Nano-CT scanning

Nano-CT scanning was carried out with a Zeiss Versa 520. Samples were analysed in pairs using a customised pine wood mount. Sample mount, X-ray source and detector geometry were kept constant throughout the first set of scans (Fig. 2). A scan resolution voxel (a 3D unit of space which varies in dimensions between CT reconstructions depending on scanning parameters) size of 2.4 µm3 was typically achieved using this set up in order to maximize the number of specimens that could be analysed in a single scan. During the second run of scans (Fig. 3) focusing on just 4 specimens (2 historic and 2 modern), X-ray source and detector were positioned to maximize resolution and a voxel size of <0.8 µm3 was achieved. Between 700–1000 individual X-ray absorption profiles of each mount were taken and combined to build a 3D rendering of the image using Avizo 9.2 software which was also used for image analysis.

The differential X-ray absorption of organic matter, pine wood, and calcium carbonate translates into different greyscale intensities in the reconstructed 3D image; therefore, it was possible to filter out pine wood and organic material from the analysed data for volume, wall thickness etc. Object volume, average thickness and surface area were determined using standard Volume Graphics analysis. In addition, dimension measurements were taken on 3D renderings of shells. For N. dutertrei and G. ruber, three dimensional measurements were taken, the width (at the widest point), the width at half shell length and length (Fig. 2a). Object volume, average thickness and surface area were determined using standard Volume Graphics analysis. For direct comparisons between shell thickness, weight and density of the old and new samples, a size filter was applied to the dataset and only individuals of the same length were used.

X-ray diffraction

Specimens of Globigerina bulloides were picked for X-ray diffraction analysis as they were relatively abundant in both Tara and Challenger samples, between 5 to 10 shells were soaked in ethanol and gently ground in an agate mortar and deposited on a circular sapphire substrate. The XRD data were collected using an ENRAF-Nonius 590 diffractometer with a Tintel curved position sensitive detector (PSD). The angular linearity of the PSD was calibrated with silicone powder and silver behenate. This apparatus collects data from 2–120 °2θ continuously throughout the experiment. Cobalt Ka radiation was selected from the primary beam by a Germanium 111 crystal monochromator with the x-ray tube operating at 40 kV and 30 mA. Horizontal and vertical slits restricted the beam to a height of 0.14 mm and width of 5 mm. Data were collected with samples spinning continuously in the plane of the sample surface and with the sample surface at an angle of 4° to the incident beam.

Detailed statistics

Neogloboquadrina dutertrei

Scan 1. In chamber 1 (the final chamber in the whorl), the preindustrial (Challenger) specimens recorded a mean shell thickness of 20.60 ± 0.56 μm, and a mean thickness of 8.93 ± 0.44 μm was recorded in the modern (Tara) specimen. A two sample t-test assuming equal variance was carried out. The t-statistic calculated was 16.593 and the two- tailed probability was 0.000. Therefore we can conclude there is a significant difference in shell thickness between the Challenger and Tara specimens.

In chamber 2, the preindustrial (Challenger) specimens recorded a mean shell thickness of 28.02 ± 1.2 μm, and a mean thickness of 18.12 ± 0.89 μm was recorded in the modern (Tara) specimen. A two sample t-test assuming equal variance was carried out. The t-statistic calculated was 6.71 and the two- tailed probability was 0.000. Therefore we can conclude there is a significant difference in shell thickness between the Challenger and Tara specimens.

In chamber 3, the preindustrial (Challenger) specimens recorded a mean shell thickness of 37.98 ± 1.30 μm, and a mean thickness of 16.19 ± 0.90 μm was recorded in the modern (Tara) specimen. A Mann Whitney U test showed that there was a significant difference (U = 170, p = 0.000) between the preindustrial and modern specimens.

Scan 2. In chamber 1 (the final chamber in the whorl), the preindustrial (Challenger) specimens recorded a mean shell thickness of 20.16 ± 0.65 μm, and a mean thickness of 4.33 ± 0.17 μm was recorded in the modern (Tara) specimen. A two sample t-test assuming unequal variance was carried out. The t-statistic calculated was 24.142 and the two- tailed probability was 0.000. Therefore we can conclude there is a significant difference in shell thickness between the Challenger and Tara specimens.

In chamber 2, the preindustrial (Challenger) specimens recorded a mean shell thickness of 22.55 ± 0.59 μm, and a mean thickness of 5.44 ± 0.27 μm was recorded in the modern (Tara) specimen. A two sample t-test assuming unequal variance was carried out. The t-statistic calculated was 26.521 and the two- tailed probability was 0.000. Therefore we can conclude there is a significant difference in shell thickness between the Challenger and Tara specimens.

In chamber 3, the preindustrial (Challenger) specimens recorded a mean shell thickness of 32.87 ± 0.90 μm, and a mean thickness of 7.89 ± 0.33 μm was recorded in the modern (Tara) specimen. A Mann Whitney U test showed that there was a significant difference (U = 432, p = 0.000) between the preindustrial and modern specimens.

Globigerinoides ruber

Scan 1 In chamber 1 (the final chamber in the whorl), the preindustrial (Challenger) specimens recorded a mean shell thickness of 16.60 ± 0.59 μm, and a mean thickness of 10.38 ± 0.59 μm was recorded in the modern (Tara) specimen. A two sample t-test assuming equal variance was carried out. The t-statistic calculated was -7.459 and the two- tailed probability was 0.000. Therefore we can conclude there is a significant difference in shell thickness between the Challenger and Tara specimens.

In chamber 2, the preindustrial (Challenger) specimens recorded a mean shell thickness of 15.74 ± 0.74 μm, and a mean thickness of 12.74 ± 1.12 μm was recorded in the modern (Tara) specimen A Mann Whitney U test showed that there was a not a significant difference (U = 95.5, p = 0.000) between the preindustrial and modern specimens.

In chamber 3, the preindustrial (Challenger) specimens recorded a mean shell thickness of 17.40 ± 0.78 μm, and a mean thickness of 14.52 ± 0.45 μm was recorded in the modern (Tara) specimen. However insufficient replicate measurements were collected (<10) to perform statistical analysis of the data.

Scan 2. In chamber 1, the preindustrial (Challenger) specimens recorded a mean shell thickness of 12.71 ± 0.25 μm, and a mean thickness of 10.01 ± 0.47 μm was recorded in the modern (Tara) specimen. A Mann Whitney U test showed that there was a significant difference (U = 58.5, p = 0.000) between the preindustrial and modern specimens.

In chamber 2, the preindustrial (Challenger) specimens recorded a mean shell thickness of 18.77 ± 0.64 μm, and a mean thickness of 15.06 ± 0.48 μm was recorded in the modern (Tara) specimen. A two sample t-test assuming equal variance was carried out. The t-statistic calculated was 4.749 and the two- tailed probability was 0.000. Therefore we can conclude there is a significant difference in shell thickness between the Challenger and Tara specimens.

Statistical analysis of the measurements taken on chamber 3 suggest that any observed difference in shell thickness is not significant.