, R. G.; Kurihara, Y. & Kiel, S. (2007) A new genus for Vesicomya inflate Kanie & Nishida, a lucinid shell convergent with that of vesicomyids, from Cretaceous strata of Hokkaido, Japan. The Veliger 50: 255-262. Ambrose, S. H. (1994): Preparation and characterization of bone and tooth collagen for isotopic analysis. Journal of Archaeological Science 17: 431-451. Baco, A. R.; Smith, C. R.; Peek, A. S.; Roderick, G. K. & Vrijen- hoek, R. C. (1999): The phylogenetic relationships of whale- fall vesicomyid clams based on mitochondrial COI DNA se- quences. Marine Ecology Progress Series 182: 137-147. Ben-Mlih, F.; Marty, J. C. & Fiala-Médioni, A. (1992): Fatty acid composition in deep hydrothermal vent symbiotic bivalves. Journal of Lipid Research 33: 1797-1806. Berg, C. J. jr.; Krzynowek, J.; Alatalo, P. & Wiggin, K. (1985): Sterol and fatty acid composition of the clam, Codakia orbicu- laris, with chemoautotrophic symbionts. Lipids 20: 116-120. Berner, R. A. (1984): Sedimentary pyrite formation: An update. Geochimica et Cosmochimica Acta 48: 605-615. http://dx.doi.org/ 10.1016/0016-7037(84)90089-9 Bishop, D. G. (1976): Lipids and lipid metabolism. Comparative animal nutrition 1: 74-98. Blank, R. J. (1986): Unusual chloroplast structures in endosym- biotic dinoflagellates: a clue to evolutionary differentiation within the genus Symbiodinium (Dinophyceae). Plant Systematics and Evolution 151: 271-280. http://dx.doi.org/10.1007/ BF02430280 92 Anne Dreier & Michael Hoppert Blumenberg, M. (2010): Microbial chemofossils in specific ma- rine hydrothermal and cold methane cold seep settings. In: Kiel, S. (ed.): The Vent and Seep Biota: Aspects from Mi- crobes to Ecosystems. Dordrecht etc. (Springer): 73-106. [= Topics in Geobiology 33] Bradshaw, S. A.; O'Hara, S. C. M.; Corner, E. D. S. & Eglinton, G. (1991): Effects on dietary lipids of marine bivalve Scrobicu- laria plana feeding in different modes. Journal of the Marine Bio- logical Association of the United Kingdom 71: 635-653. http://dx.doi.org/10.1017/S0025315400053200 Brooks, J. M.; Kennicutt, M. C.; Fisher, C. R.; Macko, S. A.; Cole, K.; Childress, J. J.; Bidigare, R. R. & Vetter, R. D. (1987): Deep-sea hydrocarbon seep communities: Evidence for energy and nutritional carbon sources. Science 238:1138-1142. http://dx.doi.org/10.1126/science.238.4830.1138 Callender, W. R. & Powell, E. N. (1997): Autochthonous death assemblages from chemautotrophic communities at petroleum seeps: palaeoproduction, energy flow and implications from the fossil record. Historical Biology 12: 165-198. http://dx.doi.org/10.1080/08912969709386562 Campbell, K. A. (2006): Hydrocarbon seep and hydrothermal vent paleoenvironments and paleontology: Past developments and future research directions. Palaeogeography, Palaeoclimatology, Palaeoecology 232: 362-407. http://dx.doi.org/10.1016/ j.palaeo.2005.06.018 Carlier, A.; Riera, P.; Amouroux, J-M.; Bodiou, J-Y.; Escoubey- rou, K.; Desmalades, M.; Caparros, J. & Grémare, A. (2007): A seasonal survey of the food web in the Lapalme Lagoon (northwestern Mediterranean) assessed by carbon and nitrogen stable isotope analysis. Estuarine, Coastal and Shelf Science 73: 299-315. Carlier, A.; Riera, P.; Amouroux, J. M.; Bodiou, J. Y.; Desma- lades, M. & Gremare, A. (2009): Spatial heterogeneity in the food web of a heavily modified Mediterranean coastal lagoon: stable isotope evidence. Aquatic Biology 5: 167-179. Carpenter, K.; Abrar, M.; Aeby, G.; Aronson, R. B.; Banks, S.; Bruckner, A.; Chiriboga, A.; Cortés, J.; Delbeek, J. C.; De- Vantier, L.; Edgar, G. J.; Edwards, A. J.; Fenner, D.; Guz- mán, H. M.; Hoeksma, B. W.; Hodgson, G.; Johan, O.; Lic- uanan, W. Y.; Livingstone, S. R.; Lovell, E. R.; Moore, J. A.; Obura, D. O.; Ochavillo, D.; Polidoro, B. A.; Precht, W. F.; Quibilan, M. C.; Reboton, C.; Richards, Z. T.; Rogers, A. D.; Sanciangco, J.; Sheppard, A.; Sheppard, C.; Smith, J.; Stuart, S.; Turak, E.; Veron, J. E. N.; Wallace, C.; Weil, E. & Wood, E. (2008): One-third of reef-building corals face elevated extinc- tion risk from climate change and local impacts. Science 321: 560-563. http://dx.doi.org/10.1126/science.1159196 Casey, M. M. & Post, D. M. (2011): The problem of isotopic baseline: reconstructing the diet and trophic position of fossil animals. Earth-Science Reviews 106: 131-148. http://dx.doi.org/ 10.1016/j.earscirev.2011.02.001 Cavanaugh, C. M.; McKiness, Z. P.; Newton, I. L. G. & Stewart, F. J. (2006): Marine chemosynthetic symbioses. In: Dworkin, M.; Falkow, S.; Rosenberg, E. & Stackebrandt, E. (eds.): Pro- karyotes. Volume 1: Symbiotic associations, Biotechnology, Applied Mi- crobiology. New York, N.Y. (Springer): 475-507. Childress, J. J. & Fisher, C. R. (1992): The biology of hydro- thermal vent animals: physiology, biochemistry, and auto- trophic symbioses. Oceanography and Marine Biology – An Annual Review 30: 337-441. Cobabe, E. A. & Pratt, L. M. (1995): Molecular and isotopic compositions of lipids in bivalve shells: a new prospect for molecular paleontology. Geochimica et Cosmochimica Acta 59: 87- 95. http://dx.doi.org/10.1016/0016-7037(94)00374-U Colaco, A.; Dehairs, F. & Desbruyéres, D. (2002): Nutritional relations of deep-sea hydrothermal fields at the Mid-Atlantic Ridge: a stable isotope approach. Deep-Sea Research (I: Oceano- graphic Research Papers) 49: 395-412. http://dx.doi.org/10.1016/ S0967-0637(01)00060-7 Colaço, A.; Desbruyéres, D. & Guezennec, J. (2007): Polar lipid fatty acids as indicators of trophic associations in a deep-sea vent system community. Marine Ecology 28: 15-24. http://dx.doi.org/10.111/j.1439-0485.2006.00123.x Coleman, D. D.; Risatti, J. B. & Schoell, M. (1981): Fractionation of carbon and hydrogen isotopes by methane-oxidizing bacte- ria. Geochimica et Cosmochimica Acta 45:1033-1037. http://dx.doi.org/10.1016/0016-7037(81)90129-0 Conway, N.; McDowe, J.; Capuzzo, J.; Fry, B. (1989): The role of endosymbiotic bacteria in the nutrition of Solemya velum: Ev- idence from a stable isotope analysis of endosymbionts and host. Limnology and Oceanography 34:149-155. Conway, N. & Capuzzo, J. McD. (1990): The use of biochemical indicators in the study of trophic interactions in animal- bacteria symbiosis: Solemya velum, a case study. In: Barnes, M. & Gibson, R. N. (eds.): Trophic relationships in the marine environ- ment. Proceedings of the 24th European Marine Biology Symposium. Aberdeen (University Press): 553-564. Conway, N. & Capuzzo, J. McD. (1991): Incorporation and utili- zation of bacterial lipids in the Solemya velum symbiosis. Marine Biology 108: 277-291. http://dx.doi.org/10.1007/BF01344343 Conway, N. M.; Howes, B. L.; Capuzzo, J.; McDowell J. E.; Turner, R. D. & Cavanaugh, C. M. (1992): Characterization and site description of Solemya borealis (Bivalvia; Solemyidae), another bivalve–bacteria symbiosis. Marine Biology 112: 601-613. http://dx.doi.org/10.1007/BF00346178 Conway, N. M.; Kennicutt; M. C. & Van Dover, C. L. (1994): Stable isotopes in the study of marine chemosynthetic based ecosystems. In: Lajtha, K. & Michener, R. H. (eds.): Stable Iso- topes in Ecology and Environmental Science. Oxford (Blackwell): 158-186. Corliss, J. B.; Dymond, J.; Gordon, L. I.; Edmond, J. M.; Her- zen, R. P. von; Ballard, R. D.; Green, K.; Williams, D.; Bain- bridge, A.; Crane, K. & Andel, T. H. van (1979): Submarine thermal springs on the Galápagos rift. Science 203: 1073-1083. http://dx.doi.org/10.1126/science.203.4385.1073 Cowen, R. (1970): Analogies between the Recent bivalve Tridacna and the fossil brachiopods Lyttoniacea and Richthofeniacea. Palaeogeography Palaeoclimatology Palaeooecology 8: 329-344. http://dx.doi.org/10.1016/0031-0182(70)90105-7 Cowen, R. (1982): Algal symbiosis and its recognition in the fos- sil record. In: Tevesz, M. J. S. & McCall, P. L. (eds.): Biotic in- teractions in Recent and fossil benthic communities. New York, N.Y. (Plenum): 431-478. [= Topics in Geobiology 3] Cramton, J. S. (1988): Comperative taxonomy of the bivalve families Isognomidae, Inoceramidae, and Retroceramidae. Pal- aeontology 31: 956-996. Crenshaw, M. (1980): Mechanisms of shell formation and disso- lution. In: Rhoads, D. & Lutz, R. (eds.): Skeletal Growth of Aquatic Organisms. New York, N.Y. (Plenum): 115-128. Dando, P. R. & Southward A. J. (1986): Chemoautotrophy in bivalve molluscs of the genus Thyasira. Journal of the Marine Bio- logical Association of the United Kingdom 66: 915-929. http://dx.doi.org/10.1017/S0025315400048529 Dando, P. R. & Spiro, B. (1993): Varying nutritional dependence of the thyasirid bivalves Thyasira sarsi and T. equalis on chemo- autotrophic symbiotic bacteria, demonstrated by isotope ratios of tissue carbon and shell carbonate. Marine Ecology Progress Se- ries 92: 151-158. Dando, P. R.; Ridgway, S. A. & Spiro, B. (1994): Sulphide ‘min- ing’ by lucinid bivalve molluscs: demonstrated by stable sul- phur isotope measurements and experimental models. Marine Ecology Progress Series 107: 169-175. Dattagupta, S.; Bergquist, D. C.; Szalai, E. B.; Macko, S. A. & Fisher; C. R. (2004): Tissue carbon, nitrogen, and sulfur stable isotope turnover in transplanted Bathymodiolus childressi mussels: relation to growth and physiological condition. Limnology and Oceanography 49: 1144-1151. Symbiont bearing molluscs during earth history 93 DeNiro, M. J. & Epstein, S. (1977): Mechanism of carbon iso- tope fractionation associated with lipid synthesis. Science 197: 261-263. http://dx.doi.org/10.1126/science.327543 Distel, D.; Baco, A.; Chuang, E.; Morrill, W.; Cavanaugh, C. & Smith, C. (2000): Do mussels take wooden steps to deep-sea vents? Nature 403: 725-726. http://dx.doi.org/10.1038/ 35001667 Distel, D. L.; Morrill,W.; MacLaren-Toussaint, N.; Franks, D. & Waterbury, J. (2002): Teredinibacter turnerae gen. nov., sp. nov., a dinitrogen-fixing, cellulolytic, endosymbiotic γ-proteobacter- ium isolated from the gills of wood-boring molluscs (Bivalvia: Teredinidae). International Journal of Systematic and Evolutionary Microbiology 52: 2261-2269. Dhont, A. V. (1983): Campanian and Maastrichtian inoceramids: A review. Zitteliana 10: 689-701. Dreier, A.; Stannek, L.; Blumenberg, M.; Taviani, M.; Sigovini, M.; Wrede, C.; Thiel, V. & Hoppert, M. (2012): The finger- print of chemosymbiosis: origin and preservation of isotopic biosignatures in the nonseep bivalve Loripes lacteus compared with Venerupis aurea. FEMS Microbiology Ecology 81: 480-493. http://dx.doi.org/10.1111/j.1574-6941.2012.01374.x Dubilier, N.; Bergin, C. & Lott, C. (2008): Symbiotic diversity in marine animals: the art of harnessing chemosynthesis. Nature Reviews Microbiology 6: 725-740. http://dx.doi.org/10.1038/ nrmicro1992 Duperron, S. & Fiala-Médioni, A. (2007): Evidence for chemo- autotrophic symbiosis in a Mediterranean cold seep clam (Bi- valvia: Lucinidae): comparative sequence analysis of bacterial 16S rRNA, APS reductase and RubisCO genes. FEMS Microbi- ology Ecology 59: 1-11. Duperron, S.; Halary, S.; Lorion, J.; Sibuet, M. & Gaill, F. (2008): Unexpected co-occurrence of six bacterial symbionts in the gills of the cold seep mussel Idas sp. (Bivalvia: Mytilidae). Envi- ronmental Microbiology 10: 433-445. http://dx.doi.org/10.1111/ j.1462-2920.2007.01465.x Duperron, S. (2010): The diversity of deep-sea mussels and their bacterial symbioses. In: Kiel, S. (ed.): The Vent and Seep Biota: Aspects from Microbes to Ecosystems. Dordrecht etc. (Springer): 137-167. [= Topics in Geobiology 33] Duplessis, M. R.; Dufour, S. C.; Blankenship, L. E.; Feldbeck, H. & Yayanos, A. A. (2004): Anatomical and experimental evi- dence for particulate feeding in Lucinoma aequizonata and Par- vilucina tenuisculpta (Bivalvia: Lucinidae) from the Santa Barbara Basin. Marine Biology 145: 551-561. http://dx.doi.org/ 10.1007/s00227-004-1350-6 Evens, J. S. (2008): “Tuning in” to mollusc shell nacre- and pris- matic-associated protein terminal sequences. Implications for biomineralization and the construction of high performance inorganic organic composites. Chemical Reviews 108: 4455-4462. http://dx.doi.org/10.1021/cr078251e Fiala-Médioni, A. & Felbeck, H. (1990): Autotrophic process in invertebrate nutrition: bacterial symbiosis in bivalve molluscs. In: Mellinger, J. (ed.): Animal nutrition and transport processes. Vol- ume 1: Nutrition in wild and domestic animals. Basel etc. (Karger): 49-69. Findlay, R. H.; Trexler, M. B.; Guckert, J. B. & White, D. C. (1990): Laboratory study of disturbance in marine sediments: response of a microbial community. Marine Ecology Progress Se- ries 62: 121-133. Fisher, C. R. (1990): Chemoautotrophic and methanotrophic symbioses in marine invertebrates. Reviews in Aquatic Sciences 2: 399-436. Fischer, C. R. (1995): Toward an appreciation of hydrothermal- vent animals: their environment, physiological ecology, and tis- sue stable isotope values. Geophysical Monograph Series 91: 297- 316. http://dx.doi.org/10.1029/GM091p0297 Fortey, R. (2000): Olenid trilobites: The oldest known chemoau- totrophic symbionts? Proceedings of the National Academy of Scienc- es of the United States of America 97: 6574-6578. http://dx.doi.org/10.1073/pnas.97.12.6574 Frémy, M. E. (1855): Recherches chimiques sur les os. Annales de Chimie et de Physique 43: 45-107. Fullarton, J. G.; Dando, P. R.; Sargent, J. R.; Southward, A. J. & Southward, E. C. (1995): Fatty acids of hydrothermal vent Ridgeia piscesae and inshore bivalves containing symbiotic bacte- ria. Journal of the Marine Biological Association of the United Kingdom 75: 455-468. http://dx.doi.org/10.1017/S0025315400018300 Gehron, M. J. & White, D. C. (1982): Quantitative determination of the nutritional status of detrital microbiota and the grazing fauna by triglycerides and glycerol analysis. Journal of Experi- mental Marine Biology and Ecology 64: 145-158. http://dx.doi.org/10.1016/0022-0981(82)90150-2 Gill, F. L.; Harding, I. C.; Little, C. T. S. & Jonathan, A. T. (2005): Palaeogene and Neogene cold seep communities in Barbados, Trinidad and Venezuela: An overview. Palaeogeogra- phy, Palaeoclimatology, Palaeoecology 227: 191-209. http://dx.doi.org/10.1016/j.palaeo.2005.04.024 Glover, E. A.; Taylor, J. D. & Rowden, A. A. (2004): Bathyaustri- ella thionipta, a new lucinid bivalve from a hydrothermal vent on the Kermadec Ridge, New Zealand and its relationship to shallow-water taxa (Bivalvia: Lucinidae). Journal of Molluscan Studies 70: 283-295. Glover, E. A. & Taylor, J. D. (2007): Diversity of chemosymbi- otic bivalves on coral reefs: Lucinidae (Mollusca, Bivalvia) of New Caledonia and Lifou. Zoosystema 29: 109-181. Goedert, J. L. & Squires, R. L. (1990): Eocene deep-sea commu- nities in localized limestones formed by subduction-related methane seeps, southwestern Washington. Geology 18: 1182- 1185. http://dx.doi.org/10.1130/0091-7631(1990)18<1182: EDSCIL>2.3.CO;2 Goedert, J. L. & Campbell, K. A. (1995): An Early Oligocene chemosynthetic community from the Makah Formation, Northwestern Olympic Peninsula, Washington. The Veliger 38: 22-29. Goedert, J. L.; Thiel, V.; Schmale, O.; Rau, W. W.; Michaelis, W. & Peckmann, J.; (2003): The Late Eocene ‘Wiskey Creek’ me- thane-seep deposit (Western Washington State). Part I: Geolo- gy, palaeontology and molecular geobiology. Facies 48: 223- 240. Goericke, R.; Montoya, J. P.; & Fry, B. (1994): Physiology of isotopic fractionation in algae and cyanobacteria. In: Lajtha, K. & Michener, R. H. (eds.): Stable Isotopes in Ecology and Environ- mental Science. Oxford (Blackwell): 189-221. Grossman, E. L.; Cifuentes, L. A. & Cozzarelli, I. M. (2002): An- aerobic methane oxidation in a landfill-leachate plume. Envi- ronmental Science & Technology 36: 2436-2442. Gubanov, A. P.; Skovsted, C. B. & Peel, J. S. (2004): Early Cam- brian molluscs from Sierra de Cordoba (Spain). Geobios 37: 199-215. http://dx.doi.org/10.1016/j.geobios.2003.04.003 Guckert, J. B.; Antworth, C. P.; Nichols, P. D. & White, D. C. (1985): Phospholipid, ester-linked fatty acid profiles as repro- ducible assays for changes in prokaryotic community structure of estuarine sediments. FEMS Microbiology Letters 31: 147-158. http://dx.doi.org/10.1111/j.1574-6968.1985.tb01143.x Guy, R. D., Fogel, M. L. & Berry, J. A. (1993) Photosynthetic fractionation of stable isotopes of oxygen and carbon. Plant Physiology 101: 37-47. http://dx.doi.org/10.1104/pp.101.1.37 Hallock, P. & Schlager, W. (1986): Nutrient excess and the de- mise of coral reefs and carbonate platforms. Palaios 1: 389-398. http://dx.doi.org/10.2307/3514476 Hallock, P. (1987): Fluctuations in the trophic resource continu- um: a factor in global diversity cycles? Paleoceanography 2: 457- 471. http://dx.doi.org/10.1029/PA002i005p00457 Hartman, M. C. & Pratt, I. (1976): Infection of the heart cockle, Clinocardium nuttalli, from Yaquina Bay, Oregon, with an endo- symbiotic alga. Journal of Invertebrate Pathology 28: 291-299. http://dx.doi.org/10.1016/0022-2011(76)90002-1 Hawkins, A. J. S.; & Klumpp, D. W. (1995): Nutrition of the giant clam Tridacna gigas (L.) II. Relative contribution of filter feeding and the ammonium acquired and recycled by symbi- 94 Anne Dreier & Michael Hoppert otic algae towards total nitrogen requirements for tissue growth and metabolism. Journal of Experimental Marine Biology and Ecology 190: 263-290. http://dx.doi.org/10.1016/0022- 0981(95)00044-R Heide, T. van der; Govers, L. L.; de Fouw, J.; Olff, H.; Geest, M. van der; Katwijk, M. M. van; Piersma, T.; Koppel, J. van de; Silliman, B. R.; Smolders, A. J. P. & Gils, J. van (2012): A Three-Stage Symbiosis Forms the Foundation of Seagrass Ecosystems. Science 336: 1432-1434. http://dx.doi.org/ 10.1126/science.1219973 Herry, M. D. & Le Pennec, M. (1989): Chemoautotrophic sym- bionts and translocation of fixed carbon from bacteria to host tissues in the littoral bivalve Loripes lucinalis (Lucinidae). Marine Biology 101: 305-312. http://dx.doi.org/10.1007/BF00428126 Hesselbo, S. P.; Gröcke, D. R.; Jenkyns, H. C.; Bjerrum, C. J.; Farrimond, P.; Morgans Bell, H. S. & Green, O. R. (2000): Massive dissociation of gas hydrate during a Jurassic oceanic anoxic event. Nature 406: 392-395. http://dx.doi.org/ 10.1038/35019044 Hoch, M. P.; Fogel, M. L. & Kirchman, D. L. (1992): Isotope fractionation associated with ammonium uptake by a marine bacterium. Limnology and Oceanography 37: 1447-1459. Hopkins, C. C. E.; Sargent, J. R. & Nilssen, E. M. (1993): Total lipid content, and lipid and fatty acid composition of the deep- water prawn Pandalus borealis from Balsfjord, northern Norway: growth and feeding relationships. Marine Ecology Progress Series 96: 217-228. Jacobs, D. K. & Jones, D. S. (1989): Photosymbiosis in Clinocar- dium nuttalli: a model for isotopic “vital effecs” with implica- tions for the fossil record of photosymbiosis. Geological Society of America, Abstracts with Program 21: A77. Jannasch, H. W. & Wirsen, C. O. (1979): Chemosynthetic prima- ry production at West Pacific sea floor spreading centers. Bio- science 29: 592-598. Johnson, K. S.; Childress, J. J.; Hessler, R. R.; Sakamoto-Arnold, C. M. & Beehler, C. L. (1988): Chemical and biological interac- tions in the Rose Garden hydrothermal vent field. Deep-Sea Re- search (A: Oceanographic Research Papers) 35: 1723-1744. http://dx.doi.org/10.1016/0198-0149(88)90046-5 Jones, D. S.; Williams, D. F. & Spero, H. J. (1988): More Light on Photosymbiosis in fossil molluscs: The case of Mercenaria “tridacnoides”. Palaeogeography, Palaeoclimatology, Palaeoecology 64: 141-152. http://dx.doi.org/10.1016/0031-0182(88)90003-X Jones, D. S. & Jacobs, D. K. (1992): Photosymbiosis in Clinocar- dium nuttalli: implications for test of photosymbiosis in fossil molluscs. Palaios 7: 86-95. http://dx.doi.org/10.2307/3514798 Jones, J. D. & Vallentyne, J. R. (1960): Biogeochemistry of or- ganic matter. I. Polypeptides and amino acids in fossil and sed- iments in relation to geothermometry. Geochimica et Cosmo- chimica Acta 21: 1-34. http://dx.doi.org/10.1016/S0016- 7037(60)80002-6 Joye, S. B.; Boetius, A.; Orcutt; B. N.; Montoya, J. P.; Schulz, H. N.; Erickson, M. J. & Lugo, S. K. (2004): The anaerobic oxida- tion of methane and sulfate reduction in sediments from Gulf of Mexico cold seeps. Chemical Geology 205: 219-238. http://dx.doi.org/10.1016/j.chemgeo.2003.12.019 Kano, Y.; Chiba, S. & Kase, T. (2002): Major adaptive radiation in neritopsine gastropods estimated from 28S rRNA sequences and fossil records. Proceedings of the Royal Society (B: Biological Sci- ences) 269: 2457-2465. http://dx.doi.org/10.1098/ rspb.2002.2178 Kauffman, E. G., (1969): Form, function and evolution. In: Moore, R. C. (ed.): Treatise on Invertebrate Paleontology, Part N, Mollusca 6(1), Bivalvia. Boulder, Colo. (Geological Society of America) & Lawrence, Kans. (University of Kansas): N129- N205. Kaplan, I. R.; Emery, K. O. & Rittenberg, S. C. (1963): The dis- tribution and isotopic abundance of sulphur in recent marine sediments off southern California. Geochimica et Cosmochimica Acta 27: 297-331. http://dx.doi.org/10.1016/0016- 7037(63)90074-7 Kawaguti, S. (1950): Observations on the heart shell, Corculum cardissa (L.), and its associated zooxanthellae. Pacific Science 4: 43-49. Kawaguti, S. (1968): Electron microscopy on zooxanthellae in the mantle and gill of the heart shell. Biological Journal of Okaya- ma University 14: 1-11. Kawaguti, S. (1983): The third record of association between bivalve molluscs and zooxanthellae. Proceedings of Japan Academic (B: Physical and Biological Sciences) 59: 17-20. Keen, A. M. (1980): The pelecypod family Cardiidae: a taxonom- ic summary. Tulane Studies in Geology and Paleontology 16: 1-40. Kennicutt, M. C.; Burke, R. A. Jr.; MacDonald, I. R.; Brooks, J. M.; Denoux, G. J. & Macko, S. A. (1992): Stable isotope parti- tioning in seep and vent organisms: chemical and ecological significance. Chemical Geology (including Isotope geoscience) 101: 293- 310. http://dx.doi.org/10.1016/0009-2541(92)90009-T Kharlamenko, V. I.; Zhukova, N. V.; Khotimchenko, S. V.; Svetashev, V. I. & Kamenev, G. M. (1995): Fatty acids as markers of food sources in a shallow water hydrothermal eco- system (Kraternaya Bight, Yankich Island, Kurile Islands). Ma- rine Ecology Progress Series 120: 231-241. Kiel, S. & Goedert, J. L. (2006a): Deep-sea food bonanzas: early Cenozoic whale-fall communities resemble wood-fall rather than seep communities. Proceedings of the Royal Society of London (B: Biological Sciences) 273: 2625-2631. http://dx.doi.org/ 10.1098/rspb.2006.3620 Kiel, S. & Little, C. S. T. (2006): Cold-Seep molluscs are older than the general marine mollusc fauna. Science 313: 1429-1431. http://dx.doi.org/10.1126/science.1126286 Kiel, S. & Peckmann, J. (2007): Chemosymbiotic bivalves and stable carbon isotopes indicate hydrocarbon seepage at four unusual Cenozoic fossil localities. Lethaia 40: 345-357. http://dx.doi.org/10.1111/j.1502-3931.2007.00033.x Kiel, S.; Amano, K. & Jenkins, R. G. (2008a): Bivalves from Cre- taceous cold-seep deposits on Hokkaido, Japan. Acta Palaeonto- logica Polonica 53: 525-537. Kiel, S. & Dando, P. R. (2009): Chaetopterid tubes from vent and seep sites: Implications for fossil record and evolutionary history of vent seep annelids. Acta Palaeontologica Polonica 54: 443-448. Kiel, S. & Tyler, P. A. (2010): Chemosynthetically-driven ecosys- tems in the Deep Sea. In: Kiel, S. (ed.): The Vent and Seep Bi- ota: Aspects from Microbes to Ecosystems. Dordrecht etc. (Springer): 1-14. [= Topics in Geobiology 33] Kiyosu, Y. & Krouse H. R. (1993): Thermochemical reduction and sulfur isotopic behavior of sulfat by acetic acid in the presence of native sulfur. Geochemical Journal 27: 49-57. Klumpp, D. W.; Bayne, B. L.; Hawkins, A. J. S. (1992): Nutrition of the giant clam Tridacna gigas (L.). I Contribution of filter feeding and photosynthates to respiration and growth. Journal of Experimental Marine Biology and Ecology 155: 105-122. http://dx.doi.org/10.1016/0022-0981(92)90030-E Krueger, D. M.; Dubilier, N. & Cavanaugh, C. M. (1996): Chemoautotrophic symbiosis in the tropical clam Solemya occi- dentalis (Bivalvia: Protobranchia): ultrastructural and phyloge- netic analysis. Marine Biology 126: 55-64. http://dx.doi.org/ 10.1007/BF00571377 Leak, D. J.; Stanley, S. H.; & Dalton, H. (1985): Implication of the nature of methane monooxygenase on carbon assimilation in methanotrophs. In: Poole, R. K. & Dow, C. S. (eds.): Micro- bial Gas Metabolism, Mechanistic, Metabolic and Biotechnological As- pects. London (Academic Press): 201-208. Symbiont bearing molluscs during earth history 95 Lee, R. W. & Childress, J. J. (1994): Assimilation of inorganic nitrogen by chemoautotrophic and methanotrophic symbioses. Applied and Environmental Microbiology 60: 1852-1858. Lee, R. W.; Robinson, J. J. & Cavanaugh, C. M. (1999): Pathways of inorganic nitrogen assimilation in chemoautotrophic bacte- ria-marine invertebrate symbioses: expression of host and sym- biont glutamine synthetase. Journal of Experimental Biology 202: 289-300. Lilley, M. D.; Butterfield, D. A.; Olson, E. J.; Lupton, J. E.; Macko, S. A. & McDuff, R. E. (1993): Anomalous CH4 and NH4+ concentrations at an unsedimented mid-ocean-ridge hy- drothermal system. Nature 364: 45-47. http://dx.doi.org/ 10.1038/364045a0 Little, C. T. S.; Campbell, K. A. & Herrington, R. J. (2002): Why did ancient chemosynthetic seep and vent assemblages occur in shallower water than they do today? International Journal of Earth Sciences 91: 149-153. http://dx.doi.org/10.1007/ s005310050273 Lonsdale, P. (1977): Clustering of suspension-feeding macroben- thos near abyssal hydrothermal vents at oceanic spreading cen- ters. Deep Sea Research 24: 857-863. http://dx.doi.org/ 10.1016/0146-6291(77)90478-7 Lorrain, A.; Paulet, Y-M.; Chauvaud, L.; Savoye, N.; Donval, A. & Saout, C. (2002): Differential d13C and d15N signatures among scallop tissues: implications for ecology and physiology. Journal of Experimental Marine Biology and Ecology 275: 47-61. MacLeod, K. G. & Hoppe, K. A. (1992): Evidence that Inocer- amid bivalves were benthic and harbored chemosynthetic symbionts. Geology 20: 117-120. http://dx.doi.org/10.1130/ 0091-7613(1992)020<0117:ETIBWB>2.3.CO;2 Mae, A.; Yamanak, T. & Shimoyama, S. (2007): Stable isotope evidence for identification of chemosynthesis-based fossil bi- valves associated with cold seepages. Palaeogeography, Palaeocli- matology and Palaeoecology 245: 411-420. http://dx.doi.org/ 10.1016/j.palaeo.2006.09.003 Majima, R.; Nobuhara, T. & Kitazaki, T. (2005): Review of fossil chemosynthetic assemblages in Japan. Palaeogeography, Palaeocli- matology and Palaeoecology 227: 86-123. http://dx.doi.org/ 10.1016/j.palaeo.2005.04.028 Marin, F. & Luquet, G. (2007): Unusually acidic shell proteins in biomineralization. In: Bäuerlein, E. (ed.): Handbook of Biominer- alization 1: 273-290. Marin, F.; Luquet, G.; Marie, B.; Medakovic, D. (2008): Mollus- can shell proteins: primary structure, origin and evolution. Cur- rent Topics in Developmental Biology 80: 209-276. McKenzie, J. D.; Black, K. D.; Kelly, M. S.; Newton, L. C.; Handley, L. L.; Scrimgeour, C. M.; Raven, J. A. & Henderson, R. J. (2000): Comparisons of fatty acid and stable isotope rati- os in symbiotic and nonsymbiotic brittlestars from Oban Bay, Scotland. Journal of the Marine Biological Association of the United Kingdom 80: 311-320. Michener, R. & Schell, D. (1994): Stable isotope ratios as tracers in marine aquatic food webs. In: Lajtha, K. & Michener, R. H. (eds.): Stable Isotopes in Ecology and Environmental Science. Oxford (Blackwell): 138-157. Minagawa, M. & Wada, E. (1984): Stepwise enrichment of 15N along food chains: further evidence and the relation between d15N and animal age. Geochimica et Cosmochimica Acta 48: 1135- 1140. http://dx.doi.org/10.1016/0016-7037(84)90204-7 Mizota, C. & Yamanaka, T. (2003): Strategic adaptation of a deep-sea, chemosynthesis-based animal community: An evalu- ation based on soft body part carbon, nitrogen, and sulfur iso- topic signatures. Japanese Journal of Benthology 58: 56-69. [in Jap- anese with English summary] Moreno, J.; Pollero, A. E.; Moreno, V. J.; Brenner, R. R. (1980): Lipids and fatty acids of the mussel (Mytilus platensis d'Orbigny) from South Atlantic Waters. Journal of Experimental Marine Biolo- gy and Ecology 48: 263-276. http://dx.doi.org/10.1016/0022- 0981(80)90081-7 Morton, B. (2000): The biology and functional morphology of Fragum erugatum (Bivalvia: Cardiidae) from Shark Bay, Western Australia: the significance of its relationship with entrained zooxanthelllae. Journal of Zoology 251: 39-52. Neff, J. (1972): Ultrastructure of the outer epithelium of the mantle in the clam Mercenaria mercenaria in relation to calcifica- tion of the shell. Tissue Cell 4: 591-600. http://dx.doi.org/ 10.1016/S0040-8166(72)80032-6 O’Donnell, T. H.; Mackob, S. A.; Choub, J.; Davis-Harttenc, K. L. & Wehmillerc, J. F. (2003): Analysis of δ 13C, δ15N, and δ34S in organic matter from the biominerals of modern and fossil Mercenaria spp. Organic Geochemistry 34: 165-183. http://dx.doi.org/10.1016/S0146-6380(02)00160-2 Ohno, T.; Katoh, T.; Yamasu, T. (1995): The origin of algal- bivalve photosymbiosis. Palaeontology 38: 1-21. Oliver, P. G.; Southward, E. C. & Dando P. R. (2013) Bacterial symbiosis in Syssitomya pourtalesiana Oliver, 2012 (Galeomma- toidea: Montacutidae), a bivalve commensal with the deep-sea echinoid Pourtalesia. Journal of Molluscan Studies 79: 30-41. Parkes, R. J. & Taylor, J. (1983): The relationship between fatty acid distributions and bacterial respiratory types in contempo- rary marine sediments. Estuarine, Coastal and Shelf Science 16: 173-189. Payne, W. J. (1973): Reduction of nitrogen oxides by microor- ganisms. Bacteriological Reviews 37: 409-452. Peckmann, J.; Thiel, V.; Michaelis, W.; Clari, P.; Gaillard, C.; Martire, L.; Reitner, J. (1999): Cold seep deposits of Beau- voisin (Oxfordian; southeastern France) and Marmorito (Mio- cene; northern Italy): microbially induced authigenic car- bonates. International Journal of Earth Sciences 88: 60-75. http://dx.doi.org/10.1007/s005310050246 Peckmann, J.; Goedert, J. L.; Thiel, V.; Michaelis, W. & Reitner, J. (2002): A comprehensive approach to the study of methane- seep deposits from the Lincoln Creek Formation, western Washington State, USA. Sedimentology 49: 855-873. http://dx.doi.org/10.1046/j.1365-3091.2002.00474.x Peckmann, J.; Thiel, V.; Reitner, J.; Taviani, M.; Aharon, P. & Michaelis, W. (2004): A microbial mat of a large sulphur bacte- rium preserved in a Miocene methane-seep. Geomicrobiology Journal 21: 247-255. http://dx.doi.org/10.1080/ 01490450490438757 Persselin, S. (1998): The Evolution of Shell Windows within the Fraginae (Bivalvia: Cardiidae) and the Origin of Algal Symbio- sis in Cardiids. MSc Thesis Publication. Mangilao, Guam: University of Guam Marine Laboratory: 49 pp. Peterson, B. J. & Fry, B. (1987): Stable isotopes in ecosystem studies. Annual Review of Ecology and Systematics 18: 293-320. Petersen, J. M. & Dubilier, N. (2009): Methanotrophic symbioses in marine invertebrates. Environmental Microbiology Reports 1: 319- 335. http://dx.doi.org/10.1111/j.1758-2229.2009.00081.x Peel, J. S. (1991): Functional morphology of the Class Helcionel- loida nov, and the early evolution of Mollusca. In: Simonetta, A. M. & Conway, S. (eds): The early evolution of Metazoa and the significance of problematic taxa. Cambridge (Cambridge University Press): 157-177. Philip, J. (1972): Paléoécologie des formations á rudistes du Cré- tacé supérier - l'example du sud-est de la France. Palaeogeogra- phy, Palaeoclimatology, Palaeoecology 12: 205-222. http://dx.doi.org/10.1016/0031-0182(72)90060-0 Piretti, M. V.; Tioli, F. & Pagliuca, G. (1987): Investigation of the seasonal variations of sterol and fatty acid constituents in the bivalve molluscs Venus gallina and Seapharca inaequivalvis (Bru- guiére). Comparative Biochemistry and Physiology (B: Comparative Bio- chemistry) 88: 1201-1208. http://dx.doi.org/10.1016/0305- 0491(87)90024-1 Purchon, R. D. (1955): A note on the biology of Tridacna crocea Lam. Proceedings of the Malacological Society of London 31: 95-110. Purich, D. L. & Allison, R. D. (2000): Handbook of Biochemical Ki- netics. New York, N.Y. (Academic Press): xxii + 788 pp. 96 Anne Dreier & Michael Hoppert Rajendran, N.; Suwa, Y. & Urushigawa, Y. (1993): Distribution of phospholipid ester-linked fatty acid biomarkers for bacteria in the sediment of Ise Bay, Japan. Marine Chemistry 42: 39-56. http://dx.doi.org/10.1016/0304-4203(93)90248-M Reitzer, L. J. & Magasanik, B. (1987): Ammonia assimilation and the biosynthesis of glutamine, glutamate, aspartate, asparagine, L-alanine and D-alanine. In: Neidhardt, F. C.; Ingraham, J. L.; Low, K. B.; Magasanik, B.; Schaechter, M. & Umbarger, H. E. (eds.): Escherichia coli and Salmonella typhimurium, Cellular and Molecular Biology. Washington, D.C. (American Society for Microbiology): 302-320. Robinson, J. J. & Cavanaugh, C. M. (1995) Expression of form I and form II Rubisco in chemoautotrophic symbioses: Implica- tions for the interpretion of stable carbon isotope values. Lim- nology and Oceanography 40: 1496-1502. Roeske, C. A. & O’Leary, M. H. (1984) Carbon isotope effects on the enzyme-catalyzed carboxylation of ribulose bisphos- phate. Biochemistry 23: 6275-6284. http://dx.doi.org/10.1021/ bi00320a058 Romanek, C. S.; Jones, D. S.; Williams, D. F.; Krantz, D. E. & Radtke, R. (1987): Stable isotopic investigation of physiological and environmental changes recorded in shell carbonate from the giant clam Tridacna maxima. Marine Biology 94: 385-393. http://dx.doi.org/10.1007/BF00428244 Ruby, E. G.; Jannasch, H. W. & Deuser, W. G. (1987): Fraction- ation of stable carbon isotopes during chemoautotrophie growth of sulfur-oxidizing bacteria. Applied and Environmental Microbiology 53: 1940-1943. Sandy, M. R. (2010): Brachiopods from ancient hydrocarbon seeps and hydrothermal vents. In: Kiel, S. (ed.): The Vent and Seep Biota: Aspects from Microbes to Ecosystems. Dordrecht etc. (Springer): 279-314. [= Topics in Geobiology 33] Sargent, J. R.; Parkes R. J.; Mueller-Harviy, I. & Henderson, R. J. (1987): Lipid biomarkers in marine ecology. In: Sleigh, M. A. (ed.): Microbes and the Sea. Chichester (Ellis Horwood Ltd.): 119-138. Sargent, J. R.; Bell, M. V.; Henderson, R. J. & Tocher, D. R. (1990): Polyunsaturated fatty acids in the marine and terrestrial food webs. In: Mellinger, J. (ed.): Animal nutrition and transport processes. Volume 1: Nutrition in wild and domestic animals. Basel etc. (Karger): 11-23. Schlager, W. (2003): Benthic carbonate factories of the Phanero- zoic. International Journal of Earth Sciences 92: 445-464. http://dx.doi.org/10.1007/s00531-003-0327-x Schneider, J. A. & Carter, J. G. (2001): Evolution and phyloge- netic significance of Cardioidean shell microstructure (Mollus- ca: Bivalvia). Journal of Paleontology 75: 607-643. http://dx.doi.org/10.1666/0022- 3360(2001)075<0607:EAPSOC>2.0.CO;2 Scott, C. L.; Kwasniewski, S.; Falk-Petersen, S. & Sargent, J. R. (2000): Lipids and life strategies of Calanus finmarchicus, Calanus glacialis and Calanus hyperboreus in late autumn, Kongsfjorden, Svalbard. Polar Biology 23: 510-516. http://dx.doi.org/10.1007/ s003000000114 Scott, K. M.; Schwedock, J.; Schrag, D. P. & Cavanaugh, C. M. (2004) Influence of form IA RubisCO and environmental dis- solved inorganic carbon on the δ13C of the clam-chemo- autotroph symbiosis Solemya velum. Environmental Microbiology 12: 1210-1219. Shank, T. M.; Black, M. B.; Halanych, K. M.; Lutz, R. A. & Vri- jenhoek, R. C. (1999): Miocene radiation of deep-sae hydro- thermal vent shrimp (Caridea: Bresiliidae): evidence from mi- tochondrial cytochrome oxidase subunit I. Molecular Phylogenet- ics and Evolution 13: 244-254. http://dx.doi.org/10.1006/ mpev.1999.0642 Shaw, N. (1974): Lipid composition as a guide to the classifica- tion of bacteria. Advances in Applied Microbiology 17: 63-108. Skovsted, C. B.; Brock, G. A.; Lindstroem, A.; Peel, J. S.; Pater- son, J. R. & Fuller, M. K. (2007): Early Cambrian record of failed durophagy and shell repair in an epibenthic mollusc. Bi- ology Letters 3: 314-317. http://dx.doi.org/10.1098/ rsbl.2007.0006 Southward, E. C. (2008): The morphology of bacterial symbioses in the gills of mussels of the genera Adipicola and Idas (Bivalvia: Mytilidae). Journal of Shellfish Research 27: 139-146. Squires, R. L. & Goedert, J. L. (1991): New Late Eocene Mol- luscs from localized limestone deposits formed by subduction- related methane seeps, southwestern Washington. Journal of Paleontology 65: 412-416. Stasek, C. R. (1961): The form, growth and evolution of the Tridacnidae (giant clams). Archives de Zoologie expérimentale et gé- nérale 101: 1-40. Sugimoto, A. & Wada, E. (1995): Hydrogen isotopic composi- tion of bacterial methane: CO2/H2 reduction and acetate fer- mentation. Geochimica et Cosmochimica Acta 59:1329-1337. http://dx.doi.org/10.1016/0016-7037(95)00047-4 Tarasov, V. G., Gebruk, A. V., Mironov, A. N. & Moskalev, L. I. (2005): Deep-sea and shallow-water hydrothermal vent com- munities: two different phenomena? Chemical Geology 224: 5-39. http://dx.doi.org/10.1016/j.chemgeo.2005.07.021 Taviani, M. (1994): The “calcari a Lucina” macrofauna reconsid- ered: deep-sea faunal oases from Miocene-age cold vents in the Romagna Apennine, Italy. Geo-Marine Letters 14:185-191. http://dx.doi.org/10.1007/BF01203730 Taviani, M. (2011): The deep-sea chemoautotroph microbial world as experienced by the Mediterranean metazoans through time. In: Reitner, J.; Queric, N-V. & Arp, G. (eds.): Advances in Stromatolite Geobiology. Berlin etc. (Springer): 277-295. [= Lecture Notes in Earth Sciences 131] http://dx.doi.org/10.1007/978-3-642-10415-2_18 Taylor, F. J. R. (1979): Symbionticism Revisited: A Discussion of the Evolutionary Impact of Intracellular Symbioses. Proceedings of the Royal Society of London (B: Biological Sciences) 204: 267-286. http://dx.doi.org/10.1098/rspb.1979.0027 Taylor, J. D. & Glover, E. A. (2000): Functional anatomy, chemosymbiosis and evolution of the Lucinidae. In: Harper, E. M.; Taylor, J. D. & Crame, J. A. (eds.): The Evolutionary Biology of the Bivalvia. Geological Society of London, Special Publi- cation 177: 207-225. Taylor, J. D. & Glover, E. A. (2006): Lucinidae (Bivalvia) – the most diverse group of chemosymbiotic molluscs. Zoological Journal of the Linnean Society 148: 421-438. http://dx.doi.org/ 10.1111/j.1096-3642.2006.00261.x Taylor, J. D.; Glover, E. A. & Williams, S. T. (2008): Ancient chemosynthetic bivalves: systematics of Solemyidae from east- ern and southern Australia (Mollusca: Bivalvia). Memoirs of the Queensland Museum (Nature) 51: 75-104 Taylor, J. D. & Glover, E. A. (2009): A giant lucinid bivalve from the Eocene of Jamaica – systematic, life habitats and chemosymbiosis (Mollusca: Bivalvia: Lucinidae). Palaeontology 52: 95 - 109. Taylor, J. D. & Glover, E. A. (2010): Chemosymbiotic bivalves. In: Kiel, S. (ed.): The Vent and Seep Biota: Aspects from Mi- crobes to Ecosystems. Dordrecht etc. (Springer): 107-135. [= Topics in Geobiology 33] Taylor, J. D.; Glover, E. A.; Smith, L.; Dyal, P. & Williams, S. T. (2011): Molecular phylogeny and classification of the chemo- symbiotic bivalve family Lucinidae (Mollusca: Bivalvia). Zoologi- cal Journal of the Linnean Society 163: 15-49. http://dx.doi.org/10.1111/j.1096-3642.2011.00700.x Trench, R. K.; Wethey, D. S. & Porter, J. W. (1981): Observa- tions on the symbiosis with zooxanthellae among the Tridac- nidae (Mollusca: Bivalvia). Biological Bulletin 161: 180-198. Symbiont bearing molluscs during earth history 97 Treude, T.; Smith, C. R.; Wenzhöfer, F.; Carney, E.; Bernardino, A. F.; Hannides, A. K.; Krüger, M. & Boetius, A. (2009): Bio- geochemistry of a deep-sea whale fall: sulfate reduction, sulfide efflux and methanogenesis. Marine Ecology Progress Series 382: 1- 21. Trust, B. A. & Fry, B. (1992): Stable sulphur isotopes in plants: review. Plant, Cell & Environment 15: 1105-1110. http://dx.doi.org/10.1111/j.1365-3040.1992.tb01661.x Van Dover, C. L. (2000): The Ecology of Deep-Sea Hydrothermal Vents. Princeton, N.J. (Princeton University Press): xx + 424 pp. Van Dover, C. L.; Aharon, P.; Bernhard, J. M.; Caylor, E.; Doer- ries, M.; Flickinger, W.; Gilhooly, W.; Goffredi, S. K.; Knick, K. E.; Macko, S. A.; Rapoport, S.; Raulfs, E. C.; Ruppel, C.; Salerno, J. L.; Seitz, Sen Gupta B. K.; Shank, T.; Turnipseed, M. & Vrijenhoek, R. (2003): Blake Ridge methane seeps: char- acterization of a soft-sediment, chemosynthetically based eco- system. Deep-Sea Research (I: Oceanographic Research Papers) 50: 281-300. http://dx.doi.org/10.1016/S0967-0637(02)00162-0 Van Dover, C. L. (2007): Stable isotope studies in marine chemoautotrophically based ecosystems: an update. In: Mich- ener, R. & Lajtha, K. (eds.): Stable isotopes in ecology and environ- mental science. [2nd ed.]. Boston, Mass. (Blackwell Publishing): 202-237. Vinther, J. & Nielsen, C. (2005): The Early Cambrian Halkeria is a mollusc. Zoologica Scripta 34: 81-89. http://dx.doi.org/ 10.1111/j.1463-6409.2005.00177.x Wallin, I. E. (1927): Symbionticism and the Origin of Species. London (Baillière, Tindall & Cox): x + 171 pp. Wakeham, S. G. & Canuel, E. A. (1988): Organic geochemistry of particulate matter in the eastern tropical North Pacific Ocean: implications for particle dynamics. Journal of Marine Re- search 46: 183-213. http://dx.doi.org/10.1357/ 002224088785113748 Weiner, S. & Traub, W. (1980): X-ray diffraction study of the insoluble organic matrix of mollusc shell. FEBS Letters 111: 311-316. http://dx.doi.org/10.1016/0014-5793(80)80817-9 Weiner, S., Talmon, Y. & Traub, W. (1983): Electron diffraction of mollusc shell organic matrices and their relationship to the mineral phase. International Journal of Biological Macromolecules 5: 325-328. http://dx.doi.org/10.1016/0141-8130(83)90055-7 Wilkin, R. T. (1995): Size distribution in sediments, synthesis, and formation mechanism of framboidal pyrite. PhD. disserta- tion, The Pennsylvania State University: 454 pp. Yoneyama, T.; Kamachi, K.; Yamaya, T. & Mae, T. (1993): Frac- tionation of nitrogen isotopes by glutamine synthetase solated from spinach leaves. Plant & Cell Physiology 34: 489-491. Yonge, C. M. (1936): Mode of life, feeding, digestion and symbi- osis with zooxanthellae in the Tridacnidae. Great Barrier Reef Expedition, 1928–29, British Museum (Natural History), Scientific Report 1: 283-321. Yonge, C. M. (1981): Functional morphology and evolution in the Tridacnidae (Mollusca: Bivalvia: Cardiaceae). Records of the Australian Museum 33: 735-777. Zhang, J. Z. & Millero, F. J. (1993): The products from the oxi- dation of H2S in seawater. Geochimica et Cosmochimica Acta 57: 1705-1718. http://dx.doi.org/10.1016/0016-7037(93)90108-9 Zhukova, N. V.; Kharlamenko, V. I.; Svetashev, V. I. & Rodi- onov, I. A. (1992): Fatty acids as markers of bacterial symbi- onts of marine bivalve molluscs. Journal of Experimental Marine Biology and Ecology 162: 253-263. http://dx.doi.org/10.1016/ 0022-0981(92)90205-O Zal, F. et al. (2000): Haemoglobin structure and biochemical characteristics of the sulphide-binding component from the deep-sea clam Calyptogena magnifica. Cahiers de biologie marine 41: 413-423. Zyakun, A. M. (1996): Potential of 13C/12C variations in bacterial methane in assessing origin of environmental methane. In: Schumacher, D. & Abrams, M. A. (eds.): Hydrocarbon migra- tion and its near-surface expression. AAPG Memoir 66: 341- 352. Cite this article: Dreier, A. & Hoppert, M. (2014): Following the traces of symbiont bearing molluscs during earth history. In: Wiese, F.; Reich, M. & Arp, G. (eds.): ”Spongy, slimy, cosy & more…”. Commemo- rative volume in celebration of the 60th birthday of Joachim Reitner. Göt- tingen Contributions to Geosciences 77: 83–97. http://dx.doi. org/10.3249/webdoc-3920