Mass occurrence of seep-specific bivalves in the oldest-known cold seep metazoan community

One of the most striking features of modern chemosynthesis-based ecosystems surrounding methane seeps is the presence of abundant chemosymbiotic bivalves. However, such accumulations have rarely been reported from Palaeozoic to mid-Mesozoic seeps, and it is widely thought that general trends in the evolution of chemosynthetic communities paralleled those typifying most marine environments, with the bivalve prevalence starting in the Mesozoic and with Palaeozoic seeps being dominated by brachiopods. Here, we report a discovery of bivalve clusters in the oldest-known methane seep that hosted metazoan fauna, dated to the late Silurian. We identify the bivalves, externally very similar to modern chemosymbiotic forms, as members of the extinct family Modiomorphidae, known previously from a younger, Devonian seep. The bivalves inhabited the seep at a stage of increased fluid flow, when they co-occurred with atrypid brachiopods, and display a set of morphological characteristics suggesting a seep-obligate lifestyle. We conclude that bivalves colonised chemosynthesis-based ecosystems at least as early as brachiopods and apparently first developed specialized lineages able to thrive in seep-related habitats for a prolonged period of time. Rather than being simple ecological successors of brachiopods, rich bivalve communities represent an ancient and recurring theme in the evolution of chemosynthetic assemblages.


The oldest seep-related bivalves
The bivalve clusters reach densities of up to 250 individuals/m 2 ; many specimens display consistent orientations (Fig. 2e,f), presumably reflecting their original alignment. Owing to their small sizes (max. 32 mm wide), the co-occurring brachiopods form dense aggregates among the bivalves (Figs. 2d-f and 3; Supplementary Fig. 1). Most of the bivalve shells were pervasively recrystallised or dissolved during diagenesis, which resulted in frequent deformations of their original outlines (Fig. 2e,f). Nonetheless, in some cases undeformed specimens can still be observed, enabling genus-level taxonomic recognition. The bivalves are large (up to 160 mm long), with highly elongated, fan-shaped posterior shell lobe (Fig. 2a-c). The ventral shell margin is indented by a broad sinus, giving the shells a characteristic, boomerang-like shape (Fig. 2d). This feature, together with the shape of the anterior lobe (Fig. 2a), strong carina ( Fig. 2b-c), and the pattern of the muscular and ligament attachments, places the El Borj bivalves in the genus Ataviaconcha, known so far only from a Middle Devonian seep in the eastern Anti-Atlas, where it also forms mass concentrations 12 . Ataviaconcha belongs to the extinct family Modiomorphidae, a group morphologically convergent with, but evolutionarily distant from, extant mussels 6,10,20 . Except for the two Moroccan assemblages of Ataviaconcha, reports of Palaeozoic seep-related bivalves are limited to scarce solemyids found at Middle Devonian 12 and lower Carboniferous seeps 21 . In addition, a putative modiomorphid has been documented from a Silurian hydrothermal vent 22 . However, none of these bivalves has been reported to form dense accumulations around Palaeozoic fluid emissions.
The peculiar morphological characteristics of Ataviaconcha from El Borj, mostly shared with their Devonian congener 12 , most likely represent advanced adaptations to a semi-infaunal lifestyle in a seep-related habitat. Similar elongated, variously incurved shells developed independently in several groups of semi-infaunal seep bivalves, including chemosymbiotic vesicomyids and bathymodiolins 5,6,23 , as well as another, Mesozoic lineage of modiomorphoid bivalves 10,24,25 . Such shells, when oriented with their anterior end shallowly buried in the sediment, and the posterior part exposed, enable simultaneous access to seawater-derived oxygen and interstitial sulphide 3,12,23 . Given the high metabolic toxicity of sulphide, the semi-infaunal strategy provides no obvious advantage to non-chemosymbiotic bivalves 5 . Thus, the elongated shell of Ataviaconcha strongly suggests close reliance on reduced compounds and oxic seawater, a physiological trait exhibited by bivalves living in symbiosis with chemoautotrophic bacteria. This is further supported by the large size of the shell, a common distinctive feature of chemosymbiotic molluscs 3,6 , which places Ataviaconcha among the largest Palaeozoic bivalves known to date. The bivalve gills appear generally well suited to acquire chemosymbionts, with no advanced morphological adaptations required, as shown by the independent development of chemosymbiosis in several groups of Bivalvia, including Solemyidae, one of the most basal bivalve groups 6,20 . Since a chemosymbiotic lifestyle was likely present in solemyid and lucinid bivalves as early as the Ordovician and the Silurian, respectively 6,20 , the seep-related modiomorphids may not, therefore, have been the most ancient bivalve lineage in which chemosymbiosis appeared.  heavy. At typical seeps, methane oxidation releases large quantities of isotopically light carbon, which results in strongly negative δ 13 C values in precipitating carbonates 2,15 . These signals have led to interpretations of the El Borj deposit as either strongly altered by diagenesis that overprinted originally more negative values 16 , or as having formed due to methane formation, rather than oxidation 11,18 . However, none of these scenarios offered a plausible explanation for the combination of palaeontological and geological features observed in the studied limestones (see Supplementary Discussion and Supplementary Fig. 3).

Habitat of the Silurian bivalve-brachiopod assemblage
The unusual isotopic signals are, in turn, explained by our stratigraphic data. The late Ludfordian (late Silurian) age established by the conodont analyses places the formation of the El Borj deposit within a time (e,f,) Field view (e) and corresponding schematic drawing (f) of the bivalve-brachiopod assemblage (black -bivalves; white -brachiopods). Note the common deformations of the bivalve shells due to their diagenetic recrystallisation and partial dissolution. Coin (e); 21 mm in diameter) for scale. (g) Thin-section view (cross-polarised light) of two bivalve shells (B). Note recrystallisation of the original shell material to a sparry calcite mosaic, with preservations of some remnants of the original multi-layered structure. The shell was partially corroded and coated with clotted micritic carbonate (arrow).
interval characterized by a very prominent positive excursion in the carbon isotope composition of seawater 26,27 . This is critical for the interpretation of the isotopic signals, since, when corrected for the δ 13 C seawater of +7 to +9‰ typical of the late Ludfordian excursion, the values measured in the early cements fall, in fact, down to 12‰ below the signatures of contemporaneous marine calcites. Although this 13 C depletion is still less significant than that characterising some modern seep carbonates 2 , such moderately negative ratios are typical of Palaeozoic seep limestones 15,18,19,21 . In the few instances where lower signals were measured, the seep carbonates contain few fossils, with both brachiopods and bivalves being notably absent 11,18 . Among other factors, it seems that many early seep-dwelling metazoans were less tolerant of the environmental toxicity of the most intense seeps, and preferred temperate emissions, at which lower hydrocarbon contents or diffuse flow resulted in less 13 C-depleted signatures of the carbonates. This appears particularly plausible for dimerelloid brachiopods, which are not known from contemporary seeps, and their presence at Palaeozoic and Mesozoic seeps is limited to carbonates characterized by moderate 13 C-depletions, with δ 13 C values typically ranging from several to −20‰, and only exceptionally exceeding −25‰ 19,21,25,[28][29][30][31][32] . While the El Borj site remains the only known seep inhabited by members of the order Atrypida, rather than Rhynchonellida and Terebratulida typical of younger, Late Devonian to Cretaceous seeps 33,34 , the preference for micrite-dominated, moderately 13 C-depleted seeps appears shared by all seep-related brachiopods.
Compared to the underlying, brachiopod-dominated facies, the bivalve-rich carbonates display more variable δ 13 C signals, and record both the lowest and highest values measured among the early cements (Fig. 4). Combined with the petrological observations, this can presumably be attributed to a transition from the stage of relatively slow seepage to a period of more vigorous, spatially and temporarily variable flow, which attracted the abundant seep-specialised bivalves. Since at modern seeps dense clusters of chemosymbiotic bivalves clear bottom waters of a significant proportion of toxic sulphide 5 , the appearance of the bivalves may have played an important role in enabling the continuous presence of the apparently less-specialised atrypids, as brachiopods are otherwise very rarely found in cement-dominated seep carbonates 28,29 .

Bivalve vs. brachiopod dominance at seeps over time
The present study confirms a previous suggestion that bivalves could have colonised seep-related ecosystems at least as early as brachiopods 12 . In fact, the Ataviaconcha modiomorphids remained present at seeps for at least 30 myr (Fig. 5) and reveal derived adaptations to reducing habitats 12 , whereas the abundant atrypids are known from an isolated occurrence. The former, therefore, can be perceived as more prominent inhabitants of the Middle Palaeozoic seeps.
The apparent disappearance of the bivalve-dominated seep ecosystems after the Middle Devonian is enigmatic, given that representatives of the modiomorphids, unlike the atrypid brachiopods, survived the Frasnian-Famennian extinctions. Despite the apparent physiological 'inferiority' often suggested for the Brachiopoda 35,36 , rich assemblages of dimerelloid brachiopods appeared at seeps in the Late Devonian, and were present in many of the late Palaeozoic to Early Cretaceous seep communities 30,33,34 . The next record of seep-related modiomorphoid bivalves occurs 170 myr later in the Late Triassic 28,31 . Subsequently, clusters of modiomorphoids re-appeared at seeps in the latest Jurassic and the Cretaceous 10 , but the relationships between the Palaeozoic and Mesozoic seep-related modiomorphoids remain dubious 10,12,24 . To some degree, the absence of rich seep bivalve assemblages from the Late Devonian to early Mesozoic may be attributed to the paucity of the fossil record as very few seeps have been reported from this period, with dense brachiopod clusters known from three of them 19,21,28 . In addition, the aragonitic shells of the bivalves are typified by much lower preservation potential than that of the low-Mg calcitic brachiopods, and, as illustrated by the present study, even at well-known seeps large and abundant, yet poorly preserved bivalves may long remain unnoticed.
The general scarcity of late Palaeozoic and early Mesozoic seeps could also have been of importance. It has been attributed to the continental configuration with restricted areas of continental margins that developed after the formation of the Pangaea supercontinent, and to the associated low levels of tectonic activity during that time 7,28 . In an ocean with rare, geographically distant seeps, a net result could have been limited advantage of advanced specialisation to seep-related habitats, creating favourable conditions for taxa applying more opportunistic strategies. The latter were probably more typical of the seep-related brachiopods, most likely devoid of chemosymbionts 12,34 . Indeed, the gradual decrease in the diversity of brachiopods in seep communities during the Jurassic and Cretaceous coincides approximately with the progressive Pangaea breakup, which was accompanied by the gradual restoration of the bivalve-dominated seep ecosystems 10,30,33 . Rather than being a typical pattern of pre-Cretaceous seep palaeoecology, the long period of the apparent brachiopod dominance at seeps may have, therefore, resulted from of a unique combination of geotectonic and palaeoenvironmental factors. As emphasised by the present study, not only in the late Mesozoic and Cenozoic, but also throughout a large portion of the middle Palaeozoic, dense clusters of large, seep-specialised bivalves could have, in turn, been a common form of chemosynthetic ecosystems. In terms of the dominant shelly fauna, contemporary seep assemblages represent a revival of a theme that first appeared in the evolution of chemosynthesis-based communities over 400 Ma.

Methods
The palaeontological and petrological analyses have been conducted on both isolated and carbonate-embedded specimens of the modiomorphid bivalve Ataviaconcha sp. and atrypid brachiopod Septatrypa lantenoisi. The S. lantenoisi brachiopods are abundant in the micrite-dominated seep carbonates, from which they weather out easily, so that the analyses included several tens of isolated individuals. The bivalves, in turn, were typically firmly embedded within the carbonate cementstone, and the specimens were prepared in the laboratory using a hand-held vibrotool to the extent possible. Petrographic investigations were carried out on a few tens of large early isopachous cements matrix internal sediment blocky cement matrix* early cement*  Figure 4. Carbon vs. oxygen isotope cross-plots for various carbonate phases found in the seep limestones hosting the monospecific brachiopod accumulation (Unit B; see text), and the bivalve-brachiopod assemblage (Unit C). The isotopic composition of contemporaneous ambient seawater (late Ludfordian isotope excursion) 26,27 and data from an earlier study of Buggisch & Krumm 18 (asterisks) are plotted for comparison. The error bars are smaller than the size of the symbols. All results are shown in ‰ V-PDB.
(7.5 × 5 cm) thin sections and polished slabs of the seep limestones. In addition to the plane-and cross-polarised, transmitted-light microscopic analyses, the thin sections were studied under cathodoluminescence (CL) with a Cambridge luminoscope system CITL 8200 mk3 ('cold cathode' type), operating under a 10-12 kV accelerating voltage and a 200-250 μA beam current. Samples for isotopic measurements were collected from slabbed rock surfaces using a microscope-mounted microdrill. Thin sections corresponding to each slabbed surface were analysed prior to sampling to assist in the accuracy of drilling. Carbon and oxygen isotope measurements were performed on powdered carbonates at the Stable Isotope Laboratory of GeoZentrum Nordbayern (Friedrich-Alexander University of Erlangen-Nürnberg). CO 2 was released from the carbonate phase at 70 °C using 103% H 3 PO 4 with an automated Gasbench II sampling device, and analysed for carbon and oxygen isotopes with a Thermo-Fisher Delta V Plus mass spectrometer. All isotopic ratios are given in the standard δ notation, in ‰ relative to the V-PDB standard. Reproducibility of the measurements was monitored by analyses of laboratory standards calibrated to international standards NBS19 (δ 13 C = 1.95‰, δ 18 O = −2.20‰) and LSVEC (δ 13 C = −46.6‰, δ 18 O = −26.7‰). The average reproducibility (1σ) was ±0.07‰ for δ 13 C and ±0.06‰ for δ 18 O.
Strontium isotope analyses were conducted on carbonate powders in the Isotope Laboratory of the Adam Mickiewicz University in Poznań (Poland). Samples (~50 mg each) were dissolved at ~100 °C in closed PFA vials with 0.75 N HCl. Sr separation was carried out following a procedure developed by Pin et al. 37 and Dopieralska 38 . Strontium was loaded with a TaCl 5 activator on a single rhenium filament and measured for isotopic ratios in dynamic collection mode on a Finnigan MAT 261 multi-collector thermal ionization mass spectrometer. During the course of this study, the NBS 987 Sr standard was typified by a 87 Sr/ 86 Sr ratio of 0.710230 ± 10 (2σ mean of twelve analyses). Total procedure blanks were <80 pg. Data availability. All data generated or analysed during this study are included in this published article (and its Supplementary Information files).  33,34 and epifaunal to semi-infaunal bivalves 10,12 in chemosynthesis-based communities throughout the Phanerozoic. Time periods for which representatives of different lineages are known to have formed mass concentrations at seeps are cross-hatched. Taxonomic relationships within the bivalve clade Modiomorphoida remain uncertain; in recent studies the Palaeozoic and Mesozoic seep-dwelling representatives of the group were placed in different families, Modiomorphidae and Kalenteridae, respectively 10,12 . The brachiopod superfamily Dimerelloidea is perceived here as including the three families of rhynchonellid brachiopods with abundant representatives at Palaeozoic and Mesozoic seeps: Halorellidae, Peregrinellidae and Dimerellidae 33,34,39 . The position of terebratulid brachiopods as seep-specialised inhabitants or opportunistic colonisers remains unclear 33,40 .