2. The discovery of a natural whale fall in the Antarctic deep sea
2.2 Methods
2.2.2 DNA barcoding of the whale fall
DNA was isolated from the caldera whale bone using liquid nitrogen to freeze the bone so that it could be ground into powder. Approximately 0.1 g of bone powder was placed in each of three vials. Extraction was done using Qiagen DNeasy Blood and Tissue Extraction Kit
following the protocol: Purification of Total DNA from Animal Tissue. Qiagen extractions were stored at -20°C. The primers used were made for a segment approximately 873 bp in length, containing the complete cytochrome b gene and part of the tRNA of the Orcinus orca Linnaeus, 1758 complete mitochondrion genome: Whale892F and Whale892R (Foote et al.,
2011)(NC_014682). The primer sequences were as follows: 5’-
GTTATAGCCACCGCATTCGT-3’ and 5’-AATTCCAGCTTTGGGTGTTG-3’. The DNA extracted from one vial of bone material (0.1 g) was used for PCR using PureTaq Ready-To-Go PCR beads (GE Healthcare). The PCR was performed in 25 µL reactions, consisting of 1 µL of each primer, 2 µL DNA template and 21 µL dH2O. The PCR amplification profile consisted of initial denaturation at 95°C for 5 minutes, 35 cycles of denaturation at 94°C for 45 seconds, annealing at 55°C for 45 seconds, extension at 72°C for 2 minutes and a final extension at 72°C for 10 minutes. Products of the PCR were confirmed by electrophoresis in a 1.5% agarose gel. Purification of the PCR products was achieved using a Qiagen PCR Purification Kit.
Sequencing was performed on an ABI 3730XL DNA Analyser (Applied Biosystems) at the Sequencing Facility at the Natural History Museum, London using the primers described above.
Overlapping sequence fragments were merged into consensus sequences using Geneious (Drummond et al., 2011) and aligned using MUSCLE (Edgar, 2004) provided as a plug-in in Geneious with default settings. The mtDNA sequences for all of the whales used in the phylogenetic analysis were obtained from GenBank (Table 2.1). Bayesian phylogenetic analyses (BA) were conducted with MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003). Analyses were run three times with the combined dataset with four chains for 2 000 000 generations, with 400 000 generations discarded as burn-in. The evolutionary models used for the molecular data in BA were obtained by running the two separate datasets in MrModelTest (Nylander, 2004), and for tRNA the optional model was K80+G. For cytochrome b, the data were partitioned into codon positions with position 1 following K80+G, position 2 following HKY, while HKY+G was used for position 3. In the combined BA, the data were partitioned into the two parts (cytochrome b and tRNA) and the evolutionary models mentioned above were applied to each partition and corresponding codon position respectively.
A natural whale fall in the Southern Ocean
46
Table 2.1 Specimen information for the mysticete and outgroup mt-genomes analysed in this study.
Scientific name English name Accession no. References
Hippopotamus amphibious Hippopotamus AP003425 Yasue et al., unpublished
Caperea marginata Pygmy Right whale NC_005269 Arnason et al., 2004
Balaena mysticetus Bowhead whale NC_005268 Arnason et al., 2004
Eubalaena australis 1 Southern Right whale NC_006930 Sasaki et al., 2005
Eubalaena australis 2 AP006473 Sasaki et al., 2005
Eubalaena japonica Northern Right whale NC_006931 Sasaki et al., 2005
Balaenoptera acutorostrata 1 North Atlantic Minke whale NC_005271 Arnason et al., 2004
Balaenoptera acutorostrata 2 AP006468 Sasaki et al., 2005
Balaenoptera acutorostrata 3 AJ554054 Arnason et al., 2004
Balaenoptera bonaerensis 1 Antarctic Minke whale NC_006926 Sasaki et al., 2005
Balaenoptera bonaerensis 2 AP006466 Sasaki et al., 2005
Eschrichtius robustus 1 Gray whale NC_005270 Arnason et al., 2004
Eschrichtius robustus 2 AJ554053 Arnason et al., 2005
Eschrichtius robustus 3 AP006471 Sasaki et al., 2005
Megaptera novaeangliae 1 Humpback whale AP006467 Sasaki et al., 2005
Megaptera novaeangliae 2 NC_006927 Sasaki et al., 2005
Balaenoptera physalus 1 Fin whale NC_001321 Valverde et al., 1994
Balaenoptera physalus 2 X61145 Arnason et al., 1991
Balaenoptera omurai Omura’s whale NC_007937 Sasaki et al., 2006
Balaenoptera edeni Bryde’s whale NC_007938 Sasaki et al., 2006
Balaenoptera borealis Sei whale AP006470 Sasaki et al., 2005
Balaenoptera musculus Blue whale NC_001601 Arnason & Gullberg, 1993
Physeter catodon Sperm whale AJ277029 Arnason et al., 2000
Orcinus orca Orca whale NC_014682 Foote et al., 2010
2.2.3
210Pb/
226Ra analysis of the whale fall
The isotopic pair (210 Pb/226 Ra) has been used previously to establish the time since cetacean death and deposition on the seafloor by Schuller et al. (2004). We tried to analyse the age since death of the caldera skeleton using the method below but were unsuccessful after two attempts. Seven other bones from skeletons of known varying ages were also analysed during this study to show the degree of error with the methodology (Table 2.2). The detailed methodology can be found below. Despite taking great care to avoid contamination, ratios of the isotopic pair 210 Pb/226 Ra were greater than unity on both occasions. Attempts are however, ongoing. As a result, only the state of decomposition of the bones could be used to gauge the age of the skeleton.
2.2.3.1 Sample Preparation
The known bone samples used in this study were collected from various museums, research institutes and whale strandings (Table 2.2). The samples were drilled using an electric hand drill and the shavings were collected, weighed and placed in plastic vials. At the National
Oceanography Centre, Southampton, 36 mL of 2:1 chloroform:methanol were added to the entire whale-bone samples, which were left overnight on a tumbler to remove lipids. The vials were centrifuged at 2000 rpm for two minutes each to allow the bone matter to settle and the lipid solute was decanted carefully to prevent loss of any bone material. This lipid-removal
process was repeated three times but samples were only left for two hours on a tumbler rather than overnight. After the lipid separations, the solutions were replaced with methanol and left overnight. The samples were then individually filtered using Whatman glass microfibre filter papers. The filtrates were then placed in a drying oven (65°C) for two hours. After, the samples were filled with 35 mL 30 % hydrogen peroxide to remove organic deposits. After ‘fizzing’ (O2 generation) had stopped, vials were then centrifuged, emptied and filled with deionised MilliQ water, gently shaken, centrifuged, and excess water decanted. The samples were then freeze- dried overnight. Each was reduced to powder form using an agate mortar and pestle and then weighed the following morning. Each powdered sample was placed in an airtight glass vial of fixed geometry with the sample number, sampling location and weight written on the vials. These were then left to allow ingrowth of Rn daughters for 20 days before being counted on a Canberra (HPGe-ULB) Ultra Low Background High Resolution Gamma Spectrometer for 200,000 seconds.
2.2.3.2 210 Pb analysis
After gamma counting, 3.5 g of each sample were removed and placed in PTFE beakers. Each was spiked with 10 µL of 133Ba from a stock solution of 1900 Bq/mL in 2M HNO
3, and 20.0 µL of 209Po from a stock solution of 0.2612 Bq/g (Ref date of 14/02/11). Two blank solutions and two standard solutions were prepared using the same spikes, however 20.0 µL of the spike 210Pb was added to each of the standard samples. 2 g of sodium dihydrogen orthophosphate
(NaH2PO4.2H2O) and 2 g of hydrated calcium chloride (CaCl2.2H2O) were also added as a substrate to each of the blank and standard samples to match the approximate chemical composition of the bone samples. Then approximately 10 mL fuming HNO3 (100 %) was added to all samples including the blanks and standards. These were left on a 90°C hot plate in a fume cupboard overnight. The following morning, a further 3 mL of fuming HNO3 was added to each sample and left until the HNO3 stopped fuming and each had evaporated to dryness. This process was done to remove all organics and carbonates from the samples. To remove the fuming HNO3 in each mixture, three washes with 3 mL of concentrated HCl were added and then evaporated until dryness. The residues were then dissolved in 10 mL of 6M HCl and filtered into a glass beaker using Whatman cellulose (No. 41) filter papers. Each PTFE container was then washed with 5 mL deionised MilliQ water and poured through the filter paper into the acid fraction. The filter paper was washed with 5 mL deionised MilliQ water. After filtration was complete, the volume of each solution was made up to around 60 mL with deionised MilliQ water diluting the acid to about 1 M plus 0.5 g of ascorbic acid (to ensure reduction of any FeIII to FeII). Silver discs were polished with silver cleaner to remove any residues, labeled and placed in PTFE holders. Each disc was then placed into the respective solutions and left for two days on a warm surface to allow the polonium to plate onto the silver
A natural whale fall in the Southern Ocean
48
discs. The silver discs were removed from the solutions, washed in deionised MilliQ water and air-dried for one hour. The discs were placed into a Canberra Quad Alpha Spectrometer Model 7404 and counted for approximately 400,000 seconds until there were approximately 1000 counts in both alpha peaks.
2.2.3.3 226 Ra analysis
The solutions that had their 209Po and 210Po removed were used to determine the 226Ra activity. The solutions were transferred to a 90°C hot plate to evaporate to dryness. The borosilicate beakers were placed in a muffle furnace for four hours at 550°C to ash. The selected
temperature allows ashing but avoids melting the glassware. The samples were allowed to cool and then the ash from each sample was leached twice using 10 mL 8M HNO3 each time. Solid and liquid fractions were separated by centrifuging (3000 rpm, 5 minutes) and the resulting clear liquid was evaporated to dryness in a 20 mL glass scintillation vial. The remaining solid residue was re-dissolved using 5 mL of 2M HNO3 and the total sample volume was set to 10 mL using MQ water. The chemical recovery for all sample digests and subsequent handlings was monitored through the use of 10 µL 133Ba tracer added (an effective chemical proxy for Ra) to each sample at the beginning of the sample digestion. Any losses were determined by
comparing the 133Ba gamma activity (HPGe gamma detector) with that from a reference solution that had not been through any chemical manipulation. Next, 10 mL of PerkinElmer ®High Efficiency Mineral Oil with Scintillator (HEMOS) was added. Each scintillation vial lid was fitted with a Viton seal to prevent any loss of radon gas. The mineral oil cocktail effectively traps Rn (insoluble in the acidic aqueous fraction but highly soluble in the oil) produced in the 226Ra decay chain and after a 20 day radioactive in-growth period secular equilibrium is achieved. The date and time of sealing were marked on the vials, and the radon was allowed to re-equilibriate with the 226Ra for a period of 20 days. The samples were then counted using a PerkinElmer® 1220 Quantulus Ultra Low Level Liquid Scintillation Spectrometer with alpha/beta signal discrimination mode switched on to count the radon and other short-lived radon progeny in the samples. The Rn daughters in the HEMOS provide a sensitive indication of the 226Ra activity.
Table 2.2 Minke-whale bone samples of known and unknown ages dated in this study using 210Pb/226Ra disequilibria.
No. Sample Type Location Bone Found Time Since Death
1 Natural whale fall Kemp Caldera, Scotia Sea F549 Unknown 2 Natural whale fall Kemp Caldera, Scotia Sea F542 Unknown
3 Natural whale fall Tjarno, Sweden - 125 m 8 years
4 Natural whale fall Koster Fjord Sweden (Fjallbacka) 4 years
5 Museum Bohuslan, Fjallbacka (Gothenburg) 13 years
6 Museum Bohuslan, Kungshamn (Gothenburg) 19 years
7 Museum Bohuslan, Goteborgs (Gothenburg) 21 years
8 Museum Skagen (Gothenburg) 100 years
A natural whale fall in the Southern Ocean
50
Figure