Dataset: CSIA-AA comparison between live polyps and outer proteinaceous skeletal material in three taxons of North Pacific deep-sea corals

Methods & Sampling
Live coral samples were collected with DSRV or ROV.  Once on-board polyps samples were removed using semi-sterile techniques and frozen until further analysis.

Isidella proteinaceous skeletons were washed first in sea then fresh water and air dried on deck.  On shore, cross-sectional disks ("cookies" in the parlance of dendrochronologists) were cut from near the basal attachment using a water-lubricated diamond band saw.  The proteinaceous nodes of Isidella were separated from the carbonate internodes with a scalpel.

Cross-sectional disks were approximately 1 cm in thickness.  Disks were mounted on glass slides and polished.

The outermost edge of the protein skeleton (~200mm radial depth, 5-7mm band parallel to the growth axis) from all three species was sampled with a computerized Merchantek micromill. For bulk δ13C analyses of skeleton, a subset of each skeleton sample was individually acid washed in 1N HCl in glass vials for four hours, rinsed three times in Milli-Q water, and dried over night at 50°C to remove calcium carbonate and isolate the organic fraction of the skeleton. Bulk δ15N analyses were conducted on non-acidified skeleton samples. Deep-sea coral polyp tissues are very lipid rich, and therefore a subset of each polyp sample was lipid extracted three times following the conventional methanol/chloroform protocol prior to δ13C analysis. Bulk δ15N analyses were conducted on non-lipid extracted polyp samples. Compound-specific stable isotopes of amino acids (CSI-AA) δ13C and δ15N analyses were conducted on lipid-extracted polyp tissue samples and acidified skeleton samples to improve chromatography.

Bulk stable carbon (δ13C) and stable nitrogen (δ15N) isotopes were measured on a 0.3 mg aliquot of each sample using a Carlo Erba 1108 elemental analyzer interfaced to a Thermo Finnegan Delta Plus XP isotope ratio mass spectrometer (IRMS) at the Stable Isotope Lab, University of California, Santa Cruz. Raw isotope values were corrected for instrument drift and linearity effects, calibrated against the in house isotopic reference materials of the Stable Isotope lab (http://emerald.ucsc.edu/~silab/), and reported in per mil (‰) relative to Vienna PeeDee Belemnite and air for carbon and nitrogen, respectively. Reproducibility of two lab standards was 0.05‰ and 0.15‰ for carbon and nitrogen isotopes, respectively.

CSIA was conducted on 3 mg of polyp tissue and proteinaceous skeleton for δ13C and 6 mg for δ15N. Samples were acid hydrolyzed in 1 ml of 6 N HCl at 110°C for 20 hrs to isolate the total free AAs and then evaporated to dryness under a gentle stream of ultra-high purity (UHP) N2. All samples were redissolved in 0.01N HCl and passed through 0.45mm Millipore glass-fiber filters followed by rinses with additional 0.01N HCl. Samples were then passed through individual cation exchange columns (Dowex 50WX* 400 ion exchange resin), rinsed with 0.01N HCl, and eluted into muffled glassware with 2N ammonia hydroxide. Dried samples were derivatized by esterification with acidified iso-propanol followed by acylation with trifluoroacetic anhydride. Derivatized samples were extracted with P-buffer (KH2PO4 + Na2HPO4 in Milli-Q water, pH 7) and chloroform three times with centrifugation (600 g) and organic phase extraction between each round. Samples were once again evaporated to dryness under a gentle stream of UHP N2 prior to neutralization with 2N HCl at 110°C for 5 min. Dried samples were acylated once again and then brought up in ethyl acetate for CSIA.

For AA δ13C analyses, the derivatized AAs were injected in split mode at 250°C and separated on a DB-5 column (50 m x 0.5 mm inner diameter; 0.25 mm film thickness; Agilent Technologies, Santa Clara, California, USA) in a Thermo Trace Ultra gas chromatograph (GC) at the University of California, Santa Cruz. The separated AA peaks were analyzed on a Finnegan MAT DeltaPlus XL IRMS interfaced to the GC through a GC-C III combustion furnace (960°C) and reduction furnace (630°C). For AA δ15N analyses, the derivatized AAs were injected in splitless mode at 250 °C and separated on a BPX5 column (60 m x 0.32 mm inner diameter, 1.0 mm film thickness; SGE Analytical Science, Austin, Texas, USA) in the same CG-C-IRMS interfaced through a combustion furnace (980°C), reduction furnace (650°C), and a liquid nitrogen trap. 

Instrument description:

For bulk isotopes:

Carlo Erba 1108 elemental analyzer interfaced to a Thermo Finnegan Delta Plus XP isotope ratio mass spectrometer 

CSIA-AA:

For AA δ13C analyses, the derivatized AAs were injected in split mode at 250°C and separated on a DB-5 column (50 m x 0.5 mm inner diameter; 0.25 mm film thickness; Agilent Technologies, Santa Clara, California, USA) in a Thermo Trace Ultra gas chromatograph (GC) at the University of California, Santa Cruz. The separated AA peaks were analyzed on a Finnegan MAT DeltaPlus XL IRMS interfaced to the GC through a GC-C III combustion furnace (960°C) and reduction furnace (630°C). For AA δ15N analyses, the derivatized AAs were injected in splitless mode at 250 °C and separated on a BPX5 column (60 m x 0.32 mm inner diameter, 1.0 mm film thickness; SGE Analytical Science, Austin, Texas, USA) in the same CG-C-IRMS interfaced through a combustion furnace (980°C), reduction furnace (650°C), and a liquid nitrogen trap.

Abbreviations:

AA  = amino acids
CSI-AA = ​Compound-specific stable isotopes of amino acids

Ala = Alanine

Asp = Asparagine + aspartic acid

Glu = Glutamine + glutamic acid

Gly = Glycine

Ile = Isoleucine

Leu = Leucine

Phe = Phenylalanine

Pro = Proline

Ser = Serine

Thr  = Threonine

Val  = Valine

Taxonomic identifiers:

Primnoa pacifica, urn:lsid:marinespecies.org:taxname:286539
Isidella, urn:lsid:marinespecies.org:taxname:125305
Kulamanamana haumeaae, urn:lsid:marinespecies.org:taxname:715097