Colour in Bivalve Shells: Using Resonance Raman Spectroscopy to Compare Pigments at Different Phylogenetic Levels

6 Colour in bivalve shells: using Resonance Raman spectroscopy to compare 7 pigments at different phylogenetic levels

9 J. Wade1, H. Pugh2,3, J. Nightingale1, J.S. Kim1, S. T. Williams2*

11 1 Imperial College London & Centre for Plastic Electronics, Blackett Laboratory, Prince 12 Consort Road, London SW7 2AZ, United Kingdom;

13 [email protected]; [email protected]; [email protected];

14 2 Natural History Museum, Department of Life Sciences, Cromwell Rd, London SW7 5BD, 15 United Kingdom;

16 [email protected]; [email protected]

17 3 Imperial College London, Department of Life Sciences, London SW7 2AZ, United Kingdom.

24 * Corresponding author. Email: [email protected]

25 Short title: Raman spectroscopy of bivalve shells

26 Key words: colour, bivalve, Raman spectroscopy, pigment, shell

28 Table of Contents

Studies have suggested that shell colour is phylogenetically distributed within Mollusca, but this is confounded by a lack of knowledge about the identity of most molluscan pigments. We use Raman J. Wade, H. Pugh, J. spectroscopy to examine Nightingale, J.S. Kim, S. T. * bivalve pigments and Williams compare spectra from taxa at different phylogenetic levels. We show that most Colour in bivalve shells: colours in bivalves are due using Resonance Raman to partially methylated spectroscopy to compare polyenes, possibly modified pigments at different carotenoids, but we were phylogenetic levels unable to detect Raman activity for green pigments suggesting this colour is due to structural elements or a different class of pigment.

30 Abstract

31 Several studies have suggested that shell colour may be phylogenetically distributed within 32 the phylum Mollusca, but this is confounded by our ignorance of its homology and lack of 33 understanding about the identity of most molluscan pigments. We use Resonance Raman 34 spectroscopy to address this problem by examining bivalve pigments producing a range of 35 colours and compare spectra from taxa at different phylogenetic levels. The spectra of most 36 shell pigments tested exhibited a skeletal signature typical of partially-methylated polyenes, 37 possibly modified carotenoids, with the strongest peaks occurring between 1501-1540 cm-1 -1 38 and 1117-1144 cm due to the C=C (1) and C-C (2) stretching modes respectively. Neither 39 pigment class nor mineral structure differentiated Imparidentia and Pteriomorphia, but the 40 most similar pigment spectra were found within a genus. Spectral acquisitions for purple 41 pigments for two species of Asaphis suggest that identical, or nearly identical pigments are 42 shared within this genus, and some red pigments from distantly related species have similar 43 spectra. Conversely, two species with brown shells have distinctly different pigments, 44 highlighting the difficulty in determining the homology of colour. Curiously we were unable 45 to detect any Raman activity for green pigments or pigment peaks for the yellow area of 46 Codakia paytenorum suggesting that these colours are due to structural elements or a 47 pigment that is quite different from those observed in other taxa examined to date. Our 48 results are consistent with the idea that classes of pigments are evolutionarily ancient but 49 heritable modifications may be specific to clades.

52 Introduction

53 Colour and pattern are often associated in the natural world with camouflage, warning, and 54 sexual selection1. Understanding how new biological colours have arisen is a fundamental 55 challenge to our understanding of evolutionary ecology and developmental biology, but 56 despite over a century of interest, the evolution of colour in molluscan shells is only just 57 beginning to be explored in detail2. Of particular interest is the observation that colour and 58 the distribution of fluorescence associated with some pigmentation both seem to be 59 distributed in a phylogenetically significant manner3-6. Grant & Williams6 mapped the 60 distribution of shell and periostracal colour onto a bivalve phylogeny and showed that the 61 phylogenetic distribution of colour is statistically significant, as are the distributions of 62 individual shell and periostracal colours. This finding, however, is complicated by the fact 63 that different pigments can produce the same colours, and as such the homology of colour is 64 unknown. In order to better understand how and why colour has evolved in shelled molluscs 65 it is essential to identify shell pigments and determine their similarity across different clades.

66 There have been a growing number of studies in the last decade investigating the pigments 67 responsible for molluscan shell colour (e.g.7-11) and the genes and pathways responsible for 68 their production (e.g.10,12-14). Techniques used to distinguish pigments in shells have included 69 high performance liquid chromatography (HPLC), mass spectrometry (MS) and UV-visible 70 spectrophotometry, but difficulties in some studies have arisen from the low concentrations 71 of pigments7,15,16. Raman spectroscopy overcomes this problem and is particularly effective 72 for detection of polyene pigments, as well as being non-destructive and non-invasive17-19. 73 The term polyene is a general chemical term for poly-unsaturated organic compounds 74 including carotenoids and tetrapyrroles, with one or more arrangements of alternating 75 double and single carbon–carbon bonds18. Such complex molecules tend to be highly 76 coloured, with strong absorption bands in the visible region of the spectrum18. Polyenes are 77 exceptionally strong Raman scatterers due to electron-phonon coupling20. Electron-phonon 78 coupling describes the interconnection of -electron density and nuclear position, which 79 means that changes in Raman spectra can provide detailed information on chemical 80 structure, polymer conformation, and also effective conjugation length. Additionally, they 81 have a large resonance Raman cross-section, which means that the Raman signature of a 82 pigment can be obtained with high selectivity from the shell itself.

83 Raman spectroscopy has previously been used in a broad range of molluscan shell pigment 84 studies (e.g.7,17,21-28.29,30) and to detect pigments in freshwater pearls (e.g.25,31,32). These 85 studies identified most shell pigments as polyenes, in some cases suggesting that molluscan 86 shell pigments are unsubstituted polyacetylenes, possibly of the carotenoid class of 87 compounds 22. However, Raman can be used to distinguish among all three of the main 88 classes of pigments known to occur in mollusc shells (melanins, tetrapyrroles and 89 carotenoids). For instance, Raman has been used to identify melanins in hair and 90 feathers33,34 and cuttlefish ink35; tetrapyrroles have been identified in molluscan shells, 91 brachiopod shells36, bird eggs37 and dinosaur eggs38; and carotenoids, carotenoproteins or

92 structurally modified carotenoids have been identified in molluscs, brachiopods and 93 corals22,31,36.

94 In this study we use Resonance Raman spectroscopy to compare spectra of shell pigments 95 across a range of bivalve taxa, with a range of different colours in order to explore the 96 pigments found in this group. We compare multiple spectra from fifteen specimens of 97 bivalves representing the taxonomic groups Pteriomorphia and Imparidentia at different 98 phylogenetic levels to determine whether pigments among clades are more similar than 99 those in different clades.

100 Experimental

101 Samples

102 Fifteen bivalve shells were selected from the Natural History Museum collections, from 103 major clades corresponding to the subclass Pteriomorphia (Alectryonella plicatula, Fig. 1M; 104 Arca zebra, Fig. 1C; Mimachlamys crassicostata (two colour morphs, Figs 1D, 1K;), Mi. 105 gloriosa, Fig. 1G; Mi. varia, Fig. 1A; Monia zelandica, Fig. 1O) and the superorder 106 Imparidentia (Acanthocardia tuberculata, Fig. 1F; Asaphis deflorata, Fig. 1I; As. violascens, 107 Fig. 1H; Chama brassica, Fig. 1N; Codakia paytenorum, Fig. 1L; Johnsonella fausta, Fig. 1J; 108 Pharaonella sieboldii, Fig. 1E; Phylloda foliacea, Fig. 1B). Shells of selected specimens were 109 relatively uniformly coloured, exhibiting either one or two vivid colours. Specimens were 110 cleaned using a sonicator with detergent and water for two minutes prior to Raman 111 measurements of the shell surface.

112 Where more than one colour occurred within a single shell these were examined separately 113 to investigate intra-individual variation: red and yellow shell colour in the lucinid Codakia 114 paytenorum (Fig. 1L) and purple and orange shell colour in Asaphis deflorata (Fig. 1I). 115 Intraspecific variation was investigated in two differently coloured individuals (red and 116 purple; Figs 1D, 1K) of scallops Mimachlamys crassicosta and interspecific variation within 117 two genera: Asaphis by comparing a purple shell from A. violascens (Fig. 1H) with a purple 118 and orange shell from A. deflorata and within Mimachlamys by comparing the shell spectra 119 of red and purple Mi. crassicosta with spectra from an orange Mi. varia (Fig. 1A) and a 120 yellow Mi. gloriosa (Fig. 1G).

121 Finally, species from different clades (Pteriomorphia and Imparidentia) with similar shell 122 colours were compared, focussing on five colours: red, orange, yellow, purple, brown and 123 green. We compared a red Mi. crassicostata shell (Fig. 1D) with the red margin on the 124 internal valve of C. paytenorum (Fig. 1L), yellow shells from Mi. gloriosa (Fig. 1G), C. 125 paytenorum and Johnsonella fausta (Fig. 1J), orange in Phylloda foliacea (Fig. 1B), Mi. varia 126 (Fig. 1A) and Asaphis deflorata (Fig. 1I), purple in As. violascens (Fig. 1H), As. deflorata and 127 Mi. crassicostata (Fig. 1K), brown in Arca zebra (Fig. 1C) and Acanthocardia tuberculata (Fig. 128 1F) and three green species Alectryonella plicatula (Fig. 1M), Monia zelandica (Fig. 1O) and 129 Chama brassica (Fig. 1N).

130

131 Raman measurement

132 Bivalve spectral acquisitions were recorded using a Renishaw inVia Raman Microscope 133 calibrated using the Silicon Raman band at 520.5 cm-1 with a spot size of 1 μm2. Unless 134 stated otherwise, shells were investigated using an argon-ion 488 nm excitation (9.0 mW, 10 135 %) for a 20-second accumulation time. Selected shells, including those with green pigments, 136 were also investigated at 457, 514, 633 and 785nm (Fig. S1, Supporting Information; data for 137 green shells not shown). The laser intensity was between 10-50% on shell, adjusted to avoid

138 damage to the specimen. The Raman spectra exhibited slight background fluorescence, so 139 to allow comparison of different pigments a background subtraction was performed. The 140 same points were chosen for each spectrum and a background polynomial curve, optimised 141 for each dataset, was fitted to these points. The corrected spectra were then normalised to 142 the Raman intensity of the carbonate peak (see Fig. S2A, Supporting Information for an 143 example).

144 As a Raman spectrum describes the molecular composition within the sample volume, 145 spectra were accumulated at different positions across the shell with the same coloration to 146 test the integrity of the chemical signature of the pigment. Several spectral acquisitions for 147 each excitation were performed on different areas of the shell on different days to confirm 148 that spectra were consistent within an area with the same colouration and to confirm that 149 spectra did not change due to degradation during repeated accumulations (see Fig. S2B, 150 Supporting Information for an example). Spectra in different phylogenetic categories and 151 colour groups were compared from a single representative spectrum per specimen -1 152 normalized by the intensity of the 1501-1540 cm C=C (1) peak, except in green shells 153 where it was normalised by the calcium carbonate peak.

154 Results & Discussion

155 Pigment spectra for coloured areas of all bivalve shells examined, other than the three green 156 shells examined (Fig. 1M-O) and the yellow area of Codakia paytenorum (Fig. 1L), exhibited a 157 very consistent signature, similar to those observed in previous studies. Over the 158 wavenumber range 600-1600 cm-1 there are four strong Raman active modes; two due to

159 pigments and two to the CaCO3 structure of the shell (Table S3 Supporting Information). The 160 first pigment peak occurs between 1501-1540 cm-1 and the second 1117-1144 cm-1, which

161 can be attributed to C = C (1) and C – C (2) stretching modes respectively in a polyenic 39 -1 162 chain (Fig. 2). Weaker peaks were also observed between 1009 – 1020 cm (4) and 1293 – -1 163 1300 cm (3).

164 Resonance Raman spectroscopy significantly enhances the Raman signal of polyene 165 pigments40,41 owing to the large oscillator strength when resonant with the electronic 166 transition, as well as Franck-Condon factors that particularly enhance modes in the 167 wavenumber range considered here. As such, intense overtones and combination bands can -1 168 be seen at high wavenumber for all species with pigment peaks at 21 (3010 – 3066 cm ), 1 -1 -1 169 + 2 (2614 – 2670 cm ) and 22 (2230 – 2279 cm ). Smaller peaks are seen at 1 + 4 (2517 – -1 -1 -1 170 2549 cm ), 2 + 3 (2416 – 2437 cm ) and 2 + 4 (2131 – 2155 cm ) (Figs S1, S4 & S5 171 Supporting Information).

172 Previous authors have suggested that molluscan shell pigments with similar Raman 173 signatures are unsubstituted (or non-methylated) polyenes, probably modified carotenoids 174 e.g. 22. However, coral pigments and molluscan shell pigments exhibit a peak at 1009-1020 -1 175 cm (4) assigned to in-plane methyl rocking motion that is not found in unmethylated 176 parrot feather pigments known as psittacofulvines or synthetic polyenes lacking methyl 177 groups42,43. This peak is slightly upshifted from 1010cm-1 in carotenoids (which are 178 tetramethylated)43 for bivalve shells examined in this study (except Acanthocardia 1 179 tuberculata which has 4 at 1009 cm- ). The similarity between the coral pigment and shell 180 pigments is mirrored in the overtone and combination bands, where peaks can be attributed 43 181 to combinations of 1 and 2 with 4 . Conversely, no combination bands involving the 4

182 or 5 band are present in the unmethylated psittacofulvines or synthetic polyenes lacking 183 methyl groups42,43. These differences have been attributed to the partial methylation of a 184 polyene in the red coral Corallium rubrum43 suggesting the possibility that shell polyenes are 185 also partially methylated. Further evidence for methylation comes from the small shoulder

186 for the peak at 21, which suggests that the 1 peak may actually consist of two peaks, as

187 methyl groups are known to induce splitting of 1 Raman bands when connected to a 188 conjugated chain of carotenoids44.

189 Molluscan shell pigments can be differentiated from pure carotenoids on the basis of their -1 45 190 2 vibration, which is shifted by 20-30 cm from carotenoids and carotenoproteins . This 191 shift has been attributed to the lack of methyl groups attached to middle of the backbone 192 carbon chain45,46. This difference can be demonstrated graphically (Fig. 3) by plotting the two

193 vibrational pigment bands of highest intensity, with the plot showing a linear pattern for 194 nearly all molluscan shells tested to date including exemplars from other studies17,22,24,30,32 195 (Fig. 3). Spectra from tert-butyl capped polyenes and some brachiopod and coral pigments 196 show a similar pattern to the molluscan shells (Fig. 3)22,24,31,36.

197 The effective conjugation length of the polyene chain can be estimated from the position of 24,27 198 the 2 and 1 vibrational modes, and the chain length has been shown to affect colour .

199 The Raman shift for the C – C vibrational mode (2) can be used to separate yellow shells in 200 our study from other colours we examined, with all our yellow shells exhibiting peaks above 201 1139 cm-1 (Table S3, Supporting Information; Fig. 3). This pattern does not hold when

202 including data from other studies. Although yellow shells have the highest 1 and 2 values, 203 they overlap with red and orange shells (Fig. 3). Pink and purple shells also overlap with red, 204 and brown shells are scattered throughout the range (Fig. 3). The shortest chains are found 205 in yellow pigments, with the longest chains in purple and brown pigments, and the brown 206 pigment in Arca zebra (Fig. 1C) estimated to have the longest chain length of all molluscs 207 included in our graph, other than a blue pigment from the mussel, Mytilus edulis 24 (Fig. 3).

208 It seems likely that the polyenes in molluscan shell pigments are based on carotenoid 209 precursors, which are known to colour soft tissues and are correlated with colour in 210 shells10,47. As most animals are incapable of producing carotenoids de novo, molluscan 211 carotenoids are likely to be dietary in origin, whereas some modifications may be genetically 212 controlled2. As such, the differently colour shells may reflect both environment (dietary 213 intake of different carotenoids) and genetics (heritable modifications of identical or differing 214 carotenoid precursors). Factors affecting the effective conjugation length, and therefore 215 colour, in carotenoids include binding to proteins, modifications to their configuration, the 216 addition of functional groups and isomerism41,48,49. Differences in aggregation of 217 carotenoproteins has also been shown to affect colour in lobsters50 and the effect of 218 orientation in the shell matrix has been considered previously with respect to molluscan 219 pigments22. Unmodified carotenoids are commonly red, yellow or orange, but the 220 complexing of proteins and carotenoids can result in other colours, including purple or blue. 221 Marine examples include the violet carotenoprotein in the starfish Asterias rubens, which 222 absorbs at 570 nm51 and blue β-crustacyanin found in lobsters, which absorbs at 630 nm 223 wavelength52.

224 In previous Raman studies of molluscan shells black and brown pigments have been 225 attributed to polyenes17,22,24,30, but other studies using HPLC, MS or spectral analysis, have 226 shown that these shell colours can also be due to melanins7,9,53 or tetrapyrroles15. The brown 227 tetrapyrrole protoporphyrin IX has been found in the pteriomorph Argopecten sp. and 228 Raman studies showed that it exhibits Raman peaks attributable to C=C bonds at 1619, 1585 -1 -1 -1 15 229 and 1339 cm ; C-C deformation at 1255 cm , and C-C and C-H3 rocking at 970 cm . 230 Raman spectroscopy of black eumelanin isolated from Sepia cuttlefish ink found two intense, 231 broad peaks at 1380 and 1580 cm-1 54. Analysis of feathers and hair samples found similar 232 results for eumelanin with three broad Raman modes at 500, 1380, 1580 cm-1, and peaks at 233 about 500, 1150, 1490 and 2000 cm-1 for phaeomelanin33. Black Recent brachiopod shell

234 pigments thought to be melanins have broad peaks at 1400 and 1592 cm-1 (fossil shells show 235 different bands)36. In this study, brown Arca zebra (Fig. 1C) has several peaks at 386, 880, -1 236 1173 and 1629 cm in addition to the peaks discussed above, and brown Acanthocardia 237 tuberculata (Fig. 1F) has peaks at 878, 1132, 1528. As such, our data are not consistent with 238 the presence of melanins or protoporphyrin in either species.

239 Our most curious results were obtained for yellow Codakia paytenorum (Fig. 1L) and all 240 green shell pigments examined (Fig. 1M-O). No Raman activity was detected at all for green 241 shells and no pigment peaks were detected for yellow C. paytenorum (Fig. 4). This is not true 242 of all yellow shells in this study (see below), nor the red portion of the C. paytenorum (see 243 below), nor green shells examined in other studies. For example, green nerite gastropod 244 shells have Raman spectra consistent with the presence of polyenes or other unidentified 245 pigments30. In the case of the yellow pigment, this could be due to lower pigment 246 concentrations, as evidenced by the pale colour (although visually the colour is no paler than 247 that in Johnsonella fausta, Fig. 1J), but at least some of the green pigmentation is very 248 intense. As such, we are unable to explain what our results mean, other than to suggest that 249 the pigments responsible are quite different to other molluscan shell pigments examined to 250 date using Raman spectroscopy, as was hypothesised by Grant & Williams6 for bivalve green 251 pigments or that the colour is a structural colour, and not the result of a pigment. Grant & 252 Williams6 noted that non-iridescent green shell colouration, as observed in these specimens, 253 is rare in bivalves (although common in some groups of gastropods where it may be due to 254 tetrapyrroles). When non-iridescent green colouration occurs on the outer side of bivalve 255 valves, it tends to be more common in the periostracum (an organic layer) than the mineral 256 shell matrix and when found on the inside of valves (as in this study), it is more common in 257 species that produce proteinaceous sheets overlying calcareous shell6. Proteinaceous sheets 258 are known to occur in some freshwater bivalves, oysters (a group that includes both Monia 259 zelandica and Alectryonella plicatula tested in this study) and corbulid clams 6. This might 260 suggest that the green coloration (whether a pigment or structural in nature) might occur in 261 the organic layer rather than the calcareous shell.

262 Shell pigments identified using Raman have usually been determined to be modified 263 carotenoids, rather than melanins or tetrapyrroles, which might be explained by the fact 264 that pigments are likely distributed across Mollusca in a phylogenetically relevant 265 manner5,6,24,55,56. Most Raman studies have focused on bivalves, caenogastropods or land 266 snails, where pigments have rarely been identified chemically, but are unlikely to be 267 tetrapyrroles, which tend to occur more frequently in Vetigastropoda (with some exceptions 268 in Bivalvia and Caenogastropoda). Alternatively, signal from carotenoid-based pigments may 269 be masking other shell pigments. Further work is needed to test these hypotheses, for 270 example using higher energy excitation wavelengths and working with species with known 271 shell pigments.

272

273 Phylogenetic comparisons

274 Multiple spectra taken across one colour within a single shell were homogeneous in all 275 species suggesting that these shell colours can each be attributed to a single pigment or 276 group of pigments. We also compared two different species with different colours on the 277 same shell: Asaphis deflorata (orange and purple; Fig. 1I) and Codakia paytenorum (yellow 278 and red; Fig. 1L) (Fig. S4, Supporting Information). In both cases, different colours within a 279 single shell were found to be due to different pigments, rather than differences in 280 concentration of the same pigment. For example, Asaphis deflorata showed a shift to a 281 lower wavenumber and broader full width at half maximum (FWHM) in the purple muscle 282 scar compared to the orange area (Table S3, Fig S4; Supporting Information). In C. 283 paytenorum, while strong pigment related peaks were observed for red areas, pigment 284 related peaks were not found in the yellow shell area as discussed above.

285 Analyses of both intraspecific (purple and red morphs of the scallop Mimachlamys 286 crassicostata; Figs 1D, 1K) and interspecific (Mi. crassicostata, Mi. gloriosa, Fig. 1G and Mi. 287 varia, Fig. 1A; and Asaphis deflorata Fig. 1I and As. violascens, Fig. 1H) variation suggest that 288 all pigments within a single genus belong to the same class of pigment (Fig. S5, Supporting 289 Information). Moreover, purple pigments in two species from the same genus (As. 290 violaescens shell and the purple muscle scar on As. deflorata) were likely the same, or a very 291 similar, pigment, having very similar peak centres and FWHM values. Pigments 292 corresponding to different shell colours, within a single species, or within a genus however 293 differed in chain length (Table S3, Supporting Information; Fig. 3).

294 Raman spectroscopy of bivalve larval shells has been shown to differentiate among different 295 families or orders28. In this study, comparison of two taxonomic groups (Imparidentia versus 296 Pteriomorphia) shows that species from both groups with the same shell colour have very 297 similar Raman signatures, with the exception of brown shells and yellow shells (Fig. 4). Most 298 similar are the red pigments in Pharaonella sieboldii (Fig. 1E) and Mimachlamys crassicostata -1 -1 299 (Fig. 1D), which have the same 2 (1131 cm ) and 1 (1521 cm ). The purple pigment in 300 Asaphis deflorata (Fig. 1I), As. violascens (Fig. 1H), Mi. crassicostata (Fig. 1K) have the same -1 -1 301 2 (1130 cm ) and similar 1 peaks (1518 and 1516 cm ). Peaks for red and orange shell -1 -1 302 colours are very close and overlap, and considered together, 1 = 12 cm and 2 = 4 cm . 303 Shells with orange and yellow pigments (excluding Codakia paytenorum Fig. 1L, which lacked -1 -1 304 pigment peaks) show a similar range: 1 = 9 cm and 2 = 4 cm for orange Mimachlamys -1 -1 305 varia (Fig. 1A) to Phylloda foliacea (Fig. 1B) and 2 = 5 cm and 1 = 7 cm for yellow Mi. 306 gloriosa (Fig. 1G) to Johnsonella fausta (Fig. 1J). Green shells also showed an identical result, 307 in that the pigments of all three species were Raman inactive. Such similarities suggest that 308 some pigments may be shared among species and even between Imparidentia and 309 Pteriomorphia suggesting an ancient origin. Conversely, brown Acanthocardia tuberculata -1 310 (Fig. 1F) (1 1526 and 2 1132 cm ) is more similar to red-brown C. paytenorum (Fig. 1L),

-1 -1 -1 311 with (1 1527 and 2 1136 cm ) than brown Arca zebra (Fig. 1C) (1 1501 cm , 2 1117 cm ). 312 These results highlight some of the difficulties in determining the homology of colour.

313

314 Mineral structure

315 Molluscan shells are composed of calcium carbonate and an organic matrix, in a highly 316 ordered arrangement, often in layers, with species dependent microstructures57,58. The 317 polymorphs aragonite and calcite are the most common mineral forms, although vaterite is 318 also found in some species e.g. 59. Generally, a single polymorph is found in any one layer, 319 although a second polymorph may be found in separate layers 57. The distribution of 320 polymorphs differs between Imparidentia, which has shells composed only of aragonite, and 321 Pteriomorphia (other than Arcoidea) which have shells with the outermost layer composed 322 of calcite58,60.

323 Surface examination of the shells in this study showed, unsurprisingly, that all shells have a 324 peak between 1085 cm-1 and 1087 cm-1 (Peak 2; Table S3, Supporting Information), which

325 can be attributed to the internal A1g symmetric stretching (1) of the carbonate ion in 326 calcium carbonate. The relative intensity of calcium carbonate peaks in green-coloured 327 areas, however, was reduced, perhaps because an increase in organic material at green 328 coloured sites masks the mineral layer 6.

329 Aragonite and calcite can be distinguished by minor Raman bands because of their different 330 lattice structures, with calcite having minor bands at ∼282 and 713 cm-1 and aragonite 331 having minor bands at ∼207 and 704 cm-1 61. Bands near 700 cm-1 are due to the in-plane 62 332 bending (4 vibration) of CO3 . All Imparidentia shell spectra obtained in this study exhibit a 333 peak at 704 cm-1 consistent with aragonitic shells (Table S3, Supporting Information).

334 Most pteriomorphs (three species of Mimachlamys and Alectryonella plicatula) had a peak 335 between 712-714 cm-1 and the latter also had a peak at 279 cm-1 consistent with calcitic 336 shells (Table S3, Fig. S6; Supporting Information). The pteriomorph Arca zebra, however, had 337 an aragonite peak at 704 cm-1 (Table S3, Supporting Information) consistent with previous 338 findings for this family60. The pteriomorph Monia zelandica had one minor peak for each 339 polymorph (704 cm-1 and 279 cm-1) (Table S3, Fig. S6; Supporting Information). This result 340 could be due to the presence of both polymorphs in a single layer on the inside of the valve. 341 Shells of the family Anomiidae (which includes Monia) although mainly calcitic, have a thin 342 aragonitic inner shell layer and aragonitic myostraca (shell layer secreted beneath the 343 adductor and pedal muscles on the inside of the valve)63.

344

345 Conclusion

346 Our spectra are consistent with previous studies of molluscan shell pigments but rather than 347 suggesting these are unmethylated polyenes, we suggest that the majority of bivalve shell 348 pigments in the species examined are partially-methylated polyenes, possibly modified

349 carotenoids, and are therefore likely affected by both diet and genetics. The most similar 350 pigments are found in a single genus, although similar pigments are also shared across 351 Imparidentia and Pteriomorphia. The greatest difference among the spectra from different 352 shell pigments are in the stretching and in-plane bending modes of the C=C and C-C bonds. 353 We did not find any evidence of melanins or tetrapyrroles in the shells examined. We did not 354 detect any Raman activity in three green shells examined nor any pigment peaks for the 355 yellow region of Codakia paytenorum and we can only speculate that colour in these areas is 356 either due to structural colour, or that the pigments responsible are quite different from 357 other molluscan shell pigments examined to date. Together these findings are consistent 358 with the idea that while classes of pigments are evolutionarily ancient, pigments may be 359 specific to particular groups and sort phylogenetically2.

360 Future studies could focus on taxa with known pigments in order to determine whether the 361 presence of one class of pigment can mask the presence of others in Raman studies, and 362 where possible aim to characterise differences among pigments. These will be combined 363 with quantum chemical calculations to help fully characterise molecular structures and allow 364 the investigation of pigment biosynthetic pathways.

365

366

367 Acknowledgements

368 We thank John Taylor for the identification of specimens and for comments on shell 369 mineralogy and Andreia Salvador and Tom White for providing access to specimens and 370 Harry Taylor for photos in Figure 1. Authors (JN, JSK) acknowledge the UK EPSRC for 371 studentships under DTG. We also thank two reviewers for their constructive comments.

372 References

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488

489 Figure Legends

490 Figure 1. Photos of 15 shells used in analyses. A. Mimachlamys varia (orange), (Kisch 491 Collection). B. Phylloda foliacea (orange). C. Arca zebra (brown and white). D. Mi. 492 crassicostata (red), NHMUK Acc. No. 2381. E. Pharaonella sieboldii (red). F. Acanthocardia 493 tuberculata (brown and white). G. Mi. gloriosa (yellow). H. Asaphis violascens (purple). I. As. 494 deflorata (purple and orange). J. Johnsonella fausta (yellow). K. Mi. crassicostata (purple), 495 (note that the right valve is figured, but the left valve was used in analysis), NHMUK Acc. No. 496 2381. L. Codakia paytenorum (yellow and red), NHMUK 20180291. M. Alectryonella plicatula 497 (green). NHMUK Acc. No. 2325. N. Chama brassica (green), within 20 mile radius of Dar-es- 498 Salaam, Tanzania. NHMUK 19920800. O. Monia zelandica (green), Port Jackson. NHMUK 499 20180286. Except where indicated, specimens are unregistered and no locality data are 500 available. Scale bars 1 cm.

501 Figure 2. Spectra for all species examined separated by taxonomic group. A. Imparidentia. B. 502 Pteriomorphia. Spectral acquisitions were recorded using a Renishaw inVia Raman 503 Microscope calibrated using the Silicon Raman band at 520.5 cm-1 with a spot size of 1 μm2 504 and an argon-ion 488 nm excitation (9.0 mW, 10 %) for a 20-second accumulation time. Keys 505 list spectra in the same order as shown in graph. Grey bars indicate non-pigment peaks 506 associated with mineralogy.

507 Figure 3. Graph of the C=C wavenumber (1) versus C–C wavenumber (2) as in other studies 508 (e.g. 22,64). Data are for molluscan polyene pigments (open triangles - colour of symbol in 509 online version reflects shell or pearl colour) from this study and exemplars from previously 510 published data 17,22,24,30,31. Reference data are: carotenoids ( ) 65,66, carotenoproteins ( ) 511 66,67, brachiopod pigments ( ) 36, coral pigments ( )31,32, feather pigments ( ) 68, and tert- 512 butyl-capped polyacetylenes ( ) 64 with the expected number of conjugated double-bonds 513 given alongside the circle (based on 64). Spectral acquisitions for this study were recorded 514 using a Renishaw inVia Raman Microscope calibrated using the Silicon Raman band at 520.5 515 cm-1 with a spot size of 1 μm2 and an argon-ion 488 nm excitation (9.0 mW, 10 %) for a 20- 516 second accumulation time. Excitation levels and other parameters varied among previous 517 studies.

518 Figure 4. Spectra for shell pigments, grouped by colour. A. Red shells: Codakia paytenorum 519 (Fig. 1L), Mimachlamys crassicostata (Fig. 1D) and Pharaonella sieboldii (Fig. 1E). B. Orange 520 shells: Asaphis deflorata (Fig. 1I), Mi. varia (Fig. 1A) and Phylloda foliacea (Fig. 1B). C. Yellow 521 shells: Codakia paytenorum (Fig. 1L), Johnsonella fausta (Fig. 1J) and Mi. gloriosa (Fig. 1G). 522 Note lack of pigment peaks for C. paytenorum. D. Green shells: Alectryonella plicatula (Fig. 523 1M), Chama brassica (Fig. 1N) and Monia zelandica (Fig. 1O). Note lack of Raman signal for 524 any shell. E. Purple shells: As. deflorata (Fig. 1I), As. violascens (Fig. 1H) and Mi. crassicostata 525 (FIG. 1K). F. Brown shells: Acanthocardia tuberculata (Fig. 1F) and Arca zebra (Fig. 1C). 526 Spectral acquisitions were recorded using a Renishaw inVia Raman Microscope calibrated 527 using the Silicon Raman band at 520.5 cm-1 with a spot size of 1 μm2 and an argon-ion 488 528 nm excitation (9.0 mW, 10 %) for a 20-second accumulation time.

529 Supporting Information

530 Figure S1. Illustrative spectra and graphs showing shift in peak position acquired at different 531 excitation wavelengths. A. Spectra for Mimachlamys crassicostata red shell. B. Spectra for 532 Codakia paytenorum red shell. C. Raman dispersion of the overtones. Note the Y-axis is not 533 continuous. Spectra recorded using a Renishaw inVia Raman Microscope calibrated using 534 the Silicon Raman band at 520.5 cm-1 with a spot size of 1 μm2 and argon-ion excitation at 535 457, 488, 514, 633 and 785 nm (9.0 mW, 10 %) for a 20-second accumulation time. Note that 536 when tested for the same range of excitation values no Raman signal was detected for green 537 shells.

538 Figure S2. Preliminary data. A. Examples of background subtraction for Arca zebra and 539 Mimachlamys crassicostata. B. Examples of spectra taken on two different days of the same 540 shell colour for Asaphis violascens and Pharaonella sieboldii.

541 Table S3. Raman wavenumbers (cm-1) for peak centres and full width at half maximum 542 (FWHM) for the four most intense peaks identified in 15 bivalve shells examined. Species are 543 grouped by taxonomic group (Imparidentia or Pteriomorphia), photos are in Figure 1, and 544 results are presented for each colour found on a shell, indicating the area of shell studied 545 (inside or outside surface of the valve). Note that no Raman activity was detected for yellow 546 shell of Codakia paytenorum or any green shell in this study and neither were mineral peaks 547 observed in green shell. Wavenumbers are rounded to the nearest cm-1. Polymorph of 548 calcium carbonate is indicated, note that both polymorphs were detected for Monia 549 zelandica. Spectral acquisitions were recorded using a Renishaw inVia Raman Microscope 550 calibrated using the Silicon Raman band at 520.5 cm-1 with a spot size of 1 μm2 and an 551 argon-ion 488 nm excitation (9.0 mW, 10 %) for a 20-second accumulation time.

552 Figure S4. Spectra for shell colours found within a single shell (intraindividual variation). A. 553 Asaphis deflorata (purple and orange; Fig. 1I). B. Codakia paytenorum (yellow and red; Fig. 554 1L). Spectral acquisitions were recorded using a Renishaw inVia Raman Microscope 555 calibrated using the Silicon Raman band at 520.5 cm-1 with a spot size of 1 μm2 and an 556 argon-ion 488 nm excitation (9.0 mW, 10 %) for a 20-second accumulation time.

557 Figure S5. Spectra for shell colours found within a single species (intraspecific variation) or 558 within a single genus (interspecific variation). A. Asaphis violascens (purple; Fig. 1H) and As. 559 deflorata (purple and orange; Fig. 1I). B. Mimachlamys crassicostata (red & purple morphs; 560 Figs 1D, 1K), Mi. gloriosa (yellow; Fig. 1G) and Mi. varia (orange; Fig. 1A). Spectral 561 acquisitions were recorded using a Renishaw inVia Raman Microscope calibrated using the 562 Silicon Raman band at 520.5 cm-1 with a spot size of 1 μm2 and an argon-ion 488 nm 563 excitation (9.0 mW, 10 %) for a 20-second accumulation time.

564 Figure S6. Spectra for unpigmented areas of green shells showing locations of peaks 565 associated with calcite and aragonite. Peaks at 207, 704 and 1085 cm-1 are typical of 566 aragonite and those at 280, 713 and 1086 cm-1 are typical of calcite. Spectral acquisitions 567 were recorded using a Renishaw inVia Raman Microscope calibrated using the Silicon Raman 568 band at 520.5 cm-1 with a spot size of 1 μm2 and an argon-ion 488 nm excitation (9.0 mW, 10 569 %) for a 20-second accumulation time.