Straight from the Neandertal's mouth

The dental plaque we so assiduously scrub off our teeth when we brush every night before bed has proved to be a goldmine for scientists that study microbes. Plaque is a thin sticky film made from proteins and microbes that forms on our teeth. Over time, it hardens to form tartar, also known as dental calculus. This is actually a combination of different forms of calcium phosphate minerals (the same stuff as our actual teeth are made from), which have different ratios of calcium (Ca), phosphate (PO₄) and water (H₂O or sometimes in the form of hydroxide, OH). Some of the minerals can also have small amounts of magnesium (Mg) or iron (Fe) incorporated into their crystal structure. As these minerals form, tiny amounts of microbial matter can be trapped inside and are preserved, for thousands, even up to millions, of years within the mineral casing.

By extracting DNA preserved within the calculus, scientists can determine the species of microbes present in the mouth of a Neandertal, and investigate what they can tell us about the diets and even the lifestyles of our closest extinct relatives. Tiny amounts of food, like proteins and starches, can also become trapped and preserved within dental calculus, providing some information about a Neandertal’s food preferences.

It is possible to find entire bacterial organisms preserved within dental calculus—scanning electron microscope images show many different types of bacteria. But a full identification of the full range of microbes present in dental calculus requires DNA extraction and sequencing.

• But how do you pull DNA from dental calculus?... you ask

  First, you need to collect your dental calculus sample. Calculus forms on top of the tooth enamel, and you have to pry or scrape it off. It can require a reasonable amount of force, so you have to take care to catch all the bits that may fly off if the calculus shatters into small pieces.

  Second, you have to decontaminate the sample, and remove any traces of modern DNA that might have found their way onto the surface sample. One larger fragment has a smaller overall surface area than several smaller fragments, so this is the optimal choice for analysis. The sample should be irradiated, then immersed in bleach, then rinsed in ethanol. All instruments should be washed in bleach and ethanol as well.

  Third, you pulverise the sample. Basically, this means hitting it with a hammer. But carefully! This is done with the sample inside a bag so all the pulverised bits don’t get lost.

  Fourth, it’s time for the DNA extraction. You need to decalcify the sample using a mixture of chemicals (ETDA, sodium dodecylsulfate and proteinase K) and then there are a few methods of actually extracting the DNA. Controls must be run by analysing empty sample tubes processed alongside the calculus samples to check for any potential contaminant material within the laboratory environment.

  Fifth and finally, you sequence the extracted DNA. Again, there are a few different ways of doing this. One is targeted amplification, a quick and cheap method that amplifies targeted sections of bacterial DNA sequences. This method is used to test for the presence of certain species, identify single species or simply check the quality of the DNA preservation of the sample. Another method that enables the identification of several species from a single sample is called amplicon sequencing. This method is particularly useful for analysing entire microbiome communities, but because ancient DNA is so fragmented and damaged it can be difficult to reliably amplify it.

  The method of choice in most labs is shotgun sequencing. This method targets virtually all types of DNA sequences present in a sample. So, as well as microbes, it can tell you about the DNA of any plants, animals, virus or fungi that might be there. This method is expensive and the data analysis can be complicated, but it gives you a broad overview  of all the material present in the sample.

  
  From Weyrich et al. 2015 Ancient DNA analysis of dental calculus Journal of Human Evolution 79, 119–124

Identification of the microbes present in Neandertal dental calculus from samples found in the Spy cave in Belgium revealed that these guys were meat-lovers, dining on woolly rhinoceros and wild sheep.

The same study also looked at calculus from two Neandertals from El Sidrón cave in Spain. They found that one chap was eating mainly mushrooms, pine nuts, moss and even tree bark.

The researchers also obtained a glimpse into the health issues that affected Neandertals 50,000 years ago. One of the Spanish Neandertals had suffered from a dental abscess, which was evident in his skeletal material. Also present in this individual’s dental calculus was the DNA of Enterocytozoon bieneusi, which is a rather unpleasant gastro parasite.

The DNA of poplar bark was also found in the same sample. Poplar bark contains salicylic acid—the basic ingredient of aspirin. Also found were traces of the Penicillium mould, which likely came from eating mouldy plant material. These ‘medicinal’ items were not found in the other sample, leading to the conjecture that this particular Neandertal was attempting to treat his health issues with pain-killers and potentially with antibiotic products.

Another aspect of the microbiome is the presence (and absence) of pathogens—disease-causing microbes—in other ancient (non-Neandertal) dental calculus. Studies have reported that microbes known to cause tooth decay that leads to cavities were much less common in the dental calculus of humans that lived around five to seven thousand years ago. The growth of these microbes is supported by sugars, so their absence indicates a Neolithic diet low in easily fermented sugars. An increase in these microbes has been found to coincide with the establishment of agriculture.

Ancient poo

Another way to study the ancient microbiome is using coprolites—the official (and polite) term for fossilised poo. Poo is chock-full of microbes—each milligram of it contains more than a million bacterial cells. This means it decomposes quickly, and it’s rare to find well preserved coprolites except in very dry, or very cold conditions. Dinosaur coprolites have been dated back to Cretaceous period (146–66 million years ago). The oldest known fossilised poo (from an animal) that’s been found so far goes back to the Paleozoic Era—around 270 million years ago.

Human coprolites are very rare, and tend to only be preserved in either very dry or frozen environments, however samples have been found that date back to the Late Paleolithic—around 22,000 years ago. Most human coprolite samples are found within latrine deposits, and so unlike with dental calculus, the microbes found in these most likely represent the microbiome of an entire community, rather than a single individual. They also tend to be quite degraded, more reminiscent of compost than fresh fecal material.

Provided they have been well-preserved, and are unaffected by contamination from soil microbes, microbial communities found in coprolites can provide information regarding ancient diets, and also the gut microbiome of whoever produced the coprolite.

One prominent study of fossilised poo describes the microbiome of coprolites found in three distinct environments, from southern USA, northern Chile and northern Mexico, and compared the findings with modern microbiome data. They found that the ancient microbiomes were a closer match to modern rural communities than to the microbiomes of people from urban environments. 

A study of coprolites found on an island of Puerto Rico from people that lived between 5 and 1170 AD found distinct communities of parasite loads, one associated with maize and fungi (Basidiomycetes), the other associated with fish species, suggesting the coprolites came from two distinct communities with very different diets.  

As with the dental calculus, coprolites can also tell interesting stories from (slightly) more modern times. Excavations for an urban development in the Belgian town of Namur discovered latrines from the Middle Ages, complete with barrels of human waste that had been sitting around for nearly 700 years. Samples from these barrels of poo showed evidence of bacteriophages. These are microbes capable of transferring genes from one type of bacteria to another, and are responsible for a number of metabolic functions, and also the development of antibiotic resistance in some bugs. Their presence in the coprolites, and the increased diversity of the genes they carried, compared to those found in modern humans’ guts suggests the natural inbuilt defences of our gut bacteria may have weakened in modern times, perhaps as a result of modern sanitation and hygiene practices.

And it’s not just microbes that can be found in coprolites. Fossilised bits of plants, animals and fish remains, both digested and undigested, can also be found in coprolites. A study of Neandertal coprolite found in the El Salt site in southern Spain found traces of chemicals that indicated Neandertals ate not only meat, but also significant amounts of plants.

Studying ancient remains provides insights into past diets and the evolution of the human microbiome—it’s amazing what scientists can decipher from the clues inadvertently left behind in our ancient relatives’ bodily gunks and waste.