Stable isotope ratios of C, N and S in Southern Ocean sea stars (1985-2017)

Study Extent

Sea stars were sampled in the Southern Ocean from 1985 to 2017 throughout multiple oceanographic campaigns.

Sampling

A double sampling strategy was set up to maximise the scope of this PhD thesis. First, sea stars were collected in the Southern Ocean during campaigns taking place in the framework of the vERSO and RECTO projects from December 2015 to March 2017. Second, suitable samples originating from multiple oceanographic campaigns and surveys from January 1985 to January 2015 were retrieved from archived collections stored in museums or institutions. Institutions that provided samples included the Université Libre de Bruxelles (Belgium), the National Museum of Natural History (Paris, France) and the Institute of Oceanology of the Polish Academy of Sciences (Sopot, Poland). Storage: Depending of the sampling campaign, sea stars were frozen, dried, stored in ethanol or fixed with formaldehyde and then stored in ethanol.

Quality Control

- Isotopic ratios were expressed in ‰ using the widespread δ notation (Coplen 2011) and relative to the international references Vienna Pee Dee Belemnite (for carbon), Atmospheric Air (for nitrogen) and Vienna Canyon Diablo Troilite (for sulfur). Sucrose (IAEA-C-6; δ13C=−10.8 ± 0.5‰; mean ± standard deviation), ammonium sulfate (IAEA-N-1; δ15N= 0.4 ± 0.2‰; mean ± SD) and silver sulfide (IAEA-S-1; δ34S = −0.3‰) were used as primary analytical standards for stable isotope ratios. Sulfanilic acid (Sigma-Aldrich; δ13C=−25.6 ± 0.4‰; δ15N=−0.13 ± 0.4‰; δ34S = 5.9 ± 0.5‰; means ± SD) was used as a secondary analytical standard for stable isotope ratios and as elemental standard. Standard deviations on multi-batch replicate measurements of secondary and internal laboratory standards (sea star tegument), analyzed interspersed with samples (one replicate of each standard every 15 analyses), were 0.3‰ for δ13C and δ15N and 0.5‰ for δ34S. - All records were validated. - Coordinates were plotted on map to verify the geographical location and locality. - All scientific names were checked for typo and matched to the species information backbone of Worlds Register of Marine Species (http://marinespecies.org/) and LSID were assigned to each taxa as scientificNameID. - Event date and time were converted into ISO 8601

Method steps

1. Sample preparation: For each sea star, one or several arms were separated from the central disc. Internal organs and podia were removed in each arm. With the exception of the already dried samples, the tegument and the podia of each arm were washed with demineralised water and oven-dried at 50°C during 48 hours. All samples were then homogenised into powder. Carbonates were removed from subsamples by exposing subsamples to 37 % hydrochloric acid vapour during 48 hours. Acidified subsamples were then kept at 60°C until further sample preparation.
2. Stable isotope analysis: The subsamples were then precisely weighed (ca 2.5-3 mg) in 5×8 tin cups with ca 3 mg of tungsten trioxide. Their elemental composition and their stable isotope values were analysed with an elemental analyser coupled to a continuous-flow isotope-ratio mass spectrometer.
3. Impact of preservation on stable isotope values: Correction factors were added to the δ13C and δ34S values of sea stars fixed with formaldehyde and/or stored in ethanol as fixation in formaldehyde and preservation in ethanol impact stable isotope values in sea stars (Le Bourg et al., 2020). For samples stored in ethanol, a correction factor of –0.6 ‰ was subtracted to δ13C values. For samples fixed with formaldehyde and then stored in ethanol, a correction factor of 0.2 ‰ was added to δ13C values to take into account the effects of both ethanol (–0.6 ‰) and formaldehyde (+0.8 ‰) on δ13C values. A correction factor of 1.5 ‰ was also added to δ34S values for samples fixed with formaldehyde.
4. Instrumentation: Stable isotope ratio measurements were performed by continuous flow–elemental analysis–isotope ratio mass spectrometry (CF-EA-IRMS) at University of Liège (Belgium), using a vario MICRO cube C-N-S elemental analyzer (Elementar Analysensysteme GmbH, Hanau, Germany) coupled to an IsoPrime100 isotope ratio mass spectrometer (Isoprime, Cheadle, United Kingdom).
5. Taxonomy and systematics: In the laboratory, sea stars were identified to the lowest taxonomic level possible either using morphological or molecular characters. Moreover, in several genera where clades showed a clear pattern of geographic or bathymetric distribution, results of genetic analyses were used as proxies to assign specimens to a probable species (Moreau 2019, Moreau et al. 2019). Bathybiaster sp. individuals sampled between 0 and 1000 m on the Antarctic continental shelf were considered as Bathybiaster loripes, those sampled deeper than 2000 m, as well as on the Kerguelen Plateau were considered as Bathybiaster vexillifer (Moreau 2019) while no species was assigned to one individual sampled in the South Sandwich Islands between 1000 and 1500 m. Chitonaster sp. individuals sampled in Western Antarctic Peninsula and South Orkney Island were considered as Chitonaster sp. 2. Diplasterias sp. individuals sampled in Western Antarctic Peninsula and South Orkney Island were considered as Diplasterias sp. 1, those sampled in South Sandwich Islands and Adélie Land as Diplasterias sp. 2 while no species was assigned to individuals sampled in the Weddell Shelf. Lysasterias sp. individuals sampled in Adélie Land were considered as Lysasterias sp. 1. Notasterias sp. individuals sampled in South Orkney Islands were considered as Notasterias sp. 1. Odontaster sp. individuals sampled on the Kerguelen Plateau were considered as Odontaster penicillatus. Psilaster charcoti individuals sampled near Bouvet Island, in the South Atlantic ecoregion, were considered as Psilaster charcoti – clade 2. By contrast, this method could not be used for genera for which no clear geographic or bathymetric patterns of distribution were recorded (e.g. Acodontaster). Consequently, these individuals remained identified down to the genus. Similarly, individuals that could not be identified further than the family were referred to by their family name (Echinasteridae and Pterasteridae). Nomenclature was thoroughly checked using the Taxon Match Tool implemented in the World Register of Marine Species, WoRMS (WoRMS Editorial Board 2021).

Echinodermata, Asteroidea

1. Asteroidea
   common name: Sea star rank: class
2. Animalia
   rank: kingdom
3. Echinodermata
   rank: phylum
4. Brisingida
   rank: order
5. Forcipulatida
   rank: order
6. Notomyotida
   rank: order
7. Paxillosida
   rank: order
8. Spinulosida
   rank: order
9. Valvatida
   rank: order
10. Velatida
    rank: order

Antarctic and Sub-Antarctic regions of the Southern Ocean