RESOLVING THE FISHER-FARMER CONUNDRUM IN PREHISTORIC EUROPE USING BIOMOLECULAR APPROACHES (AquaNeo)

在现代人类进化的大部分时间里，人类依赖于他们狩猎、采集和捕鱼的食物资源。在向新石器时代过渡的过程中，人类的生存方式发生了重大变化，人类的饮食中引入了驯化的植物和动物。许多沿海地区的早期农民远离海洋，不吃海洋食品。人们对内陆地区的早期农民以及他们对河流和湖泊水生资源的利用知之甚少。除牛奶外，水产资源是农民可获得的唯一维生素D3来源的食物，在大多数暴露于中波紫外线不足的欧洲地区，维生素D3对骨骼健康至关重要。因此，水产食品可能在农民的饮食中发挥了关键的营养作用，特别是在没有大量开采牛奶的地区。然而，捕鱼和诱捕技能是狩猎采集者生活方式的重要组成部分。因此，农业社区对水生资源的利用很可能表明，这些技能是从剩余的狩猎采集者群体传给早期农民的（文化假设）。早期农民对水生资源的利用，除了能方便地进入有生产力的河流和溪流之外，据推测还受到营养需求和获得文化技能的影响。水生骨骼遗骸非常小且脆弱，使用人骨胶原检测淡水资源消耗的传统方法极具挑战性。我们的目标是陶器碎片，以检测早期（内陆）农业遗址淡水资源的使用情况。事实上，被困在陶瓷孔隙中的脂质（或脂肪）为陶瓷容器中烹饪的东西提供了证据。通过使用最先进的分析方法表征脂质，我们将能够检测出水生资源加工过程中众所周知的化合物（或生物标志物）特征。我们将开发一种新方法，使用高灵敏度的仪器来检测陶器提取物脂质组合中极少量的此类生物标志物。我们还将使用水生生物标志物和动物脂肪中普遍存在的化合物（C16:0和C18:0脂肪酸）的碳同位素特征来建立混合模型，以量化每个陶罐中淡水衍生动物脂肪的数量。关于食物中的蛋白质是否能在考古陶器碎片上存活，存在很大的不确定性——如果是这样，它们将为脂质提供非常补充的见解，因为它们是物种特异性的（而脂质不是）。我们将通过在仿制品陶器中烹饪各种食物（包括不同种类的鱼）来测试食物蛋白质的稳定性。然后，我们将对这些容器的部分进行取样，并将它们埋在两种不同条件下的堆肥中，为期18个月，以模拟考古降解。然后，我们将使用古蛋白质组学方法分析这些碎片，以评估食物蛋白质是否存活以及存活的程度。我们在布里斯托尔收藏了大约1500个考古脂质提取物和碎片，这些脂质提取物和碎片来自我们欧洲资助的neommilk项目。我们将选择来自欧洲大陆不同养殖地点的提取物和碎片来研究水生脂质生物标志物和蛋白质。这将使我们能够评估这些地点使用淡水资源的水平和类型。这些新数据对于检测未用于烹饪淡水资源的碎片将是非常有价值的——它们将被用来证明保存在其中的脂质可以追溯到14碳年代，并被用来建立可靠的年表，因为它们不受“旧碳”效应的影响。最后，我们将使用模型来研究早期农民对淡水资源的利用与生态（水体的可及性）、营养（牛奶的使用、狩猎猎物的开发）和文化（与狩猎采集者群体的接触）参数之间的联系。该项目将强调淡水生态系统在过去、现在和未来对人类的重要性。

For most of the modern human evolution, humans were depending on food resources they hunted, gathered and fished. The transition to the Neolithic saw a major change in subsistence, with the introduction of domesticated plants and animals in human diet. Many coastal populations of early farmers turned their back from the sea and were not consuming marine foodstuffs. Little is known about early farmers from inland locations and their use of aquatic resources from rivers and lakes. Together with milk, aquatic resources are the only foodstuff available to farmers that are a source of vitamin D3, crucial for bone health in most of Europe where exposition to UVB are insufficient. Aquatic foods may thus have played a key nutritional role in farmers' diet, particularly in regions where milk was not greatly exploited. However, fishing and trapping skills are very much part of the hunter-gatherer life-ways. Aquatic resource use in farming communities is thus likely to show that these skills were transmitted from residual hunter-gatherer populations to early farmers (cultural hypothesis). Beyond the mere ease-of-access to productive rivers and streams, the use of aquatic resources by early farmers is hypothesised to have been shaped by nutritional necessity and access to cultural skills. Aquatic skeletal remains are very small and fragile, and traditional methods for the detection of consumption of freshwater resources using human bone collagen is extremely challenging. We are targeting pottery sherds to detect the use of freshwater resources at early (inland) farming sites. Indeed lipids (or fats) trapped in pottery pores provide evidence for what was cooked in ceramic vessels. By characterising lipids using state-of-the-art analytical methods, we will be able to detect well-known compounds (or biomarkers) characteristic of aquatic resource processing. We will develop a novel method using a highly sensitive instrument to detect extremely low amounts of such biomarkers in the lipid assemblage of pottery extracts. We will also use the aquatic biomarkers and the carbon isotope signature of ubiquitous compounds found in animal fats (C16:0 and C18:0 fatty acids) to build a mixing model to quantify the amount of freshwater-derived animal fats in each pottery vessel. There is very much uncertainty into whether proteins from foodstuffs would survive on archaeological pottery sherds - if so, they would provide very complementary insights to lipids as they are species-specific (while lipids are not). We will test the stability of foodstuff proteins by cooking diverse foodstuffs (including different types of fish) in replica pottery. We will then sample parts of the vessels and bury them in compost under two different conditions for 18 months to mimic archaeological degradation. We will then analyse the sherds using palaeo-proteomics methods to assess whether foodstuff proteins have survived and how well. We have >1,500 archaeological lipid extracts and sherds curated in Bristol that were obtained from our European-funded NeoMilk project. We will select extracts and sherds from various farming sites across mainland Europe to investigate aquatic lipid biomarkers and proteins. That will enable us to assess the level and type of freshwater resource use at those sites. This novel data will be extremely valuable to detect sherds that were not used for cooking freshwater resources - they will be used to demonstrate that lipids preserved within can be 14C-dated and be used to build robust chronologies as they are not affected by an "old-carbon" effect. Finally, we will use models to study the link between the use of freshwater resources by early farmers and ecological (accessibility to water bodies), nutritional (milk use, exploitation of hunted game) and cultural (contact with hunter-gatherer populations) parameters. This project will highlight the importance of freshwater ecosystems to human populations in the past, present and future.