Ancient mysteries – The world of palaeogenomics

Ancient mysteries – The world of palaeogenomics

Sampling lake sediment cores for Woolly Mammoth DNA. Photo: Andre Soares

By Caroline Wood 

“ Contrary to what many people think, it’s not all about recreating Jurassic Park!”, so said one of the opening speakers at the session Palaeogenomics and Ancient DNA at the SEB 2017 Annual Meeting in Gothenburg. Nevertheless, ancient DNA is proving to be the master key for unlocking a whole host of mysteries,including human migrations, the spread of disease, the disappearance of mammoths and the environmental effects of past climate change. Crucially, lessons from the past may even help us adapt to the rapidly changing world for the future.

Cultural Diffusion

One of the greatest cultural revolutions in the history of modern man was the transition from hunter-gatherer to agriculturalist, but how this occurred is far from straightforward. “The merging of hunter gatherer and farming cultures in Europe unfolded over thousands of years and in different ways in different areas”, said Ryan Schmidt (University College Dublin, Ireland). “Besides migration, cultural processes such as trade and exchange, as well as assimilation, were also at work. For example, in the Late Mesolithic (9600 – 4000 BC), prior to the migration of agricultural populations from Anatolia, the dental plaques of hunter-gatherers show evidence of domesticated cereal consumption, meaning some aspects of agriculture must have traded via social networks.”

Ryan’s current focus is the agricultural transition of the Cucuteni-Trypillian people (c. 5200 to 3500 BC), who lived in modern day Ukraine, Romania and Moldova. This culture is distinct for producing highly stylized anthropomorphic figurines and for establishing a number of ‘proto-cities’, with estimated populations of over 10,000. Despite this, human Trypillian remains are rare as few burial sites are known. An exception is Verteba Cave in Western Ukraine, where the remains of 12 individuals were recovered from a single chamber and radiocarbon dated to 3950–2573 BC. The DNA extracted from these remains was assessed for genome-wide Single Nucleotide Polymorphisms (SNPs: genetic variations involving a single DNA nucleotide base) to compare against other ancient European populations. The results demonstrated that “most of the samples were closely related to Early Neolithic farmers (4000–2900 BC) from Iberia, Central Europe and Anatolia. This suggests that hunter-gatherers in this area were replaced by migrating farmers from Southern Europe and the Near East, contrary to previous studies which indicate biological continuity with local hunter-gatherers”. Curiously, the DNA extracted from the only female sample showed greater similarity with Mesolithic hunter-gatherers. “This indicates that sociocultural forces likely attracted many hunter-gatherers to join farming communities”, said Ryan. “Perhaps some of the late foragers decided they could improve their social position by adopting the Neolithic way of life”.

Unintended Trade

Human migrations spread more than just ideas and, during the session, Ammielle Kerudin (University of Manchester, United Kingdom) demonstrated how palaeogenomics can be used to assess the role of human activity in transmitting diseases to new populations, including Mycobacterium leprae, the causative agent of leprosy. To source ancient M. leprae samples, Ammielle used tibiae and fibula bone samples recovered by the Biological Anthropology Research Centre (University of Bradford) from a medieval hospital in Chichester, UK (dated 1300 to 1700 AD) and Raunds Furnells, an Anglo-Saxon cemetery in Northamptonshire, UK (dated 900–1100 AD). “These bone samples showed new bone formation indicative of leprosy infection”, she said.

Interestingly the samples from Chichester contained M. leprae subtype 3I, which is associated with most leprosy cases in North, Central and South America today. “This suggests that the introduction of leprosy to America was most likely the result of European immigrants or colonizers, rather than early humans via the Bering Straits”, said Ammielle. However, the sample from Raunds Furnells contained the first known UK sample of the 3K subtype, now modernly associated with China, Japan and South- East Asia. “This suggests that the northern route of M. leprae transmission followed the Silk Road trade route to China and subsequently to Japan”, said Ammielle. Curiously the Black Death, caused by the bacterium Yersinia pestis, is also thought to have been transmitted along the Silk Road but in the other direction, having originated in China. “For leprosy, these results indicate an opposite route of transmission, with the 3K subtype originating in Europe and then spreading via trading routes to the Far East”, concluded Ammielle.

Marine-ated DNA

Skeletal remains have limited value in reconstructing complete prehistoric environments so one alternative is to use sedimentary DNA, where free ancient DNA is trapped in lake sediments deposited over millennia in seas and lakes. “Marine environments are like nature’s fridge for preserving DNA”, said Roselyn Ware (University of Warwick, United Kingdom). “Water is densest at 4°C, and thus sinks to form stratification of the marine environment by temperature, meaning that deposited sediments show great thermostability which is ideal for DNA preservation”. However, care has to be taken when dating such samples as Roselyn explained: “Normally, characteristic DNA breakdown patterns, including strand fragmentation and cytosine deamination, are used to authenticate the age of the ancient DNA. But recent work has found that this fragmentation process is not linear: it starts relatively fast then subsequently slows”. Part of Roselyn’s work involves understanding how different abiotic and biotic conditions, such as salinity, affect this breakdown process so that sedimentary DNA can be aged more accurately. In the meantime, “it is imperative that sedimentary DNA work is carried out with parallel evidence from geology to understand the environments they represent.” Peter Heintzman (UiT – The Arctic University of Norway), explained how this can be done: “Along with DNA, lots of small plant and animal fragments are also deposited in the sediments” he said. “These small fragments can be radiocarbon dated, and we can use this information to understand the age of the sediment at each depth.”

Using this approach, Peter has used lake sediment cores to determine when exactly one of the last surviving populations of Woolly Mammoths went extinct. Long after these mighty behemoths disappeared from the mainland 11,500 years ago, small populations remained isolated on tiny islands. The longest known surviving group was that of Wrangel Island in the Arctic Ocean, which endured until 4,000 years ago, coinciding with the arrival of humans. However, another late-surviving population existed on St. Paul Island in the Bering Sea, which, according to dated fossil remains, endured until 6,500 years ago, long before the island was first occupied by humans in the 1700s. As the time of extinction was based on a limited number of fossils, the mammoths could have potentially survived much longer than the available fossil evidence suggests. To find out when exactly they disappeared, Peter’s team used radiocarbon dating to develop a precise age-depth model for a sediment core from the largest and oldest lake on the island. DNA samples were extracted, sequenced and compared against the Woolly Mammoth genome, which was itself only sequenced in 2015. “Our results show that mammoth DNA disappears from the sediment record 5,600 years ago, around a millennium later than the youngest published radiocarbon date from a St. Paul island mammoth fossil.”, he concluded. This result was neatly confirmed by t he marked disappearance of coprophilous (dung-loving) fungi from the sediment at exactly the same time as the mammoth ancient DNA and with radiocarbon dates from newly discovered fossils¹.

Lake Hill, a freshwater lake on St Paul Island that used to be visited by Woolly Mammoths, now yields clues to their disappearance through DNA captured in sediment cores. Photo: Peter Heintzman

A view to the future

Besides solving riddles of the past, DNA in sediment cores could help us predict how the environment will respond to our changing climate, particularly when sourced from areas that have experienced pronounced climatic changes in the past. One such site is Bol’shoy Lyakhovsky, a treeless island in the Arctic Ocean, one of the few areas of the former Beringian landbridge between Eurasia and the Americas that is not covered by the sea today. “Climate models project an annual average temperature increase of about 3–5°C in the terrestrial Arctic until the year 2100, which corresponds to the reconstructed temperatures of the last interglacial period (130–110 kyr BP)”, said Heike Zimmerman (Alfred-Wegener-Institute for Polar and Marine Research, Germany). “Our goal was to reconstruct the interglacial vegetation on Bol’shoy Lyakhovsky to provide an analogue for the future”. Heike extracted DNA from four permafrost sediment cores and performed PCR to select sequences containing a universal plant plastid marker.“The results showed that during the last interglacial period, this region supported evergreen spruce, deciduous larch and poplar trees”, said Heike. She explained how the movement of trees northwards could result in a positive feedback cycle: “In treeless tundra, the wind flows freely, leading to very shallow snow cover. However, trees and shrubs act as a barrier, allowing snow to accumulate on the ground.” A thicker snow layer insulates the soil, so that it does not freeze so much in winter. This means that during the summer months, warmer temperatures can penetrate further into the soil. “If this happened, then organic matter frozen within the deeper layers of the soil may begin to thaw and be decomposed by microorganisms. This would release methane and carbon dioxide into the atmosphere, accelerating climate warming” said Heike.

Bolshoy Lyakhovsky Island – formerly part of the Beringia land bridge between Europe and North America. Photo: Sebastian Wetterich

On the move

Like most sediment studies so far, Heike used DNA metabarcoding amplification, which identifies specific species of interest from short, standardised sections of the genome. But a more recent alternative called shotgun sequencing could allow researchers to capture the full range of genomic data from their samples as Mikkel Pedersen (University of Cambridge, United Kingdom) explained:“In shotgun sequencing you do not amplify up and only analyse specific genes, but the full pool of DNA is completely and blindly sequenced. It is more unbiased as you have no a priori assumption about which organisms should be there, so it can reveal the whole diversity of taxonomic groups present”. Mikkel has already used this pioneering approach to address a highly debated scientific question: the route by which early humans entered America from Beringia (modern Siberia). It has long been thought that humans migrated along a 1,500 km long ice-free corridor formed by retreating ice sheets in Western Canada. But for this to have been possible, the landscape must have supported enough plant and animal life to sustain a roving human population.

To assess this, Mikkel used shotgun sequencing on lake sediment cores which measured back to the glacial retreat. “The data surprised us as it captured not only organisms living within the lake but also terrestrial species from plants to small insects and even Woolly Mammoths”, he said. Crucially, the results indicated that plants and animals only began to appear in the region 13,000 years ago. “It was only 400 years later that the tundra was properly established, including sagebrush, bison and mammoths, an environment that could have sustained human migration”, said Mikkel. However, recent archaeological evidence indicates that humans were present in the South Americas as early as 14,000 years ago. “This route therefore could not have been used by the first humans coming to America south of the retreating ice. They must have used an alternative route, probably along the Pacific Coast”, concluded Mikkel².

A burning issue

Yet even the most sophisticated techniques are limited by the quality of the starting material. For those researching the development of prehistoric agriculture, this remains a problem as Terry Brown (University of Manchester, United Kingdom) explained: “In arid environments, plant remains such as seeds can be preserved by desiccation, however virtually all ancient crop remains in Europe are charred material, having been preserved by burning, for example the accidental combustion of a grain store”. Up to now, such charred samples have been unsuccessful in sequencing studies, with most reads being identified as bacterial in origin, and assumed to be contaminants. But Terry’s work suggests this may not be the case: “We really don’t understand the charring process so we performed simple ‘cooking experiments’ by heating barley seeds in an oven at over 200 °C for up to 5 hours. Over time, the barley sequences started to fragment and accumulate miscoding lesions”. Given that genomic databases contain thousands of bacterial sequences, these errors can cause the barley sequences to be mis-assigned as contaminants. “A better understanding of these degradation processes could enable palaeogenomic material to be obtained from charred material, which could be of real interest to modern plant breeders” said Terry. “The spread of agriculture can be thought of as ‘enforced climate change’ for crops. When plants were moved from the Fertile Crescent in South West Asia to Northern Europe, they had to adapt to a colder, wetter climate with a longer growing season”. Understanding the changes which facilitated this, including changes in both coding sequences and gene expression levels, could help to breed crops with resilience to modern climate change. “If we could follow the genetic changes in charred grain samples in real time from different ages and parts of Europe, we could go a long way towards understanding the breeding and genetic engineering that needs to be done with modern crops” said Terry.  

As techniques for extracting and sequencing ancient DNA improve, even more riddles of the past may become open for investigation. Besides satisfying our curiosity about mass migrations and prehistoric beasts, this could be invaluable towards helping us prepare for the future. As the old English proverb says: “You don’t know where you’re going until you know where you’ve been”.

1. Graham, Russell W., et al. “Timing and causes of mid-Holocene mammoth extinction on St. Paul Island, Alaska.” PNAS 113.33 (2016): 9310–9314.
2. Pedersen, Mikkel W., et al. “Postglacial viability and colonization in North America’s ice-free corridor.” Nature 537.7618 (2016): 45–49.