Detection of intact polyene pigments in Miocene gastropod shells

Polyene pigments represent a major class of pigments in present‐day organisms. Their occurrence in fossils has been frequently discussed, but to date no spectroscopic evidence has been found. Here, we use in situ Raman spectroscopy to examine the chemistry of exceptionally well‐preserved gastropod shells with colour preservation from the Middle Miocene of the Vienna Basin (Austria, Hungary). Raman signals indicative of the presence of intact (i.e. non‐hydrogenated) polyene pigments were obtained from fossil shells with reddish colour patterns, thus revealing the first record of intact polyenes in fossils. The observed Raman values are in good agreement with those of unmethylated (non‐carotenoid) polyenes. Fossil polyene pigments were detected in representatives of the superfamily Cerithioidea, but not in representatives of other gastropod families with colour preservation found at the same localities, demonstrating that the occurrence of polyene pigments is taxon‐specific. Our results show that Raman spectroscopy represents a valuable tool for the non‐destructive screening of rare fossils with colour preservation for the occurrence of polyene pigments.

P O L Y E N E pigments are widely distributed in the three domains of life and are responsible for most yellow, orange and red colours observed in nature (Yabuzaki 2017;Maia et al. 2021).The dominant group among the polyene pigments are the carotenoids, but non-carotenoid polyenes such as the psittacofulvins have increasingly become the subject of recent research (Maia et al. 2021).The occurrence of polyene pigments in fossils has been discussed especially in the context of molluscan shell colour (Williams 2017; Wolkenstein 2022), plumage pigments (Thomas et al. 2014a(Thomas et al. , 2014b) ) and recent palaeocolour reconstructions (McNamara et al. 2016;Roy et al. 2020;Vinther 2020).However, because of the known susceptibility of polyenes to oxidation (Britton et al. 2008), the preservation potential of intact polyenes in sediments and fossils is generally considered to be low (Sinninghe Damsté & Koopmans 1997;Williams 2017), and to date no spectroscopic evidence for the preservation of intact polyene pigments in fossils has been reported.
Colour pattern preservation can be commonly found in Cenozoic molluscan shells (Hollingworth & Barker 1991).Many shell colour patterns that have faded to some extent have also been revealed using UV light (e.g.Krueger 1974;Dockery 1980;Caze et al. 2010Caze et al. , 2011;;Schneider et al. 2013;Hendricks 2015).By contrast, obvious colour preservation (i.e.preservation of distinct colours in fossils that goes beyond the usual brown tones) is rare.An outstanding example is the small gastropod Pithocerithium rubiginosum (Cerithiidae), representing one of the most abundant gastropod species of the Sarmatian (Middle Miocene) of the Central Paratethys (Harzhauser & Piller 2010).Many specimens of P. rubiginosum show a distinct red colouration of the beads of the spiral cords (Fig. 1A).This red colouration has been well known since the 19th century (Hörnes 1848) and it is so conspicuous that the species was named for it (from the Latin for 'rusty red') by Eichwald (1830Eichwald ( , 1853)).In addition to P. rubiginosum, colour preservation can be found in a number of other gastropods from the Vienna Basin.However, despite the often distinct colouration, until now no attempt has been made to chemically analyse the pigments of fossil gastropods.
A survey of the literature shows that even the pigments of present-day molluscs are merely known (Williams 2017).Some results concerning the pigments of present-day molluscs have been obtained using Raman analysis.Although Raman spectroscopy cannot be considered as a method for structure elucidation in the narrower sense, it provides valuable information on the molecular framework and the nature of bonding of unknown compounds.In situ Raman spectroscopy has suggested that polyene pigments are distributed in many present-day molluscs (Merlin & Delé-Dubois 1986;Barnard & de Waal 2006;Hedegaard et al. 2006;Ishikawa et al. 2019;Wade et al. 2019).Polyenes are polyunsaturated organic compounds that contain at least two conjugated carbon double bonds and include natural products such as isoprenoid carotenoids and unmethylated polyenes such as the psittacofulvins (Maia et al. 2021).Based on Raman data, those polyenes found in modern molluscs have generally been assigned as unmethylated polyenes (Merlin & Delé-Dubois 1986;Barnard & de Waal 2006;Hedegaard et al. 2006;Ishikawa et al. 2019).
In recent years, Raman spectroscopy has gained increasing popularity in the analysis of fossil samples, because it is a non-destructive method and measurements can be easily performed.On the other hand, some limitations and pitfalls have to be considered with the Raman analysis of fossil samples.In particular, the high autofluorescence of many fossils represents a serious problem, often making analysis impossible (Olcott Marshall & Marshall 2015;Geisler & Menneken 2021).In addition to inorganic compounds, Raman spectroscopy has been successfully applied to fossil organic materials such as amber (Winkler et al.In order to explore the chemical nature of colourful fossil gastropod shells from the Vienna Basin as nondestructively as possible, we decided to investigate a set of representative specimens with Raman spectroscopy.This technique appears particularly suitable for the analysis of potential fossil polyene pigments, since polyenes are known to be strong Raman scatterers (signal enhancement by the resonance Raman effect) (e.g.Maia et al. 2021), thus allowing for the detection of even small amounts of pigment.For comparison we used a specimen of the modern Cerithiidae, Thericium vulgatum without ostracum from the Mediterranean Sea (Malacological Collection of NHMW, NHMW 2023/0105/0001).All specimens used in this study are stored in the collection of the Natural History Museum Vienna (NHMW).

MATERIAL AND METHOD
Samples were cleaned with acetone and coloured as well as non-coloured areas of the shell were subjected to in situ Raman spectroscopy.Raman spectra were acquired using a Horiba Labram HR800 UV instrument equipped with an Olympus BX41 microscope.The excitation wavelength was the 488 nm line of a diode laser with a reduced laser power (10%) of about 0.45 mW at the sample surface.The use of a 100× objective (long working distance objective Olympus MLPlanFl 100× with numerical aperture (NA) of 0.8) for focusing the laser on the sample surface and a confocal hole of 100 μm yielded a spatial resolution of about 1 μm lateral and 5 μm in depth.The scattered light was dispersed by a grating with 600 lines mm À1 and detected with a charge-coupled device (CCD) detector with 1024 × 256 pixels.Raman spectra were acquired in the range of 100-2100 cm À1 and 100-4200 cm À1 with a spectral dispersion of about 2 cm À1 per pixel and final spectra were averaged from 16 spectra with 15 s exposure time each (with exception of the spectrum of Tiaracerithium from the Miocene of St Margarethen, which was averaged from 24 spectra with 10 s exposure time, because of the high fluorescence of the sample).The spectrometer was calibrated against the Si band at 520.4 cm À1 .During the measurement, the Horiba intensity correction system (ICS) was applied to correct the instrument specific spectral range sensitivity and quasi-periodic 'ripples' that can be induced by the Rayleigh filters (edge filters) of the Raman spectrometer, in particular for spectra with high fluorescence intensity (Casadio et al. 2016;Alleon et al. 2021).After measurement, adaptive background correction was applied using SpectraGryph 1.2 (coarseness: 10%, offset: 0).Uncorrected Raman spectra that show the elevated fluorescence background especially of the fossil samples are shown in the Supporting Information (Figs S2,S3).
UV light-induced autofluorescence of samples was documented using a Canon PowerShot A700 digital camera and a Benda NU-8 KL 8W UV lamp (366 nm), with a distance between the light source and the fossils of about 10 cm.The exposure time for all UV photographs was 2 s.
A further specimen of Pithocerithium rubiginosum from Nexing with preservation of red dots (see Fig. 2E) was ground to a fine powder and stirred overnight in EDTA solution (pH 8.0) to dissolve the shell without destroying potential polyene pigments (which might be degraded by acids).Following centrifugation, the supernatant was removed and the orange-brown residue was extracted with distilled water.The yellowish solution was subjected to solid-phase extraction.The sorbent (Bondesil C18, 40 μm) was conditioned with acetonitrile, followed by water.The sample solution then was loaded onto the column, and the sorbent was washed with water.The yellow pigments were eluted with acetonitrile and the solvent was removed by evaporation under a stream of nitrogen at 40°C.Pigments were transferred to a CaF 2 disc.Raman spectra were acquired as described above, however, using a standard objective Olympus MPlan 100× with NA of 0.9 and a strongly reduced laser power (1%) of about 0.045 mW at the sample surface to avoid any deterioration of the organic sample.

RESULTS
The majority of specimens of Pithocerithium rubiginosum found at the localities Nexing and Hautzendorf (Fig. S1) show a distinct orange to red colouration of the beads of the spiral cords (Fig. 1A).Orange to red colouration can also be observed in many shells of Tiaracerithium pictum from all four investigated localities (Fig. 1B).In the shells of Potamides disjunctus from St Margarethen and Fertőrákos, spiral lines with a tan to reddish colour can be found (Fig. 1C), although in general the colouration in Potamides is less distinct than that of Pithocerithium and Tiaracerithium.In contrast to Tiaracerithium and Potamides, specimens of Sarmatigibbula podolica with colour preservation from Fertőrákos show only a tan colour (Fig. 1D).Specimens of Megalotachea sylvestrina from Nexing show yellow spiral bands (Fig. 1E).
Raman spectra indicate preservation of the original aragonite shell (several signals between 125 and 225 cm À1 , but no signal at about 280 cm À1 that would be indicative for calcite) in all investigated gastropod specimens (Figs S2,  S3).Further signals characteristic of carbonates occur at 704 cm À1 (in-plane band ν 4 of CO 2À 3 ) and 1085 cm À1 (symmetric stretching band ν 1 of CO 2À 3 ), the latter being the most intense carbonate signal (Figs 2, S2, S3).From the Raman spectra, it can also be excluded that the reddish colouration of the shells is due to iron oxides such as haematite, because no corresponding signals (c. 225, 290, 410, 610, 1320 cm À1 ) (de Faria et al. 1997;Marshall & Olcott Marshall 2011) were found.
Whereas no signals in addition to the carbonate bands were obtained from non-coloured shell areas of the gastropods (Fig. 2D), Raman spectra of coloured areas of the fossil gastropods Pithocerithium, Tiaracerithium and  2A).The ν 1 signals of the red coloured Pithocerithium are shifted by about 20 cm À1 to higher wavenumbers compared to the brown coloured Thericium (Table 1).This shift can be explained by the conjugation length of the polyene backbones, which is higher for brown polyenes than for red polyenes (Ishikawa et al. 2019).
Remarkably, taxonomic differences in the composition of pigments are still recorded in the gastropod shells.Although evidence for polyene pigments was found in gastropods from all investigated localities in the Vienna Basin, not all species with preservation of colour patterns from Nexing (e.g.Megalotachea sylvestrina) show the presence of polyene pigments (Fig. S3; Table 1) (intense signals at about 1100 und 1500 cm À1 are missing).No polyene pigments were also found in Sarmatigibbula with colour pattern preservation from Fertőrákos (Fig. S3; Table 1).The presence or lack of polyenes in the gastropod shells (fossil and modern) is also in agreement with the fluorescence properties of the shells as observed under UV light.Whereas gastropods that yielded Raman signals characteristic for polyenes show no fluorescence of the pigments, the colour patterns of Megalotachea and Sarmatigibbula show distinct yellow or red fluorescence, respectively (Fig. S4), suggesting the presence of other pigments than polyenes (in the case of Sarmatigibbula possibly porphyrines, which are known for their characteristic red to purple fluorescence).
Following dissolution of the shell, the pigments of a specimen of Pithocerithium rubiginosum with preservation of red dots were extracted and isolated (Fig. 2E).Raman analysis of the isolated orange pigments (note that the carbonate signals at 704 and 1085 cm À1 have disappeared) yielded all characteristic polyene signals (Fig. 2B; Table 1) that were previously observed in the in situ Raman spectrum of the pigmented gastropod shell (Fig. 2C; Table 1) with even better signal-to-noise ratio, indicating that the Raman signals are due to the orange pigments.

DISCUSSION
The specific Raman signals of the fossil gastropod pigments show the presence of C-C and C=C bonds, which have to be arranged at least to some extent in the form of conjugated double bonds, as is obvious from the orange to red colour of the compounds, thus indicating the presence of original, still intact (non-hydrogenated) polyene The discovery of still intact polyene pigments in gastropod shells of Middle Miocene (Sarmatian) age is highly surprising, since the preservation potential of unsaturated polyene pigments such as carotenes in contrast to hydrogenated carotenoids is very low (Sinninghe Damsté & Koopmans 1997).It should be also noted that no indications for the presence of hydrogenated polyenes (no signal at 1455 cm À1 ; Marshall & Olcott Marshall 2010) can be found in the Raman spectra of the fossil gastropods.The previously oldest intact polyenes, all of them found in sediments, not in macrofossils, were the carotenoid isorenieratene from a Late Miocene (Messinian) marl from Italy (Keely et al. 1995) and an unspecified diaromatic carotenoid from a Lower Miocene clay from the Blake Bahama Basin in the Western North Atlantic Ocean (Cardoso et al. 1978) (Sinninghe Damsté & Koopmans 1997;Hopmans et al. 2005).
The exact wavenumber positions of the polyene Raman bands (in particular of the ν 1 and ν 2 bands) depend on the length of the polyene chain, substituents attached to the chain, its molecular configuration and potential binding to other molecules (e.g.proteins) (Merlin & Delé-Dubois 1986;Schaffer et al. 1991;Hedegaard et al. 2006;Maia et al. 2021).It has been shown that the ν 2 band can be used as a reliable indicator for the presence or absence of methyl groups in polyenes (Merlin & Delé-Dubois 1986;Barnard & de Waal 2006;Maia et al. 2021).Whereas methylated polyenes such as carotenoids have ν 2 values of about 1150-1158 cm À1 (Barnard & de Waal 2006), those of unmethylated polyenes such as polyanes (psittacofulvins) have ν 2 values that are about 20-30 cm À1 lower than those of the methylated polyenes (Maia et al. 2021).Therefore, the ν 2 values observed in the present work (1132-1134 cm À1 ) (Table 1) rather point to the occurrence of unmethylated polyenes in fossil and modern representatives of the Cerithioidea.This is also in agreement with a generally observed wide occurrence of unmethylated polyenes in modern molluscs (Merlin & Delé-Dubois 1986;Barnard & de Waal 2006;Hedegaard et al. 2006;Ishikawa et al. 2019).
Furthermore, it has been demonstrated that with increasing length of the conjugated chain in unmethylated polyenes the wavenumber position of the ν 1 band decreases (Schaffer et al. 1991).These data obtained on synthetic polyenes have been successfully applied to the characterization of unmethylated polyenes from differently coloured modern molluscan shells (Hedegaard et al. 2006;Ishikawa et al. 2019).By comparison of the Raman band maxima measured in the present work with those of the synthetic polyenes (Schaffer et al. 1991), the number of conjugated double bonds of the polyenes in the fossil cerithioidean gastropods and the modern Thericium can be estimated to be n = 9 and n = 12, respectively, corresponding very well to the observed orange-red and brown colour of the gastropod shells.It is also remarkable that the polyene signals in the Raman spectrum of Thericium are broader than those signals in the spectra of the fossil representatives, suggesting a broader range in the number of conjugated double bonds in the pigments of the modern gastropod.
It is well known that polyene pigments such as carotenoids in nature often occur as carotenoproteins, which have different colours to the free carotenoids (Britton et al. 2008).Interaction of unmethylated polyenes (psittacofulvins) with the protein keratin has been reported too (Stradi et al. 2001).However, although proteinaceous material has been reported from shells of the Miocene gastropod Ecphora (Nance et al. 2015), in the present work, no indications were found that the polyenes of the cerithioidean gastropods could be bound to proteins.Nevertheless, it has to be considered that the actual colour of the polyene-containing Miocene gastropods during lifetime may have been different to orange-red, if they were bound to proteins.
A prerequisite for the exceptional colour preservation in the investigated fossil gastropods is certainly the generally low thermal maturity of organic material from the Neogene sediments in the Vienna Basin (Sachsenhofer 1992).A low degree of diagenetic change is also indicated by the preservation of the original aragonite shell.Furthermore, the occurrence of the pigments within the carbonate matrix of the gastropod shells has obviously contributed to the exceptional preservation.
It is very interesting that reddish colouration of shells and corresponding Raman signatures for intact polyenes were only found in fossil members of the superfamily Cerithioidea (Pithocerithium, Potamides, Tiaracerithium), but not in members of other gastropod families with colour preservation from the same localities such as Sarmatigibbula (superfamily Trochoidea) and Megalotachea (superfamily Helicoidea) (Fig. 1; Table 1).This indicates that the occurrence of polyene pigments in ancient gastropods is related to certain taxonomic groups.Residual colour patterns have even been found in representatives of the Cerithioidea from the Late Jurassic Cordebugle Lagerstätte (Calvados, France) (Caze et al. 2015).However, it still has to be analysed whether these pigments survived unaltered and were related to polyenes.

CONCLUSION
Important information on the chemistry of organic compounds in fossils can be obtained from Raman bands.Whereas typically D and G bands of disordered carbon are obtained from thermally mature material, more characteristic bands can be obtained from thermally immature samples.In situ Raman spectroscopy indicates the preservation of intact polyene pigments in about 12 millionyear-old orange to red coloured gastropod shells from the Middle Miocene of the Vienna Basin.As far as known, these pigments represent the first record of intact polyene pigments in fossils.Moreover, the preservation of these pigments is observed especially within the Cerithioidea, but not in two other tested gastropod superfamilies.The results show that in situ Raman analysis enables the nondestructive analysis of rare fossils with colour preservation and thus provides a valuable tool for the screening of further fossils on the occurrence of polyene pigments.
2001) and fossilized latex (Lönartz et al. 2023), although it should be considered that not all organic molecules are equally suitable for Raman analysis.A limitation for organic compounds is that especially in thermally more mature samples commonly only D and G bands of carbon are obtained (Peteya et al. 2017; Wolkenstein 2022).Moreover, in some recent Raman studies on fossil samples (e.g.Wiemann et al. 2018; McCoy et al. 2020) numerous quasi-periodic signals have been obtained over the full spectral range, which, however, are supposed to represent instrumental artefacts generated by interferences from intense background fluorescence at the edge filters (Alleon et al. 2021).

F
I G . 2 .Raman analysis of shell pigments of the Miocene gastropod Pithocerithium and the modern gastropod Thericium.A, in situ Raman spectrum of the brown coloured shell of the modern Thericium vulgatum (specimen shown in Fig.1F).B, Raman spectrum of the isolated orange polyene pigment of Pithocerithium rubiginosum from the Miocene of Nexing (see F). C, in situ Raman spectrum of the red coloured shell of Pithocerithium rubiginosum from the Miocene of Nexing (specimen shown in Fig.1A).D, in situ Raman spectrum of the non-coloured shell of Pithocerithium rubiginosum from the Miocene of Nexing (specimen shown in Fig.1A).E, specimen of Pithocerithium rubiginosum from the Miocene of Nexing used for extraction of pigments (height is 1.5 cm).F, isolated orange polyene pigments of Pithocerithium rubiginosum (see E) on CaF 2 disc (diameter of disc is 2 cm).Raman spectra are background corrected and normalized.The signals at 704 and 1085 cm À1 are due to carbonate.Potamides from different localities show distinct signals at about 1100 and 1500 cm À1 (Figs 2C, S2; Table 1), different from common signals of D and G bands (at about 1350 and 1600 cm À1) of disordered carbon (e.g.Pasteris & Wopenka 2003).Both orange and red colouration show the same Raman signals, differing only in the intensity of the signal.This indicates that only the concentration of pigments is different in the individual shells.In those shells with the most intense colouration (Pithocerithium and Tiaracerithium) further signals at about 1000, 1300, 2200, 2600 and 3000 cm À1 (Fig. 2C; Table 1) can be observed.All these signals are characteristic of polyene pigments (Merlin & Delé-Dubois 1986; Barnard & de Waal 2006; Hedegaard et al. 2006) and can be interpreted as corresponding to known Raman bands of linear polyenes (trans-polyacetylenes).The signals of highest intensity at about 1500 and 1100 cm À1 can be assigned to C=C (ν 1 ) and C-C stretching bands (ν 2 ), respectively, and the weaker signals at about 1300 and 1000 cm À1 to CH=CH (ν 3 ) and CH bending bands (ν 4 ) (Merlin & Delé-Dubois 1986; Barnard & de Waal 2006).The signals above 2000 cm À1 represent overtone and combination bands (mainly of the intense ν 1 and ν 2 bands).The specific polyene signals are also observed in the Raman spectrum of a dark brown coloured present-day Thericium vulgatum (Fig. A L L E O N , J., M O N T A G N A C , G., R E Y N A R D , B., B R UL É, T., T H O U R Y , M. and G U E R I A U , P. 2021.Pushing Raman spectroscopy over the edge: purported signatures of organic molecules in fossil animals are instrumental artefacts.BioEssays, 43, 2000295.B A R N A R D , W. and d e W A A L , D. 2006.Raman investigation of pigmentary molecules in the molluscan biogenic matrix.Journal of Raman Spectroscopy, 37, 342-352.B R I T T O N , G., L I A A E N -J E N S E N , S. and P F A N D E R , H. 2008.Carotenoids Vol.4: Natural functions.Birkhäuser, 370 pp.C A R D O S O , J. N., W A R D R O P E R , A. M. K., W A T T S , C. D., B A R N E S , P. J., M A X W E L L , J. R., E G L I N T O N , G., M O U N D ,D.G. and S P E E R S , G. C. 1978.Preliminary organic geochemical analyses; site 391, leg 44 of the deep sea drilling project.Initial Reports of the Deep Sea Drilling Project, 44, 617-623.C A S A D I O , F., D A H E R , C. and B E L L O T -G U R L E T , L. 2016.Raman spectroscopy of cultural heritage materials: overview of applications and new frontiers in instrumentation, sampling modalities, and data processing.Topics in Current Chemistry, 374, 62. C A Z E , B., M E R L E , D., P A C A U D , J.-M. and S A I N T M A R T I N , J.-P. 2010.First systematic study using the variability of the residual colour patterns: the case of the Paleogene Seraphsidae (Mollusca, Gastropoda, Stromboidea).Geodiversitas, 32, 417-477.C A Z E , B., M E R L E , D., S A I N T M A R T I N , J.-P. and P A C A U D , J.-M. 2011.Contribution of residual colour patterns to the species characterization of Caenozoic molluscs (Gastropoda, Bivalvia).Comptes Rendus Palevol, 10, 171-179.C A Z E , B., M E R L E , D. and S C H N E I D E R , S. 2015.UV light reveals the diversity of Jurassic shell colour patterns: examples from the Cordebugle Lagerstätte (Calvados, France).PLoS One, 10, e0126745.C U R R Y , G. B. 1999.Original shell colouration in Late Pleistocene terebratulid brachiopods from New Zealand.Palaeontologia Electronica, 2 (2), 10A.D O C K E R Y , D. T. 1980.Color patterns of some Eocene molluscs.Mississippi Geology, 1, 3-7.v o n E I C H W A L D , C. E. 1830.Naturhistorische Skizze von Lithauen, Volhynien und Podolien in Geognostisch-Mineralogischer, Botanischer und Zoologischer Hinsicht.Zawadzki, Wilna, 256 pp.v o n E I C H W A L D , C. E. 1853.Lethaea Rossica ou Paléontologie de la Russie.Vol. 3. Schweizerbart, Stuttgart, 533 pp.
T A B L E 1 .Raman signals (cm À1 ) of shell pigments of Miocene gastropods compared to those of the modern gastropod Thericium.., not determined; because of lower signal intensity compared to other samples, for these samples Raman spectra were acquired only in the range of 100-2100 cm À1 .pigments.The Raman signals of the pigments of the fossil Cerithioidea are very similar to those obtained from the pigments of the modern representative Thericium and to those of many other modern molluscan pigments that have been attributed to polyenes.However, to the best of our knowledge such Raman signals have not yet been reported from the fossil record.Raman spectroscopy has been used to search for carotenoid pigments in amberpreserved feathers, but distinctive carotenoid-informative bands (at 1530 and 1153 cm À1 ) have been only obtained for present-day, and not for fossil feathers (Thomas et al. 2014a).Putative carotenoproteins have been reported for the brachiopod Calloria from the Late Pleistocene of New Zealand (Curry 1999) and the gastropod Ecphora from the Middle Miocene of North America (Nance et al. 2015), but both studies mainly focused on the characterization of the proteinaceous material or the content of amino acids with very little information on the associated pigments.Otherwise, only hydrogenated carotenoids (non-coloured carotanes) have been reported from fossil organisms, such as β-carotane that was detected in Pseudofagus leaves from the Miocene of Clarkia, Idaho(Giannasi & Niklas 1981).