Melanin in fossil ink sacs of coleoid cephalopods (Vampyromorpha)

In this work various samples from fossilized Vampyromorpha ink sacs were directly measured for characterizing the melanin spectra. The samples originate from the Cenomanian Fish Shale (Fischschiefer) in Lebanon (L1, L2), the Tithonian Solnhofen Plattenkalk (S1, S2) and the Toarcian Posidonienschiefer in Holzmaden, Germany. The color of the melanin in the ink sacs varies from yellowish in the sample from Lebanon, over brownish, red in the samples from the Solnhofen Plattenkalk to black in the samples from the Posidonienschiefer.

As melanin is very opaque, spectra were tried to record with all available excitation wavelengths. Thereby, clearest bands could be recorded with excitation in the UV (244 nm), whereas in the NIR (785 nm) no signal could be detected at all. In the low visible range (488 nm) only the main carbon vibrations could be recorded, whereas in the higher visible range (633 nm) the complete spectra were masked by fluorescence. In the following spectra recorded with 244 nm excitation will be shown from Lebanon (L1), and the Solnhofen Plattenkalk (S1), as well as from the Posidonienschiefer (M1, M2) with additional spectra recorded with 488 nm excitation. Due to technical limitations spectra in the UV can only be recorded with a minimum Raman shift of 500 cm^-1, whereas the spectra in the visible range already start at a Raman shift of 200 cm^-1.

In most of the spectra additional bands due to the carbonate matrix can be observed. Especially in the range around 1085 cm^-1 the main CO3

vibration is visible. The most prominent bands for melanin arise around 1600 cm^-1 due to C-C stretching of aromatic units and around 1400 cm^-1 due to aromatic C-N bonds (Horiba, application note: http://www.horiba.com/fileadmin/uploads/

Scientific/Documents/Raman/bands.pdf; Socrates, 2001; Smith & Dent, 2005).

Furthermore, bands or shoulders can be recognized around 1330 and 1200 cm^-1.

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In the Fish Shale sample from Lebanon always a very strong carbonate signal (1085, 1413 and 1749 cm^-1) from the surrounding rock was visible (Fig. 10). The melanin signal mainly was concentrated on the 1600 cm^-1 region. The aromatic C-N bonds are only visible in a very weak shoulder around 1400 cm^-1, whereas the stronger band at 1433 cm^-1 belongs to the carbonate stretching vibrations.

Around 700 cm^-1 a broad band appears probably due to additional sulfur in the melanin structure. In this range stretching vibrations of aliphatic C-S bonds occur (Smith & Dent, 2005).

Figure 10: Melanin spectrum from the fossil ink sac of Vampyromorpha from the Cenomanian Fish Shale (Lebanon). High signals from the background carbonate are prominent. Main melanin signals occur due to C-C vibrations at 1600 cm^-1, C-N vibrations at 1402 cm^-1 and C-S vibrations at 704 cm^-1.

The melanin spectra from the Solnhofen Plattenkalk (Fig. 11) are dominated by broad bands centered at 1602 cm^-1 and 1420 cm^-1, which belong to C-C and C-H vibrations, respectively (Socrates, 2001; Smith & Dent, 2005). According to Powell et al. (2004) vibrations of hydroquinone would be expected at 1425 cm^-1, and could also account to the broad band at 1420 cm^-1. Significant band also arise at 963 cm^-1 and 1075 cm^-1. Bands between 950 and 980 cm^-1 were also observed by Samokhvalov et al. (2004) and are supposed to represent ring modes of the phenyl group. However, this assignment is not quite clear. The band at 1075 cm^-1 is predicted to occur in semiquinone (Powell et al., 2004), but

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also C-S aromatic stretching vibrations appear in that area (Smith & Dent, 2005).

Figure 11: Melanin spectrum from the fossil ink sac of Vampyromorpha of the Tithonian Solnhofen Plattenkalk. Main signals belong to vibrations of C-C and C-H molecules. But also vibrations of the key monomers hydroquinone (HQ) and semiquinone (SQ) can be assigned.

Additionally, ring modes of the phenyl group can be observed.

In the spectra from the Posidonienschiefer from Holzmaden clear differences can be seen between excitation with 244 nm and with 488 nm (Fig. 12). With 244 nm almost only the main carbon stretching vibrations centered at 1600 cm^-1 are visible. In the sample M2 additionally signals from the carbonate matrix appear. On the other hand the spectra recorded with 488 nm excitation show aside to the C-C vibrations another dominant area between 1300 and 1400 cm^-1, which is only slightly denoted when excited with 244 nm. This pattern was observed by several working groups as the typical melanin spectrum, whereby an analysis of the broad band areas with Gaussian functions reveals several different bands which can be assigned to vibrations of the key monomers of eumelanin (see Capozzi et al., 2005). Another interesting area again is again more obvious in the spectra recorded with 488 nm excitation wavelength. The broad band between 550 and 600 cm^-1 can be interpreted as an enrichment of Fe (III) in the melanin. This phenomenon is described by Samokhvalov et al.

(2004) who observed an increase of a band centered at 570 cm^-1 with increasing Fe (III) content.

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Figure 12: Melanin spectra of the fossil ink sac of Vampyromorpha from the Toarcian Posidonienschiefer in Holzmaden. Clear differences can be seen between excitation with 244 nm and with 488 nm wavelength. HQ, IQ and SQ stand for the key melanin monomers hydroquinone, indolequinone and semiquinone.

The results of those Mesozoic fossils show that vibrations which can be assigned to melanin can be reproduced. However, Raman spectra of melanin are much more unspecific (cf. Samokhvalov et al., 2004; Capozzi et al., 2005;

Perna et al., 2005) as spectra of standard materials as shown in chapter 2.

Nevertheless, differences between the sampled specimens can be related to their different color, which is influenced by the occurrence of sulfur in the melanin structure (Hackman & Goldberg, 1971). Therefore, the melanin spectra from the yellowish Fish Shale sample from Lebanon show bands related to C-S vibrations and the black samples from the Posidonienschiefer are influenced by higher Fe content.

The comparison of the results with 244 nm and 488 nm excitation has shown that with 488 nm excitation the signal to noise ratio of the spectra is much better. Additionally, a better analogy to the predicted key monomers (HQ, IQ, SQ) of melanin reported by Powell et al. (2004) could be found with an excitation wavelength of 488 nm. Therefore, in the following the detection of melanin was only performed with this excitation wavelength.