Kromdraai_n Alkaine Study

Aim/rationale:
Plant wax lipid molecules, chiefly normal (n-) alkanes and n-alkanoic acids, are frequently used as proxies for understanding paleoenvironmental and paleoclimatic change. These are regularly analyzed from marine and lake sediments and even more frequently in archaeological contexts, enabling the reconstruction of past environments in direct association with records of past human behavior (Patalano et al., 2020). Carbon and hydrogen isotope measurements of these compounds are used to trace plant type and water-use efficiency, relative paleotemperature, precipitation, evapotranspiration of leaf and soil moisture, and other physiological and ecological parameters. Plant wax lipids have great potential for answering questions related to hominin-environment interactions, being for the most part chemically inert and easily recoverable in terrestrial sediments, including those dating back millions of years. The growing use of this technique, and comparison of such data with other paleoenvironmental proxies such as pollen and phytolith analysis and soil carbonate and tooth enamel isotope records, make it essential to establish consistent, best-practice protocols for extracting n-alkanes and n-alkanoic acids from archaeological sediments to provide comparable information for interpreting past climatic, ecosystem, and hydrological changes and their interaction with early hominins.
The Kromdraai site presents a unique opportunity to track the paleoecological shifts associated with the evolution from Paranthropus to Homo within a single stratigraphic sequence. This sequence spans the Pliocene-Pleistocene boundary and encompasses Units O and P, which contain abundant fossilized teeth of carnivores, cercopithecoids, and bovids. Consequently, the site is well-suited for employing the innovative approach of analyzing plant wax lipid molecules to comprehend paleoenvironmental and paleoclimatic changes (Patalano et al., 2020). This methodology holds promise for providing deeper insights into the paleoenvironmental transformations observed at Kromdraai, thereby facilitating the interpretation of the contextual significance of its significant hominin remains.
Patalano R, Zech J, Roberts P. 2020 Leaf Wax Lipid Extraction for Archaeological Applications. Curr Protoc Plant Biol. 5, e20114.

- detailed information on the research project behind it & methodology including expected outcomes (i.e., the reason for export);

The external surfaces of vascular plant leaves are coated with protective waxes that contain a wide range of organic compounds. These waxes preserve the water balance of the plant and minimize damage to leaf cells from fungal and insect attack, wind abrasion, and excessive ultraviolet radiation. The lipid component of the leaf wax is, for the most part, chemically inert and resistant to biodegradation in sediments over geologic time, making these lipids an excellent proxy measure for paleoclimatic and paleoenvironmental conditions. Palaeoclimatological research extending back to the Miocene, and even the early Eocene, highlights the long-term molecular fingerprinting capability of plant biomarkers. Plant wax lipids pass into the environment as leaf debris and can be transported long distances by wind and water before the intact molecules are deposited. Traditionally, geographers and earth scientists have sought to recover plant waxes from terrestrial, ocean, and lake sediments in order to reconstruct biotic and abiotic factors of past ecosystems, using solvent extraction of ancient sediments or rocks followed by qualitative and quantitative profiling through a combination of chromatographic separation and mass spectrometry techniques. For example, the characterization of biomarker chemical structures to assess biosynthesis is now complemented by isotopic analyses of individual compounds to study ancient biodiversity or climatic conditions. There are growing attempts to extract the same compounds from archaeological sites, providing a record of past environments that can be directly compared to sequences of past human technology, culture, and settlement.

The two main plant wax biomolecules used as paleoenvironmental proxies are normal (n-) alkanes and n-alkanoic acids. Through their carbon (δ13C) and hydrogen (δD) isotope ratios, these water-insoluble compounds can be used to research the relationship between hydrological patterns (e.g., rainfall amount, aridity vs. humidity) and plant ecology (e.g., C3 vs. C4 ecosystems, canopy structure) and, when applied in archaeological situations, to answer questions about cultural adaptive responses to ecological changes, site-use patterns across diverse plant landscapes, and the human selectivity and usage of specific plants within a wider ecological biome. The rising use of this technique in archaeological contexts and in the study of past human-environment interactions necessitates the development and presentation of appropriate standardized protocols to an archaeological audience. Nevertheless, published protocols remain variable, are rarely compared or presented in detail, and are generally lacking in the archaeological literature.

Methodology (short):
The method involves using small samples of soil (~50 g).
Total lipid extraction (TLE) is a technique based on the like-dissolves-like principle of solvent separation, whereby solvents readily dissolve analytes of approximately the same polarity. Typically, an azeotrope of solvents is refluxed through a ground plant, rock, or unconsolidated soil or sediment sample to isolate leaf waxes. There are multiple ways to extract lipids, of which the most common are the use of a Soxhlet apparatus, accelerated or pressurized solvent extraction (ASE or PSE), and ultrasonic extraction. Although Basic Protocol will focus on PSE, the different techniques share inherent principles in that the goal of each is to ensure high analyte recovery while reducing time and solvent consumption and avoiding carryover or cross-contamination between samples. Each extraction technique requires samples to be homogenous and dry and equipment to be precleaned, so sample pretreatment and cleaning recommendations are included in each protocol.
A PSE apparatus is an automated system for extracting organic compounds from a variety of solid and semisolid samples. Although these are still relatively rare pieces of equipment in many labs, they are quickly being adopted because of their efficiency in extracting compounds over short time periods. PSE uses elevated temperature and pressure in combination with organic solvents to increase the efficiency of the extraction process. During the procedure, an extraction cell containing the sample is filled with solvent and then heated and pressurized to maintain the solvent in a liquid state for a predetermined amount of time. After this hold time, the extract is discharged from the cell into a collection vessel, and then the process is repeated. PSE has major benefits over other methods in terms of speed and overall compound recoverability. First, higher temperatures increase the extraction speed and the solubility of lipid analytes. Elevated pressure allows temperatures above the solvent boiling points to be used, which is essential given that the boiling points of organic solvents and target lipid compounds are often low. Operating at elevated pressure also expedites and enhances the overall extraction, as pressurized solvent is forced into and through the pore spaces of the sediments, improving biomarker recovery.
The type of solvent used has a major impact on the extraction, and it is best to choose solvents with polarities similar to those of the target lipids; in this instance, the target analytes are nonpolar (n-alkanes) and slightly polar (n-alkanoic acids as fatty acid methyl esters). Mixtures of solvents with different polarities often provide better recoveries. A temperature of 100°C, a pressure of 1500 psi (103 bar), and a 9:1 volume/volume mixture of dichloromethane and methanol have been shown to be good starting points for a wide range of extraction applications.

The reason for export six small samples of soil (~50 grams) is because the method presented here is new and no research lab in South Africa can conduct this kind of analysis.