Introduction
Sea anemones are ecologically important inhabitants of shallow-water and deep-sea habitats globally, and exhibit high taxonomic and morphological diversity (Daly, Reference Daly2006; Xiao et al., Reference Xiao, Brugler, Broe, Gusmão, Daly and Rodríguez2019; McFadden et al., Reference McFadden, Quattrini, Brugler, Cowman, Dueñas, Kitahara, Paz-Garcia, Reimer and Rodriguez2021). They are considered ‘habitat providers’ (Briones-Fourzán et al., Reference Briones-Fourzán, Pérez-Ortiz, Negrete-Soto, Barradas-Ortiz and Lozano-Álvarez2012) and often establish associations with other organisms such as endosymbiotic algae, sponges, crustaceans and fish (Rodríguez et al., Reference Rodríguez, Barbeitos, Brugler, Crowley, Grajales, Gusmão, Häussermann, Reft and Daly2014; Xiao et al., Reference Xiao, Brugler, Broe, Gusmão, Daly and Rodríguez2019; McFadden et al., Reference McFadden, Quattrini, Brugler, Cowman, Dueñas, Kitahara, Paz-Garcia, Reimer and Rodriguez2021). Generally, anemones are suspension feeders, and can utilize a large range of nutritional sources, from detritus to small vertebrates (Xiao et al., Reference Xiao, Brugler, Broe, Gusmão, Daly and Rodríguez2019).
The giant anemone, Relicanthus daphneae (Daly, Reference Daly2006), initially described as Boloceroides daphneae, is known for its remarkable size and long tentacles. Column diameters up to 1 m (Daly, Reference Daly2006) and individual tentacles approximately 2 m in length (Cantwell and Newman, Reference Cantwell and Newman2016) have been estimated from in situ observations. The tentacles are strongly tapered with trailing threadlike tips that are often curled (Daly, Reference Daly2006; Xiao et al., Reference Xiao, Brugler, Broe, Gusmão, Daly and Rodríguez2019). The tentacles bear primarily adhesive functioning spirocysts that are thought to enable R. daphneae to capture large prey (Daly, Reference Daly2006). Living organisms are pale pink to purplish-red in colour, with darker coloration towards the mouth parts.
Relicanthus daphneae has been observed in various habitats across the Pacific and Southern Oceans (Figure 1). It was initially observed and sampled on the East Pacific Rise (EPR) (Gage and Tyler, Reference Gage and Tyler1991; Daly, Reference Daly2006), but has been observed since throughout the Pacific Ocean including the Galapagos Islands (Xiao et al., Reference Xiao, Brugler, Broe, Gusmão, Daly and Rodríguez2019) (observation excluded from Figure 1 as no coordinates were available), the Clarion Clipperton Zone (CCZ) (Amon et al., Reference Amon, Ziegler, Dahlgren, Glover, Goineau, Gooday, Wiklund and Smith2016) and the Mariana trench (Glickson et al., Reference Glickson, Amon, McKenna and Elliott2017). Additionally, it was observed on the East Scotia Ridge (ESR) in the Southern Ocean (Magalhães et al., Reference Magalhães, Linse and Wiklund2017; Xiao et al., Reference Xiao, Brugler, Broe, Gusmão, Daly and Rodríguez2019), and in the South China Sea (Song et al., Reference Song, Lyu, Xiaodi, Ruthensteiner, Ahn, Pastorino, Wang, Gu, Ta, Sun, Liu, Han, Ke and Peng2021). On the EPR and ESR, R. daphneae occurred on the periphery of active hydrothermal vent fields (Daly, Reference Daly2006; Magalhães et al., Reference Magalhães, Linse and Wiklund2017; Xiao et al., Reference Xiao, Brugler, Broe, Gusmão, Daly and Rodríguez2019), within nodule fields in the CCZ (Amon et al., Reference Amon, Ziegler, Dahlgren, Glover, Goineau, Gooday, Wiklund and Smith2016), and around mud volcanoes and cold seeps at the Mariana trench (Glickson et al., Reference Glickson, Amon, McKenna and Elliott2017). In the South China Sea, juveniles of R. daphneae were settled on plastic debris (Song et al., Reference Song, Lyu, Xiaodi, Ruthensteiner, Ahn, Pastorino, Wang, Gu, Ta, Sun, Liu, Han, Ke and Peng2021).
(a) Overview of global locations where Relicanthus daphneae has been observed (orange) or collected (purple). Stars represent where molecular analysis has been conducted. The black box outlines the data presented in this study. (b) Three sites where R. daphneae was observed within the BGR contract area in the Indian Ocean. SWIR, Southwest Indian Ridge; CIR, Central Indian Ridge; RTJ, Rodriguez Triple Junction; SEIR, Southeast Indian Ridge. Bathymetry map provided by: GEBCO Compilation Group (2020) GEBCO 2020 Grid (doi:10.5285/a29c5465-b138-234d-e053-6c86abc040b9).
In this study, we extend the known global distribution of R. daphneae presenting the first observations and DNA barcoding results in the Indian Ocean. We provide 16S, 18S and COI sequences, and present physiological and environmental data associated with the documented individuals. In addition, we report megafauna observed in proximity to R. daphneae and describe, for the first time, a feeding behaviour exhibited during an expedition in 2022.
Materials and methods
Study area and sample and imagery collection
Annual expeditions have been surveying the Federal Institute for Geosciences and Natural Resources (BGR) contract area for massive polymetallic sulphide deposits since 2013 as part of the Indian Ocean Exploration (INDEX) Project. The BGR contract area includes the Rodriguez Triple Junction (RTJ), and portions of the Central Indian Ridge (CIR) and the Southeast Indian Ridge (SEIR). Photographs of R. daphneae were obtained using a remotely operated vehicle (ROV) and video sledge aboard the R/V Sonne (2017) and R/V Pelagia (2021, 2022) (Table 1).
Metadata of eight Relicanthus daphneae individuals recorded in this study, including imagery tool (Platform), transect name, depth, substrate to which specimen was attached and distances to nearest active and inactive hydrothermal site
sCIR, southern Central Indian Ridge; RTJ, Rodriguez Triple Junction; SEIR, Southeast Indian Ridge; STR, STROMER (video sledge); ROV, remotely operated vehicle.
Minimum distance to active and inactive hydrothermal site was measured in QGis 3.10 to the centre of the nearest known active chimney complex of an active hydrothermal vent site and to the nearest inactive hydrothermal deposit.
The multi-functional video sledge STROMER (German abbr. for ‘Simpler TauchROboter, Multifunktional ERweiterbar’), developed by the BGR, recorded continuous HD-video imagery and photographs using a downward looking Canon PowerShot G15 (4000 × 3000 pixels, 180 dpi) or SONY ILCE-9 (6000 × 4000 pixels, 350 dpi) camera, illuminated by a LED lamp, high-intensity discharge lamp and flashlights. Two lasers spaced 30 cm apart were projected onto the seafloor in the field of view of the camera for scaling. The ROV ROPOS (Remotely Operated Platform for Ocean Science; Canadian Scientific Submersible Facilitation) recorded continuous HD-video imagery and manually captured high-resolution DSC photographs using a SONY ILCE-9 (6000 × 4000 pixels, 350 dpi) digital still camera. Two lasers spaced 10 cm apart were projected into the field of view for scaling.
In total, eight specimens were observed (Table 1), but only six could be confidently identified (Figure 2). The other two specimens could not be confidently identified due to high vehicle altitude and low image resolution and, therefore, were not used for any measurements (Supplementary Figure S1). Four of the six identified individuals were seen on the southern CIR (on transects I22-071ROV, I22-073ROV), one on the RTJ (transect I21-093STR) and the other on the SEIR (transect I17-94STR) (Table 1).
Relicanthus daphneae individuals observed within the BGR contract area in the Indian Ocean during three INDEX expeditions between 2017 and 2022. Relicanthus daphneae was recorded on the Southeast Indian Ridge (SEIR) (a), the southern Central Indian Ridge (sCIR) (b–e), and the Rodriguez Triple Junction (f). The specimen in panel (d) was photographed after sampling a tentacle for molecular analysis and shows the other tentacles retracted and curled at the distal end. Scale bars are 30 cm length (photograph copyright BGR).
Morphological measurements and geological distances
The column diameter and maximum tentacle length of each R. daphneae was measured from the imagery in ImageJ (Schneider et al., Reference Schneider, Rasband and Eliceiri2012). Distance from each specimen to the nearest active venting chimney complex and to the nearest inactive sulphide occurrence, as observed during INDEX expeditions (vent field names and coordinates not published), was measured in QGIS3.10 using navigation data collected by STROMER and ROPOS during deployments.
Sample acquisition and genetic analysis
A portion of a tentacle from one individual was sampled with the manipulator arm of ROV ROPOS. On board, the sample was assigned a unique reference number (voucher specimen code: I22_0227), the tissue was fixated in 96% ethanol and stored in −20 °C. Total genomic DNA was extracted at the Senckenberg am Meer, German Centre for Marine Biodiversity Research – DZMB, and sequenced by Macrogen in Amsterdam, the Netherlands. The DNA extraction was carried out using 30 μl Chelex (InstaGene Matrix, Bio-Rad) according to the manufacturer's protocol. Approximately 1800 bp of 18S, 750 bp of 16S and 630 bp of cytochrome c oxidase subunit I (COI) were amplified using primers listed in Table 2. PCR reactions were conducted using an Eppendorf Mastercycler Pro S (Eppendorf, Hamburg, Germany) and each PCR reaction contained: 13.0 μl of AccuStart II PCR ToughMix (2X) (QuantaBio), 9.0 μl of H2O, 0.5 μl of each primer (10 pmol/μl) and 2 μl of DNA template in a mixture of total 25 μl. The PCR amplification profile for each marker is listed in Table 3. PCR products were visualized after electrophoresis on 1% agarose gels and the reactions which produced bands were sent to be sequenced in both directions using either the primers themselves or the M13 sequence tails (Table 2).
List of primers and tails used for amplification and sequencing, including related literature
PCR amplification profiles used in this study
Double stranded sequences were assembled and checked with Geneious version 2023.1.1 (Kearse et al., Reference Kearse, Moir, Wilson, Stones-Havas, Cheung, Sturrock, Buxton, Cooper, Markowitz, Duran, Thierer, Ashton, Meintjes and Drummond2012). When possible, sequences were translated into amino acids prior to analysis to ensure that no gaps or stop codons were present in the alignment. Species identification was performed using the BLAST search from Geneious version 2023.1.1 (Kearse et al., Reference Kearse, Moir, Wilson, Stones-Havas, Cheung, Sturrock, Buxton, Cooper, Markowitz, Duran, Thierer, Ashton, Meintjes and Drummond2012) and BOLD system (Ratnasingham and Hebert, Reference Ratnasingham and Hebert2007) tree-based identification.
Results and discussion
Occurrence and habitat properties
Prior to this study, there were only 19 published records of R. daphneae. Here, we contribute six new observations (Figure 2), expanding the database by more than 30%, totalling 25 records, and extend the global species distribution to the Indian Ocean. These are the only records of R. daphneae in the BGR contract area since the launch of the INDEX program (up to and including the 2022 field season). Compared to the depth covered by INDEX expeditions since 2013 (~1600–3700 m), R. daphneae was observed over a relatively narrow depth range of 2993–3088 m (Table 1) which is within the globally observed depth range for this species (2400–4400 m) (Daly, Reference Daly2006; Amon et al., Reference Amon, Ziegler, Dahlgren, Glover, Goineau, Gooday, Wiklund and Smith2016; Magalhães et al., Reference Magalhães, Linse and Wiklund2017). On the EPR (Pacific Ocean) and East Scotia Ridge (Southern Ocean), R. daphneae occurs at a shallower depth (2400–2650 m) (Daly, Reference Daly2006; Magalhães et al., Reference Magalhães, Linse and Wiklund2017) while on the Mariana Trench and the CCZ (Pacific Ocean), it occurs at a greater depth (3300–4400 m) (Amon et al., Reference Amon, Ziegler, Dahlgren, Glover, Goineau, Gooday, Wiklund and Smith2016; Glickson et al., Reference Glickson, Amon, McKenna and Elliott2017). In the South China Sea, juveniles were found on plastic debris that was collected at 1890 m depth (Song et al., Reference Song, Lyu, Xiaodi, Ruthensteiner, Ahn, Pastorino, Wang, Gu, Ta, Sun, Liu, Han, Ke and Peng2021). Although this is the shallowest record of R. daphneae, it is possible that the debris was transported post settlement and does not truly represent the depth at which the species occurs.
All individuals observed on the Indian Ridge were seen less than 1 km away from both active and inactive hydrothermal deposits (Table 1). We do not believe that the occurrence of R. daphneae is limited to the periphery of hydrothermal vent fields exclusively, but rather that enhanced food availability is likely beneficial. We measured the closest reported distance of this species at 66 m from an active hydrothermal vent, shorter than the previously known distance of >100 m (Daly, Reference Daly2006; Xiao et al., Reference Xiao, Brugler, Broe, Gusmão, Daly and Rodríguez2019). Four of the six identified specimens occurred around a single active hydrothermal vent field, while the remaining two specimens were observed at two different active vent fields.
Despite the wide range of substrate types identified along imagery transects (sediments, basalts [distinguishable as ridges, pillows, fault scarps, sheet flow or talus], blocky gabbro or sulphide deposits), all observed specimens were attached to exposed basalt or hydrothermally altered basalt at the edge of a vertical fault scarp or on elevated pillows (Figure 2; Table 1). On the EPR, R. daphneae also colonized large basalt outcrops, cliffs and boulders and were oriented with tentacles into the current (Daly, Reference Daly2006). It is possible that giant anemones position themselves to maximize food availability in the current.
Morphological and behavioural observations
Consistent with existing descriptions of R. daphneae (Daly, Reference Daly2006), all specimens in this study were solitary with distances of >50 m between individuals. Epimegafauna in the immediate vicinity of R. daphneae included other anemones (Actiniaria fam. indet. [DZMB_2021_0020, Gerdes et al., Reference Gerdes, Kihara, Martínez Arbizu, Kuhn, Schwarz-Schampera, Mah, Norenburg, Linley, Shalaeva, Macpherson, Gordon, Stöhr, Messing, Bober, Guggolz, Christodoulou, Gebruk, Kremenetskaia, Kroh, Sanamyan, Bolstad, Hoffman, Gooday and Molodtsova2021]), a hydrozoan (Candelabrum sp. indet.) and stalked poriferans. Three scale worms, likely of the family Polynoidae, were observed moving on the tentacles and disc of a specimen of R. daphneae (Supplementary Video S1, Neufeld et al., Reference Neufeld, Meyn, Kihara, Martínez Arbizu and Kuhn2024). These scale worms are similar to others (Polynoidae gen. indet.) that have been observed on Actiniarian anemones (Actinostolidae gen. indet.) within the INDEX area (Gerdes et al., Reference Gerdes, Kihara, Martínez Arbizu, Kuhn, Schwarz-Schampera, Mah, Norenburg, Linley, Shalaeva, Macpherson, Gordon, Stöhr, Messing, Bober, Guggolz, Christodoulou, Gebruk, Kremenetskaia, Kroh, Sanamyan, Bolstad, Hoffman, Gooday and Molodtsova2021).
Column width ranged from 4 to 22 cm (n = 6 specimens) (Table 1) which was within the lower range of previously reported in situ measurements (2–100 cm) (Daly, Reference Daly2006), while the maximum tentacle length ranged from 56 to 197 cm although it was difficult to obtain accurate measurements. In most cases, tentacles were partially cut out of the image or retracted, resulting in an underestimation of the length.
During the sampling procedure of the tentacle for molecular analysis, no autotomy, either of the sampled or of any other tentacles, could be observed as reported by Daly (Reference Daly2006). However, we only sampled a single tentacle, and the observed autotomy was reported when sampling entire R. daphneae specimens. The disturbance caused by sampling provoked a retraction behaviour of almost all tentacles, causing the tips to curl and widening of the tentacle base (Figure 2D).
We observed for the first time, R. daphneae capturing live prey (Supplementary Video S2, Neufeld et al., Reference Neufeld, Meyn, Kihara, Martínez Arbizu and Kuhn2024), the shrimp Rimicaris kairei Watabe and Hashimoto, 2002. A single live R. kairei was stuck to the side of a tentacle of R. daphneae, moving its antennae, pleopods and legs. The movement of the partially entangled shrimp seemed to provoke R. daphneae to wrap its tentacle around the captured specimen, presumably to prevent it from escaping. It has been previously suggested that the unusually large size of the tentacles and spirocysts (organelles specialized for adhesion) enable these anemones to capture large free-swimming fauna (Daly, Reference Daly2006). However, specimens have never been observed with prey, either captured or partially digested.
Molecular results
Three sequences were obtained from the analysed material and fragment lengths ranged from 639 to 1786 bp. All sequences were barcode compliant and placed in BIN BOLD: AEI2630. Comparing our COI barcoding result with GenBank and BOLD databases, we obtained 100% similarity score with: Cnidaria, Anthozoa, Actiniaria, Relicanthidae, Relicanthus, R. daphneae – GenBank MN055612 (South China Sea; Song et al., Reference Song, Lyu, Xiaodi, Ruthensteiner, Ahn, Pastorino, Wang, Gu, Ta, Sun, Liu, Han, Ke and Peng2021) and MK947129 (Pacific and Southern Oceans; Xiao et al., Reference Xiao, Brugler, Broe, Gusmão, Daly and Rodríguez2019).
In addition, genetic analysis also revealed a high degree of similarity (≥99.9%) between the sequenced 16S and 18S genes and existing sequences in the GenBank database. The 16S showed a 99.9% similarity score to MK947129 (Pacific and Southern Oceans; Xiao et al., Reference Xiao, Brugler, Broe, Gusmão, Daly and Rodríguez2019). Similarly, the 18S exhibited 100% similarity to KJ483028 (Pacific Ocean; Rodrígues et al., Reference Rodríguez, Barbeitos, Brugler, Crowley, Grajales, Gusmão, Häussermann, Reft and Daly2014) and 99.9% similarity to MN059669 (South China Sea; Song et al., Reference Song, Lyu, Xiaodi, Ruthensteiner, Ahn, Pastorino, Wang, Gu, Ta, Sun, Liu, Han, Ke and Peng2021).
While molecular data suggest a widespread distribution of R. daphneae across the Pacific, Southern and Indian Oceans (Figure 1a), it is important to consider these findings in conjunction with morphological observations and a larger sample size. Although the analysed specimens exhibited high genetic similarity to R. daphneae sequences from various locations, subtle morphological variations may exist between populations. Therefore, further investigation, incorporating both morphological examinations and a more extensive molecular dataset is needed to confirm the species identification and fully understand the biogeographic patterns of R. daphneae with potential regional variations.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0025315424001127.
Acknowledgements
Many thanks to the ROPOS ROV team and the crew and captain of RV Pelagia and RV Sonne for their support and expertise during the expedition to the INDEX area. Pictures and sample information presented in this study originate from the INDEX exploration project for marine polymetallic sulphides by the Federal Institute for Geosciences and Natural Resources (BGR) on behalf of the German Federal Ministry for Economic Affairs and Climate Action. Exploration activities are carried out in the framework and under the regulations of an exploration license with the International Seabed Authority. We are grateful to the one anonymous reviewer for their valuable feedback. This publication has the number 100 from the Senckenberg am Meer Metabarcoding and Molecular Laboratory.
Data availability
The photographs analysed during the current study and the generated tables containing aerial estimation and annotated occurrence data are available from the corresponding author on reasonable request. Sequences were deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank/) under accession numbers: COI – PP264562, 16S – PP264564 and 18S – PP264563. Additional relevant specimen information, taxonomic classifications, photos, DNA barcodes, used primer pairs and trace files were uploaded to the Barcode of Life Data Systems (BOLD; www.boldsystems.org; Ratnasingham and Hebert, Reference Ratnasingham and Hebert2007), project “Indian Ocean Hydrothermal Vent Fauna – INDEX”, subproject “Indian Ocean Hydrothermal Vent Megafauna” in the public dataset “INDEX_Megafauna_2024_2” with dataset code: DS-INMEG242 and DOI: dx.doi.org/10.5883/DS-INMEG242. Supplement videos are available on PANGAEA (https://doi.org/10.1594/PANGAEA.967443).
Author contributions
T. C. K. and P. M. A. conceived and designed research. T. C. K. and K. M. collected the data. M. N., T. C. K. and K. M. analysed the data. M. N. wrote the manuscript. T. K. is the principal investigator of the INDEX project through which data was collected. All authors read and approved the manuscript.
Financial support
This research was (partly) funded through contractual work assigned by the Federal Institute for Geosciences and Natural Resources (BGR).
Competing interest
None.
Ethical standards
All necessary permits for sampling and observational field studies have been obtained by the authors from the competent authorities and are mentioned in the acknowledgements, if applicable. The study is compliant with CBD and Nagoya protocols.
Open access
