Paleogenomic Studies of Written Artefacts of Different Origin
2019–2025
RFA04
The project opened new perspectives for the complex material analysis of plant-based artifacts. For the first time, a combination of palaeoecological analysis, high-resolution microscopy, and molecular biology techniques (such as historical plant DNA extraction, metabarcoding, targeted capture, and next-generation sequencing) was applied to the study of palm-leaf manuscripts (PLMs).
1. Studies on historical plant DNA
Palm-leaf manuscript (PLM) production is a traditional practice in many South and Southeast Asian countries, yet almost completely ceased and until the present time poorly documented. Various plants, specific to regions, historical times, or manufacturers, are involved in the multi-step PLM preparation process. Each step of the process involves close contact of the palm leaves (mainly of Borassus flabellifer L. and Corypha umbraculifera L.) with other plants – from seasoning and boiling with various fruits and herbs to brushing (e.g. with grasses, sand, marine shells), oiling, inking, and applying plant-based solutions for deemed protection against insects. Despite the known main steps, the detailed history of PLM production craft remains mostly unstudied, neither in terms of plants used at different preparation steps nor in terms of the rationale underpinning plant selection.
We applied a metabarcoding approach to reveal which plants were used in the production of Indian manuscripts. Surface swabs as well as the fallen-off fragments of historical manuscripts were analysed. About 100-250 different plants were revealed per manuscript sample. Although the studied manuscripts date from the 18th to the beginning of the 20th centuries CE, the plant DNA fragments were typically short and ultra-short molecules with extensive chemical damage that is typical of ancient DNA. Furthermore, the authenticity of the plant DNA remains largely uncertain, as it is unclear whether the plants whose DNA is present in small amounts were actually used in the PLM preparation process or if this DNA represents historical contamination associated with the craft.
About 30-35% of the plant DNA records in each case were from plants not known from South Indian flora. These exotic plants in the historical DNA, commonly referred to as molecular data misinterpretation attributed to machine failure of the name-sequence matching, in our case can actually provide important ethnobotanical data. For example, saffron (Crocus sativus L., Iridaceae family), an expensive spice and important conservation plant, referred to in the literature for seasoning palm leaves in India and Sri Lanka, is registered in our material only twice in 180 samples. In contrast, commonly known surrogates of saffron, e.g. Tagetes sp., Carthamus tinctorius L. (the both plants come from Asteraceae family), Curcuma longa L. (Zingiberaceae) are identified in 50 analysed samples and repeatedly revealed in 4-6 of 8 PCR repeats, all showing 100% match to reference sequences.
Another example of the apparent use of botanically unrelated plants under the same name, is Couroupita guianensis Aubl. (Lecythidaceae), introduced to India and is often venerated as ‘sal tree’, which is also the local name of Shorea sp. (Dipterocarpaceae family). Both examples show that misinterpretation here is caused not by problems of DNA analysis but by incorrect naming of the plants actually used for PLM production, when two (or more) unrelated plants are confused or deliberately named wrong. As literature on the PLM preparation is mainly superficial, non-academic, subjective, and difficult to verify, these cases of incoherence may serve us as important sources of data on what plants were actually used historically (and revealed analytically) and which of them were just mentioned for various reasons but not used in fact. Along with historical DNA studies, a 2-year-long in-laboratory monitoring on the modern plant DNA contamination was performed in all facilities used for the project. Based on the results, appropriate measures were taken in order to reduce possible environmental contamination and to avoid using substandard facilities.
Possible reasons for plant confusion in the PLM production and preservation literature may include:
- Unintentional errors in plant identification (i.e. honest misapprehension).
- Accurate citation of incorrect information found in a primary source.
- Taxonomic mislabeling of a correctly identified plant (e.g. assigning a plant to an incorrect botanical family).
- Incorrect translation of plant names, especially when translating from a locally used language into English.
- Ambiguous identification statements, where a lack of clarity leads, or may lead, to misinterpretation.
- Miswriting, misspelling, or misreading.
- Financially or reputationally motivated deliberate manipulation, such as the intentional use of surrogate plants instead of the declared ingredients (i.e. fraud).
- Substitution of unavailable but desired plants with undeclared alternatives.
Our studies are only the first attempts to perform analytical studies on such complex and fascinating subject as historical palm-leaf manuscripts. A lot more and much more detailed studies are required.
2. Palaeoecological studies of PLMs: a novel approach brings promising results
We studied freshly collected, dried and herbarized leaf fragments of two palms, namely Borassus flabellifer L. and Corypha umbraculifera L., most commonly used for palm-leaf manuscript (PLM) production in South (S) and Southeast Asia (SE) in order to reveal differences in their phytolith assemblages. For each of the two palms, 25 leaf samples were taken from the two Indian states of Kerala and Tamil Nadu. Dried leaf material was obtained from the fresh one by drying the leaves in air. Herbarium samples were obtained from two independent herbaria, specimen origin comprises S and SE Asia with the main focus on South India and Sri Lanka. Additionally, 25 manuscripts made of Borassus flabellifer leaves and 25 manuscripts made of Corypha umbraculifera leaves were investigated for phytoliths. All manuscripts are preliminary dated back to between the 16th and the beginning of the 20th century CE; most of them assumedly were produced in S India (Tamil Nadu and Kerala), Sri Lanka, Burma or Indonesia. Phytolith assemblages significantly differed between fresh, dry and herbarized palm leaves in comparison to PLM material, both qualitatively and quantitatively (mean r2 = - 0.61 ± 9.3 for Borassus samples and r2 = - 0.75 ± 5.3 for Corypha samples, at p < 0.001). Fifty-three phytolith types described for PLM material were not observed in any of the fresh, dry or herbarized palm-leaf samples. Geographical analysis of PLM-specific phytoliths suggests that the combination of those phytoliths could be region-related. In this paper, we prove that the methods of palaeoecological reconstructions based on detailed microscopy of the PLMs surface and phytolith analysis applied in combination with methods of mathematical and computer data analysis can contribute to answer the questions posed by material codicology by revealing lost manuscript production recipes and by studying manuscript provenance in terms of the geographical origin of the artefacts. Our approach can potentially open a new perspective for palaeoecological studies expanding their traditional scope and making them applicable to a new research field.
People
Project lead: Anastasia Poliakova