In our investigation for the potential for combined palynological and molecular analysis of medieval wax seals, we found that the results of the DNA and pollen analysis were limited, yet the lipid analysis could hold more potential for future work on a material such as sealing wax.
The lipid profile and abundance of compounds in DA177 agrees with previous research of Apis mellifera beeswax [16, 28, 29], thus providing unambiguous evidence for the use of beeswax in the seal. Although beeswax is typically resistant to large changes in chemical composition due to its hydrophobic nature, degradation may occur through exposure to UV, high temperatures or microbial attack [31]. As a result, modern and historic beeswax evidence different lipid profiles that may be characterised by the absence of n-alcohols (C24–C38) arising from the hydrolysis of wax monoesters; this is often accompanied by the loss of low weight n-alkanes [31, 32]. With this in mind, the absence of n-alcohols in DA177 may indeed suggest the sample is generally well-preserved although when compared with modern beeswax, the distribution of n-alkanes seems to favour higher-weight homologues as seen by the absence of n-tricosane (C23) and the low abundance of n-pentacosane (C25).
What is also interesting is the absence of terpenic acids in the sample, that presumably would be there due to the rosin traditionally added to the sealing wax mixture. However, this could indicate that the sample DA177 has either a very low, undetectable amount of rosin or none at all, where the latter fits in with previous knowledge on the composition of ‘white seals’ being mostly of beeswax [7, 15].
The use of organic and inorganic additives to modify the properties of beeswax is well-reported in wax seal recipes [33]. To harden and extend the working life of seals, pine resin (rosin) was historically added to beeswax and may be identified by pimarane and abietane acids [34]. Venice turpentine, an exudate of the European larch (Larix decidua) is also cited in wax seal recipes [35]. Like rosin, turpentine is typically dominated by abietane and pimarane acids but can be distinguished by the presence of larixol and larixyl acetate [36]. Shellac is another common additive that originates from the Kerria lacca insect [37]. Its chemical composition differs from that of rosin and turpentine and is characterised by jalaric, laccijalaric, aleurtic and butolic acids. The absence of these biomarkers in DA177 suggests that these common admixtures were not employed during the production of the seal, instead, it is more likely the seal was manufactured from pure beeswax.
Overall, our analysis shows that the DNA content in the samples subjected to investigation was very low after extraction, and that only one sample (DA118) contained enough DNA to build a shotgun library. This could be a consequence of the efficient way that beeswax was refined (cleaned) by melting in hot water, allowing the water to cool and recovering purified wax floating on the surface. This method would be very efficient at stripping polar molecules (such as DNA and proteins) from the wax, because once the wax is melted, these molecules are free to dissolve in the water. Furthermore, at least one sample (DA121) very likely contained enzymatic inhibitors that affected the library preparation step, as evidenced by the obtained Ct value during the qPCR screening, which was higher than the corresponding value obtained for the negative control (Fig. 3). This consequently restricted the sample from downstream amplification and sequencing.
The lack of enough DNA for sequencing in most of the analysed samples and the high proportion of ultra short sequencing reads in DA118 points to very poor conditions for DNA preservation in the wax seals, or to the overall low amount of bee-related DNA possibly present in beeswax to begin with. Consequently, this hampered the downstream taxonomic identification of sequencing reads. However, our analysis indicates that plant DNA can be sequenced from these samples, even if no pollen can be identified. It is striking that we find so little bee or human DNA, which would be expected from material that had been in contact with these taxa. As only refined wax is used for seals, this would explain how the wax is sterile from bee-related DNA. This ultimately questions whether our findings of plant and fungal DNA can even be traced to the original sample or is environmental contamination. If our findings of DA118 are representative for wax seal samples in general, it will prove difficult to obtain useful and reliable information through DNA analysis. Despite the high number of reads sequenced, the degraded state results in low success rate in taxonomic identification.
In addition, it should be noted that technical improvements such as extraction and library preparation could aid future studies on medieval wax samples. This could be especially relevant in terms of circumventing the effect of enzymatic inhibitors in the samples that could stem from wax additives such as plant resins as well as employing library methods with high efficiency and low background noise. Thus, while the prospects for future success of retrieving DNA from wax samples do not seem good, we emphasize that the present study relies on a small number of samples and that technological improvement could also help. When analysing ancient DNA from heritage objects such as medieval seals, it is imperative to extract and sequence relatively high amounts of DNA in order to capture the endogenous DNA that is present with much larger amounts of DNA from contaminants. Hence there is a need for efficient protocols for DNA extraction and library preparation.
Pellets after DNA extraction of the seals did not prove to be the best source of pollen for pollen analysis, yet the numerous pollen preserved in the Herefordshire sample, despite its small size, was an encouraging result. Additionally, the sample was not completely dissolved, and showed up as lumps in the sample slides, contributing to the challenges in identifying pollen from the samples. This residue could have been removed with a few steps following a pollen preparation protocol (acetolysis), that was not included in this study in order to prevent sample loss. Nevertheless, because of the lack of pollen preparation steps, the fungal spores were preserved in the pellets [38].
Overall, the samples yielded little pollen which could be due to several reasons. Firstly, the sealing wax material could have been fairly sterile from pollen to begin with. Additionally, one or more steps in the procedure for DNA extraction could rid the sample of pollen, i.e. there may have been more pollen present in the sample originally but some of that may have been lost during the extraction process. There may have been a low concentration of pollen in the sealing wax, and the sample analysed was too small to detect it: during the setting of the warm wax, pollen may have been concentrated on certain areas [10], and this sample amount was too small to detect it. Despite these challenges, the information we found could prove to be useful and the potential of analysing a larger sample of the seals should be investigated in the future.
For example, the small fungal spore (group including Aspergillus) that was found in most of the seal samples could be an infection from storage in the archive. Aspergillus has been hypothesised as being one fungi responsible for the biodeterioration of sealing wax [1]. However, there is also a possibility that the fungal spores were accidentally or intentionally brought by the bees to the hive and embedded in the wax [39]. Are we analysing the micro-organism from the original source e.g. hive, the environment of the object or a part of the deterioration?
Additionally, investigating the high protein content of pollen [40] could bear an interesting approach to wax seals. However, as the proteins are inside the pollen, accessing the proteins would potentially require breaking the pollen grain with e.g. bead-beating, which would subsequently prevent the morphological identification of the pollen. Due to this and for the high accuracy of palynological identification, we prioritised the morphological identification of the pollen grains.
A material can go through either a chemical or a physical change during its ageing process. A chemical change can also act as a trigger for further mechanical damage, e.g. crystallisation breaking a structure [1]. The tendency of white seals to form a flaky, ‘biscuit structure’ is the main concern for conservators attempting to save the seals, as this makes the seals extremely prone to mechanical damage [41]. One of the causes of the deterioration of beeswax and the flaking or dryness phenomena is alterations in the crystallisation of the wax, leading to structural changes. As the polymorphic components change in the seal, this can lead to a more stratified structure and deterioration. Porosity can also be increased by the loss of hydrocarbons and volatiles during the ageing [8, 42]. This can also lead to the crystallisation of hydrocarbons to the surface of the seal, causing an effect known as “wax bloom” that may look like fungus [43, 44]. Analyses of the chemical composition of beeswax seals has revealed that these ‘blooms’ share nearly all their compounds with that of modern beeswax, interestingly however, when compared to historical wax seals the proportion of unsaturated hydrocarbons was found to be greater; this has since led to the proposal that the migration of alkenes is indeed a causative mechanism for wax blooming [43].
In addition to this, previous suggestions for the lamellar structure of beeswax have been fungus-like bacteria, and the technique in which the seals were made: to incorporate all of the materials evenly to the mixture, it was kneaded by hand. When dealing with a natural product such as beeswax, it is always possible that even after thorough cleansing and filtering, some fine traces of pollen, bees and honey are left in the wax. Microorganisms, such as the wax-decaying bacteria, can feed on these in addition to the hydrocarbons of the wax, causing microbiological degradation. Some notes on the appearance of micro-organism, especially on the deteriorated surface, have been made and suggested as the clear cause of the deterioration [13, 15]. Our findings of Aspergillus sp. in both spores and as identified DNA, may support the hypothesis of fungi being a critical part of the biodeterioration mechanism of seals, yet to confirm this a wider set of samples should be analysed in tandem with the lipidic composition.