Stable isotope evidence for identification of chemosynthesis-based fossil bivalves associated with cold-seepages

Fossil evidence of chemosynthesis-based organisms has so far been circumstantial and completely based on association of fossilized organisms with ¹³C-depleted authigenic carbonates. In this study, the stable isotopic signatures of preserved organic matter, conchiolin, in the fossil shell interior were used to obtain additional evidence for chemosynthesis-based bivalves, since the carbon, nitrogen, and sulfur isotopic compositions of soft tissues can distinguish chemosynthesis-based animals from marine photosynthesis-based animals. The carbon and nitrogen isotopic compositions of both soft tissues (the mantle) and conchiolin from modern chemosynthesis-based bivalves belonging to the genera Calyptogena and Bathymodiolus had small difference (Δ¹³C_{conchiolin-mantle} ≤ + 3‰, Δ¹⁵N_{conchiolin-mantle} ≤ - 1‰ with the exception of - 7.5‰ for a Calyptogena specimen). Sulfur isotope compositions of the conchiolin from Calyptogena whose lifestyle is almost entirely infaunal were significantly depleted in ³⁴S, while epifaunal Bathymodiolus showed relatively small differences between the δ³⁴S values of chonchiolin and soft tissues. Thus, a systematic difference in sulfur isotope compositions has not been observed for chemosynthesis-based bivalves (Δ³⁴S_{conchiolin-mantle} = - 16.1 to + 2.6‰). Moreover, we were able to extract conchiolin from fossil bivalves to determine its isotopic compositions. The δ¹³C and δ¹⁵N values of the conchiolin obtained from the fossil Vesicomyidae were - 28.5 and - 1.4‰, respectively. The δ¹³C and δ¹⁵N values of lost soft tissues were estimated at approximately - 31‰ and - 1‰, respectively, by subtracting the Δ_{conchiolin-mantle} values determined for modern soft tissues and conchiolin (Δ¹³C_{conchiolin-mantle} = + 3‰, Δ ¹⁵N_{conchiolin-mantle} = - 1‰). Such values are expected for clams harboring thioautotrophic symbionts, suggesting that the fossil was dependent on chemosynthesis during its lifetime. In contrast, the carbon and nitrogen isotopic ratios of the conchiolin from the fossil Mytilidae were - 23.1‰ and + 4.2‰, respectively. The estimated δ¹³C and δ¹⁵N values of its lost soft tissues were approximately - 26‰ and + 5‰, respectively. These values are depleted in ¹³C and ¹⁵N relative to non-symbiont bearing marine organisms suggesting that this fossil likely supported chemosynthetic and/or methanotorophic symbionts and was also dependent on photosynthetically derived organic matter. Our study provides additional and more direct evidence for putative chemosynthesis-based bivalves, whether or not they harbor symbionts.