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，人类都依赖于他们狩猎、采集和捕捞的食物资源。在向新石器时代的过渡中，随着人类饮食中引入驯化的植物和动物，生存方式发生了重大变化。许多沿海地区的早期农民远离海洋，不食用海洋食物。关于早期来自内陆地区的农民以及他们对河流和湖泊水生资源的利用，人们知之甚少。与牛奶一起，水产资源是农民可获得的唯一食物，是维生素D3的来源，在欧洲大部分暴露于UVB不足的地方，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，特别是在牛奶没有被大量开发的地区。然而，捕鱼和诱捕技能是狩猎采集者生活方式的一部分。因此，农业社区对水资源的利用很可能表明，这些技能是从残余的狩猎采集者传给早期农民的（文化假设）。除了容易进入生产性河流和溪流之外，早期农民对水产资源的利用被假设为是由营养需求和获得文化技能所塑造的。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）农业用地对淡水资源的使用。事实上，陶器气孔中的脂质（或脂肪）为陶器中的烹饪提供了证据。通过使用最先进的分析方法对脂质进行表征，我们将能够检测出水产资源加工过程中的已知化合物（或生物标志物）。我们将开发一种新的方法，使用高灵敏度的仪器来检测陶器提取物的脂质组合中极少量的这种生物标志物。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一个混合模型，以量化每个陶器中淡水来源的动物脂肪的量。食物中的蛋白质是否能在考古陶器碎片上存活下来还存在很大的不确定性-如果是这样，它们将为脂质提供非常互补的见解，因为它们是物种特异性的（而脂质不是）。我们将通过在复制陶器中烹饪各种食物（包括不同类型的鱼）来测试食物蛋白质的稳定性。然后，我们将对容器的部分进行取样，并在两种不同的条件下将它们埋在堆肥中18个月，以模拟考古学降解。然后，我们将使用古蛋白质组学方法分析碎片，以评估食物蛋白质是否存活以及存活情况如何。我们在布里斯托有超过1,500个考古脂质提取物和碎片，这些都是从我们欧洲资助的NeoMilk项目中获得的。我们将选择来自欧洲大陆各个养殖场的提取物和碎片，以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。这一新的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被14 C测年，并用于建立强大的年表，因为它们不受“老碳”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使用与生态（水体的可及性），营养（牛奶的使用，狩猎游戏的开发）和文化（与狩猎采集人口的接触）参数之间的联系。该项目将突出淡水生态系统在过去、现在和将来对人类的重要性。

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.