1 Introduction

Marine cold-seep ecosystems are characterized by the buildup and/or emission of methane across the sediment–water interface and accumulation of very high interstitial hydrogen sulfide concentrations near the sediment–water interface at low-temperature sea-bed conditions (Levin 2005; Levin et al. 2016). The cold-seep ecosystem occurs as patches of variable shape and size across the ocean floor depending upon the areal extent of hydrocarbon conduits like fractures and faults (Panieri et al. 2017). The biotic communities thriving at the cold-seeps are characterized by an endemic ecosystem comprised of chemosynthetic and heterotrophic fauna. H2S and CH4 fluxes at the sediment–water interface (Portail et al. 2015) primarily controls the faunal diversity and spatial distribution of cold-seep ecosystems. On the other hand, a steady flux of methane and hydrogen sulfide gases control the growth and sustenance of cold-seep ecosystems.

Cold-seeps and associated ecosystems are recorded from numerous sites across the world ocean (Sibuet and Olu-Le Roy 2002; Levin 2005; Vanreusel et al. 2009; Olu et al. 2010; Mazumdar et al. 2019; Feng et al. 2020), including Hikurangi basin (off New Zealand); Congo-Angola and Nigeria margins (off West Africa), Nankai Trough (Japan); upper, middle and lower Lousiana slope/Florida escarpment (Gulf of Mexico); Gulf of Cádiz and areas of Nile deep Sea site (Eastern Mediterranean); Barbados Trench; Nordic margin; Makran coast (off Pakistan); off Papua New-Guinea; South China Sea; Queen Charlotte Basin (off the Pacific north coast of British Columbia); Krishna–Godavari basin (Mazumdar et al. 2019); Arctic sediment (Astrom et al. 2020) and below the Larsen Ice Shelf off the Antarctic (Niemann et al. 2009). The global attention to the genesis and sustenance of methane cold-seeps is attributed to interest in evolutionary biology, bioprospecting of extreme ecosystems, the contribution of methane emissions to global warming, and application in methane hydrate exploration (Le Bris et al. 2016).

Here we report for the first time association of Lamellibrachia tubeworm with methane seep related chemosynthetic ecosystem from the Indian Ocean. The seep site is identified off Mannar Basin (within the EEZ of India). The observed ecosystem was developed on a carbonate chemoherm without active gas flares. However, methane flares were observed at other locations (Peketi et al. under review). The sites are also characterized by chemosymbiont bearing and associated heterotrophic biotic assemblages and shallow methane hydrates (2–3 mbsf). The seep site is located at 1644-m water depth (pressure: 13.9 MPa and temperature: 4.95°C).

2 Geology

The Mannar basin (figure 1A), located between India and Srilanka, is a pull-apart sub-basin of the Cauvery–Mannar Basin along the eastern continental margin of India. The basin came into existence in the Late Jurassic/Early Cretaceous due to fragmentation of the Gondwanaland and drifting of the India–Srilanka landmass system away from Antarctica/Australia continental plate (Yoshida et al. 1992; Premarathne et al. 2016). Numerous deep extensional faults developed in the NE–SW direction during rifting had initiated active subsidence that resulted in the formation of graben and horst blocks (Bastia and Radhakrishna 2012; Yanqun et al. 2017). Differential subsidence resulted in the formation of sub-basins within the Cauvery basin. The Gulf of Mannar sub-basin is further divided into western and eastern segments by the NE–SW-aligned Mandapam–Delft ridge (Rao et al. 2010). Curray (1984) opined that the first rifting between India, Sri Lanka, and Antarctica occurred through the Cauvery–Palk Strait–Gulf of Mannar zone but this rift did not progress into the seafloor spreading stage.

Figure 1
figure 1

(A) Location of cold-seep sites in the Cauvery–Mannar Basin (image from Google Earth); (B) A doughnut-shaped pockmark observed in the Mannar basin. The inner and outer diameters of the pockmark are 50 and 300 m, respectively.

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Sediment thickness of more than 6 km has been reported from the deep-waters of the Mannar basin (Ratnayake et al. 2017). Seismic and drill well studies indicate six sedimentary sequences including a thick Early Cretaceous rift fill sequence (principal source horizon) overlain by a complete succession of Tertiary sequence (Rao 2006; Biswas 2012).

3 Methodology

The expedition (SSD-070) was carried out in the Mannar basin onboard ORV Sindhu Sadhana from 13th to 22nd February 2020. The multi-beam bathymetry shows several pockmark-like features in the Mannar basin; one such pockmark was investigated in the expedition SSD-070 (figure 1B). The water-column (WC) images produced by the multibeam echosounder (Atlas-Hydrosweep DS) did not detect any gas flares over the pockmark. Seabird CTD profiler and sound velocity profiling (SVP) data were used to generate water column images. Salinity, temperature, and SVP data were used to carry out the depth corrections for multibeam data. We collected seabed samples at the center of the pockmark (lat: 7°51.37086′N; long: 78°36.45660′E; water depth: 1644 m) using a spade-corer (48 cm (L) × 48 cm (B) × 44 cm (H)) and a gravity corer (PVC liner; inner diameter: 10 cm). Handpicked organisms from the spade cores were stored in either isopropyl alcohol or in –20°C refrigerator for the shore-based analysis. Authigenic carbonates and hard shells were cleaned, dried at room temperature for petrographic/chemical analyses, and taxonomic identification. Sediment pore-fluid/gas-extraction and preservation for onshore analyses of concentrations and isotope ratios (Mazumdar et al. in prep.) were carried out onboard. We recovered gas hydrate samples from the gravity core, which were stored in gas-tight tubes for on-shore carbon isotope ratio measurements.

4 Results and discussion

4.1 Biotic association

4.1.1 Polychaete tubeworm: Lamellibrachia sp. and ecological significance

Two whitish polychaete tubeworms (Lamellibrachia sp.) were recovered in a spade core from the pockmark (SSD-070-5). The tubes were firmly anchored into the authigenic carbonate crust (figure 2A). The length of tube-1 and tube-2 are 93.5 and 57 cm, respectively, and are found to lie 50 and 26 cm above the carbonate crust. The posterior part of the tube is translucent and sinuous (figure 2B). The external surface of the tube is relatively smooth due to a lack of sharply projecting collars (figure 2C) and resembles (supplementary figure S1) the appearance of Lamellibrachia columna (Southward 1991) and Lamellibrachia anaximandri (Southward et al. 2011). The growth rings are discernible in the top 32 mm. The maximum diameter of tubes 1 and 2 are 13.4 and 15 mm, respectively. The vestimentiferan tubes are composed of giant β-chitin crystallites embedded in a protein matrix (Gaill et al. 1992) and the thickness of the chitin wall is ~2.5 mm at the anterior end. The diameter of the tubes decreases downwards and tapers at the posterior end. Goose barnacles (Neolepus sp.; Mazumdar et al. 2019) and byssus threads are attached to the outer surface of the tubes (figure 2C).

Figure 2
figure 2

(A) Chitin tubes of Lamellibrachia sp. associated with carbonate crust recovered in a spade core from the study site; (B) anterior and posterior ends of the Lamellibrachia tubes; (C) a magnified view of the anterior end show growth rings or collars; (D) a magnified view of the anterior tube opening showing branchial plume and sheath lamellae. Goose-barnacles (Neolepas sp.) are attached to the exterior surface of the tube; and (E) soft body of the polychaete extracted from the tube.

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Tube-1 was dissected to observe the soft body of the worm. The different segments of the polychaete worm body like branchial plume (l = 0.84 cm), vestimentum (l = 4.7 cm), trophosome (l = 2.89 cm), and trunk (l = 9.9 cm) are marked in figure 2(D, E). The width of the branchial plume and trophosome are 1 and 0.8 cm, respectively, whereas, the trunk part tapers from 0.8 to <0.1 cm over a length of 9.9 cm. The red colour of the worm is attributed to the presence of hemoglobin within the vascular blood and coelomic fluid which can carry both oxygen and hydrogen sulfide essential for the survival of the chemosynthetic organisms (Arp et al. 1987; Fisher et al. 1988). Unlike hydrothermal vent associated tubeworms, where the H2S enters the worm’s body through the branchial plume (a set of highly vascularized gills), in the cold-seep tubeworms, H2S is transported into the cold-seep tubeworms through the root-like posterior extension of the body (Julian et al. 1999) which is buried deep inside the sediment. H2S is produced in the sediment via the sulfate reduction–AOM pathway (Vossmeyer et al. 2012). The tubeworms lack a mouth or gut and host an endosymbiotic chemolitho-autotrophic γ-proteobacterium (S-bacteria) inside the bacteriocyte cells hosted in the vascularized trophosome sac (Duperron et al. 2009). The polychaete worms acquire the symbionts from the ambient environment at the larval stage (Harmer et al. 2008). Through H2S oxidation, the symbionts in the bacteriocyte cells fix CO2 into organic molecules thus fulfilling the nutritional requirements of the polychaete worm. Sulfate ion, a by-product of HS− oxidation, and H+ produced via other metabolic processes are released from the coelomic fluid across the root into the sediment interstitial waters (Dattagupta et al. 2006). The SO42− elimination is accompanied by anion (HCO3−) uptake across the root epithelium. The sulfate–sulfide recycling process is energetically economical and plays an important role in the long-term sustenance of the organisms (Dattagupta et al. 2006). Additionally, Thiel et al. (2012) reported two different carbon fixation pathways for the endosymbiont namely, the Calvin–Benson–Bassham (CBB) and reductive tricarboxylic acid (rTCA) cycles.

Vestimentiferan tubeworms associated with methane seeps are slow-growing organisms and individuals may be older than 200 years (Cordes et al. 2005, 2007), which may be attributed to stable environments without resource limitation and lack of lethal predation. Assuming a conservative growth rate of 0.77 cmy−1 (Fisher et al. 1997), the estimated age of the longer tube (tube A) in our study is 121 years.

The genus Lamellibrachia Webb, 1969 is one of the few Siboglinidae worms with a broader geographic and habitat distribution such as seeps, vents, and whale falls in the Pacific, Atlantic, Caribbean, and Gulf of Mexico (Feldman et al. 1998; Bright and Lallier 2010; Nishijima et al. 2010; Watanabe et al. 2010; Kobayashi et al. 2015). Before this report, there was no record of this genus from the Indian Ocean. Figure 3 shows the global distribution of eight species of the genus Lamellibrachia Webb polychaete tubeworms (modified after Mccowin and Rouse 2018). The present discovery (marked as a star in figure 3) is the first report of Lamellibrachia sp. from the Indian Ocean. Earlier Mazumdar et al. (2019) reported polychaete tubeworm belonging to Sclerolinum sp from the Krishna–Godavari methane seepage sites.

Figure 3
figure 3

Global distribution of Lamellibrachia polychaete tubeworm (modified after Mccowin and Rouse 2018). The discovery from the Cauvery–Mannar Basin is marked by a star.

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4.1.2 Squat lobsters, gastropod and relict Calyptogena shells

Two morphotypes (I and II) of squat lobster (figure 4A–D) belonging to the genus Munidopsis were recovered from the pockmark site SSD-070-5. The morphotypes reported here are distinct from those reported earlier from the K–G basin (supplementary figure S2) cold-seep sites (Mazumdar et al. 2019). Numerous species (~260) of the genus Munidopsis have been reported across the globe from cold-seeps, hydrothermal vents, deep-sea decomposing natural sunken woods, and whale carcasses (Martin and Haney 2005; Marin 2020). The morphotype I (figure 4A and B) and morphotype II (figure 4C and D) show distinctly different carapace, abdominal, cheliped, telson, and sternum structures. Although the lengths of carapace and abdomen segments in morphotype I (11 and 5.5 mm) are comparable to that of morphotype II (12 and 4 mm), the rostrum in morphotype I (6 mm) is significantly bigger than that of morphotype II (3.5 mm). Morphotype I shows prominent transverse grooves and serrate granules on the carapace which is missing in morphotype II, whereas latter has setae on the carapace. The triangular abdomen part with broad ridges and groves in morphotype I is distinct from the rounded form in morphotype II. The segmentation patterns of the telson, sternum, and chelipeds are also different in the two morphotypes. The morphotype I and II are heterotrophs and their food sources include chemosynthetic bacteria, meiobenthos, and soft bodies of chemosymbiont bearing mussels (Macavoy et al. 2008; Thurber et al. 2011; Tsuchida et al. 2011).

Figure 4
figure 4

(A) Dorsal and (B) ventral view of squat lobster morphotype I belonging to the genus Munidopsis; (C) dorsal and (D) ventral view of squat lobster morphotype II belonging to the genus Munidopsis; (E) Gastropoda shell belonging to the family Buccinedae with the soft body (foot part); (F) whole shell of the genus Calyptogena showing growth layers; (G) fracture filling methane hydrate shown by arrows; and (H) methane hydrate recovered from the fractures.

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Gastropod (figure 4E) belonging to the family Buccindae (Kantor et al. 2013) known to be associated with cold-seeps and hydrothermal vents were recovered from the study sites. The 5 cm long gastropod sample has a 3-cm high spire while the last whorl is 2 cm long. Individual whorls (5 in number) have rounded margins with smooth axial ribs. The siphoncal canal is elongated with a broad inner lip. The foot part (soft body) is also visible. Cold-seep gastropods show multiple feeding habits like grazing, filter-feeding, predation, and scavenging. Additionally, Sasaki et al. (2010) reported the presence of symbiotic bacteria intracellularly in the ctenidium, on the exterior of the gill, and fine-grained pyrite grains in the diminished digestive system indicating possible chemosynthetic feeding habit as well.

Large-sized (up to 15 cm long) relict shells of Calyptogena sp. (figure 4F) are recorded from the study site. Calyptogena shells are also present as part of carbonate chemoherm in association with the tubeworms. In contrast, Bathymodiolus shell fragments were scarce; Calyptogena sp. are chemosynthetic organisms and use H2S for the production of energy and body mass following the haemoglobin–H2S–O2–CO2 biochemical pathway commonly observed in thiotrophic chemosynthesis as discussed for the tubeworms (Duperon et al. 2013). More than 60 species of Calyptogena are reported from the cold-seep and vent regions (Sibuet and Olu 1998; Barry et al. 2007); Calyptogena sp. may grow at a rate >4 cm year−1 at some locations (Turekian and Cochran 1981; Turekian et al. 1983). Earlier, Mazumdar et al. (2019) reported the presence of large Calyptogena shells from cold-seeps off the K–G basin.

4.2 Occurrence of methane hydrate, interstitial H2S, and role of AOM

Methane hydrate was observed as fracture fillings within the sediments (figure 4G and H). The thawing of methane hydrate in the network of cracks results in the brittle and fragmented nature of the muddy sediment. The presence of ethane and propane along with methane in the hydrate phase and δ13\({\text{C}}_{\text{CH}_4}\) values (–28.4 to –79.5: Mazumdar et al. in prep.) suggest a contribution of deep-seated thermogenic methane likely transmitted through fault-fracture systems. Thermogenic methane in the surface sediments was earlier reported by Rasheed et al. (2014) from the SW end of the Mannar basin. The remarkable range of the δ13\({\text{C}}_{\text{CH}_4}\) values of the methane-hydrates shows a complex system characterized by micro and macro fractures responsible for advection of thermogenic gases (δ13C > −50‰ VPDB: Whiticar et al. 1999) and production of biogenic methane (<−50‰) below the SMTZ (~30–35 cmbsf). The bottom water P–T conditions based on the CTD data support the stability of methane hydrate within the sediments.

High concentrations of hydrogen sulfide (3800–12932 µM: Mazumdar et al., in prep.) are reported below the sediment–water interface. The production of hydrogen sulfide in the sediment can be explained by microbially mediated sulfate reduction processes dominated by anaerobic oxidation of methane pathway (Knittel and Boetius 2009) represented by equation (1):

$$ {\text{CH}}_{4} + {\text{SO}}_{4}^{2 - } \to {\text{HCO}}_{3}^{ - } + {\text{HS}}^{ - } + {\text{H}}_{2} {\text{O}}. $$
(1)

The HCO3− ion is consumed by the precipitation of CaCO3 (equation 2) and forms the chemoherm (figure 5A and B).

$$ {\text{HCO}}_{3}^{ - } + {\text{Ca}}^{2 + } \to {\text{CaCO}}_{3} + {\text{H}}^{ + }. $$
(2)
Figure 5
figure 5

(A, B) Authigenic carbonate crust (chemoherm) with embedded Calyptogena shells (CL).

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The interstitial HS− (H2S) is consumed via multiple pathways, e.g., (i) precipitation of Fe-sulfide or sulfurized organic matter in the sediments, (ii) consumption by chemosynthetic organism including microbial mats, and (iii) reoxidation at or above the sediment–water interface via oxidative recycling to S0/SO42− using oxidants like O2 and NO3 (Fernandes et al. 2020; Volvoikar et al. 2020).

4.3 Association of tubeworm and carbonate crust

The association of the tubeworm with hard substratum (carbonate crust: figures 2A, 5A and B) may be attributed to the settling of larval vestimentiferans gregariously on exposed rocks which eventually form dense colonies (Fisher et al. 1997). The roots of the tubeworms extend deeper below the hard substratum, where H2S concentrations are enough for their sustenance. The posterior end of the tubes is reported to be thin (~70 µm) and quite permeable (permeability coefficient at 20°C of 0.41 × 10−3cm s−1) to H2S (Julian et al. 1999). It is interesting to note that although plenty of large shells could be recovered (figure 5A and B), no live Calyptogena sp. could be found along with the tubeworm. The lack of live Calyptogen sp. and the absence of active gas flare suggest low H2S concentration near the sediment–water interface which is essential for sustenance of the Calyptogena to survive and grow. However, the tubeworm’s posterior part could penetrate to the depth below the seafloor (~30–40 cmbsf) where H2S is in sufficient concentrations for growth and proliferation of the tubeworms. Association of tubeworm, carbonate bioherm and Calyptogena/Bathymodiolus have also been reported globally (Teichert et al. 2005; Bayon et al. 2009; Bowden et al. 2013; Feng et al. 2013).

5 Conclusion

We report for the first time presence of Lamellibrachia sp. and associated chemosynthetic ecosystem from the Indian Ocean. The chemosynthetic fauna associated with the methane cold-seeps and shallow hydrate were discovered from the Mannar basin located between India and Srilanka. The tubeworms are attached to the authigenic carbonate crust enriched with large relict shells of Calyptogena sp., a chemosymbiont bearing bivalve. Squat lobsters and gastropod belonging to the Munidopsis sp. and Buccinidae family, respectively, are integral components of the chemosynthesis-based ecosystem. The Lamellibrachia polychaete also bears sulfide oxidizing endosymbionts in their trophosome which is responsible for the energy and body mass synthesis. High concentrations of H2S at some depths below the sediment–water interface are responsible for the sustenance of the tubeworms based on the chemosynthesis based ecosystem. Anaerobic oxidation of methane coupled with sulfate reduction in the sediment is responsible for the interstitial H2S generation. Carbon isotope ratios of methane and the presence of ethane and propane in the headspace gas suggests a mixing of thermogenic and biogenic methane at the study site. A detailed RoV-based survey would be essential to understand the distribution of the chemosynthetic communities and their relationships to geological and chemical properties of the cold-seep sites.