Fig 1.
(A) Clanculus margaritarius A. Shell from Kitahama, Japan. Note that the specimen is sub adult. Specimen # 3. (B) Clanculus margaritarius B. Shell of an unlocalised specimen, NHMUK collection. Specimen # 10. (C) Clanculus pharaonius. Rose Reef, Thuwal, Saudi Arabia. Specimen # 4. (D) Calliostoma zizyphinum. Shetland Islands, United Kingdom. Specimen # 1. Scale bars = 5 mm, except in (D), where scale bar = 1 cm.
Fig 2.
Shells under visible and ultraviolet (UV) light.
(A, B) Clanculus margaritarius A (specimen #7), NHMUK 20150502. (C, D) Clanculus pharaonius (specimen #2). NHMUK 20160357. (E, F) Calliostoma zizyphinum (specimen #1). NHMUK 20160315. (A, C, E) Visible light. (B, D, F) UV light. Note that both the pink-red and yellow-brown pigmentation of Clanculus shells fluoresce red under UV light. The Ca. zizyphinum shell does not fluoresce under UV light.
Fig 3.
Confocal images showing uroporphyrin distribution in areas of pink-red and yellow-brown pigmentation on Clanculus margaritarius shell.
Shell images on each line are of a single shell and are in approximately the same orientation and magnification. (A-C) Top of shell, Clanculus margaritarius A (specimen #2). (A) Visible pink-red pigmentation. (B) Shaded volume 3D projection of a spectrally mixed image of the same shell showing fluorescent red areas congruent with visible pink-red pigment. (C) Maximum intensity projection of a spectrally unmixed image showing distribution of uroporphyrin I and III. (D-F) Clanculus margaritarius B (specimen #10). (D) Visible pink-red pigmentation. (E) Maximum intensity projection of a spectrally mixed image showing fluorescent red areas under UV light congruent with pink-red pigment. (F) Maximum intensity projection of a spectrally unmixed image showing distribution of uroporphyrin I and III. (G-L) Two different areas with yellow-brown, black and white colouration on shell of C. margaritarius A (specimen #2). (M-O) Yellow brown areas on shell of Clanculus margaritarius B (specimen #10). (G, J, M) Visible pigmentation for approximately the same areas as used in analyses. (H, K, N) Maximum intensity projection of spectrally mixed images showing red fluorescent areas under UV light congruent with yellow-brown pigmentation. Panel N is shown in 3D. (I, L, O) Maximum intensity projection of spectrally unmixed images showing distribution of uroporphyrin I and III. Panel O is shown in 3D. Note that in unmixed images green corresponds to uroporphyrin I and red to uroporphyrin III and yellow to areas where both co-occur. Note also that fluorescence is quenched in black spots and nearby white areas. Visible pigmentation is marked on each image: pr–pink-red, bl–black spot, w–nearby white area, yb–yellow-brown. Panels (G-I) include a small strip of blu-tack used to locate coloured areas.
Fig 4.
HPLC analyses for porphyrins in Clanculus.
(A) Chromatogram of nine porphyrin standards. (B, C) Clanculus margaritarius A. (B) Chromatogram of shell extract (specimen #3, 1:20 dilution). (C) Chromatogram of extract from coloured foot tissue (preserved in RNALater) (specimen #3). (D) Chromatogram of C. margaritarius B shell extract (specimen #9, 1:5 dilution). (E) Chromatogram of shell extract of Clanculus pharaonius (specimen #2). (F) Chromatogram of extract from foot tissue of Clanculus pharaonius (specimen # 1). Note that (B) and (C) were run on the same day as the standards shown in (A); other samples were run on different days.
Fig 5.
UV-visible absorption spectra of the Na-EDTA aqueous extracts from C. margaritarius A (specimen #5) and C. pharaonius (specimen #5) collected at 2 hours and 13 days after extraction.
Positions of λmax (Soret band) and Q bands Q(I)–Q(IV) are marked.
Table 1.
Concentration of melanin markers in Clanculus shells determined by HPLC.