Archives For anthropological genetics

In 1941, an archaeologist named Glenn Black excavated a site called Angel Mounds just east of Evansville Indiana. Angel Mounds (AD1050-1400) belonged to the Mississippian culture, which was found throughout the Midwest and Southeast in the centuries just prior to European contact.

When excavating a region of the site dense in children’s graves, Black uncovered a grave which contained two babies buried together in a very unusual manner: heads facing away from each other, legs intertwined, hands joined:

From Marshall et al. 2011, figure 2, showing the burial position of the two children.  Note that this is not a photograph but rather a sketch image. While it’s important to show the disposition of the skeletal elements in order to illustrate the scientific background, out of respect for descendent communities I (and others) feel it is inappropriate to post actual photographs of human remains without permission.

From Marshall et al. 2011, figure 2, showing the burial position of the two children.
Note that this is not a photograph but rather a sketch image. While it’s important to show the disposition of the skeletal elements in order to illustrate the scientific background, out of respect for descendent communities I (and others) feel it is inappropriate to post actual photographs of human remains without permission.

He interpreted this burial as “flesh-joined” twins, as they didn’t have any fused skeletal elements. Conjoined twinning* occurs when a single fertilized egg splits only partially into two fetuses (as opposed to complete splitting in monozygotic twins). The rate of conjoined twinning in the United States is approximately 1/ 33,000-165,000 births, but the frequency of conjoined twinning in ancient societies is unknown.

The children’s remains, along with those of other people excavated from Angel, were taken to be cared for by the Glenn A. Black Laboratory of Archaeology at Indiana University, Bloomington.

Seventy years later, my colleague Dr. Charla Marshall became interested in the children, and in Black’s hypothesis that they were conjoined twins. With permission of the curators at the Glenn Black laboratory, she undertook a comprehensive analysis of the children.**

Dr. Charla Marshall, doing ancient DNA work. Note the protective clothing designed to minimize risk of contamination from modern DNA.

Dr. Charla Marshall, doing ancient DNA work. Note the protective clothing designed to minimize risk of contamination from modern DNA.

She and her colleagues found that the two children (designated W11A60 and W11A61) were approximately 3 months old, and had evidence for poor health, but otherwise saw no skeletal evidence that could either support or reject the hypothesis that they were conjoined twins.

Fortunately, Dr. Marshall happened to be an expert in the one method that would definitively tell whether the children were twins or not: ancient DNA analysis. Because mitochondrial DNA is maternally inherited, siblings (and twins) MUST have the same mitochondrial sequence.

Both children, despite having been dead for nearly a thousand years, had ancient DNA still preserved. By extracting the DNA and sequencing it, Dr. Marshall was able to determine their mitochondrial lineages (haplogroups). [I give a little bit of background into how ancient DNA research is done here and here].

Surprisingly, they were different! In the table below, you can see the mutated DNA base positions for each child listed in the third column (under ‘haplotype’). The particular combination of mutations for each child means that they belonged to two different haplogroups: A and C.

Marshall et al. 2011, Table 3.

Marshall et al. 2011, Table 3.

Therefore, the “conjoined twins” were neither twins nor siblings, nor maternal relatives of any kind. Black’s 70 year old hypothesis was wrong.

Why were they buried in such a peculiar way? Dr. Marshall and her colleagues (Cook et al., 2012)*** presented a paper last year at the Midwest Archaeological Conference in which they discussed possible interpretations for this burial practice.

Perhaps, they suggest, the children were non-maternal relatives (maybe half-siblings who shared a father?), who died at the same time and were buried together to reflect this close relationship. Or perhaps the arrangement of the babies’ bodies was entirely symbolic.

Twins play a special role in Eastern Native American iconography, and different Native American societies treat twins in different ways; in some cases they are regarded as having special spiritual power, in other ancient societies they were thought to be negative. Perhaps the co-burial of two maternally un-related children of the same age was meant to be symbolic of twinship, rather than having a literal meaning.

In general, co-burial of individuals was a pretty common practice among the ancient Mississippians, and typically archaeologists have interpreted the co-buried individuals as being related to each other. However, those of us doing ancient DNA research in the Midwest have been testing this hypothesis on co-burials and finding that they’re almost never maternally related. Because no ancient Y-chromosome DNA has yet been recovered from Midwestern co-burials, we don’t know if they might be paternally related.

The motivation for Mississippians to bury people together, and these two children at Angel Mounds in particular, continues to be a mystery. However, the approach of Dr. Marshall and colleagues is a very good example of how persistent research can disprove a long-standing, wrong hypothesis. It may be that future generations of students will be able to solve this mystery with additional genetic evidence.
*The more popular term, “Siamese twins”, was introduced by P.T. Barnum to refer to Eng and Chang Bunker (, who were members of his circus. “Siamese twins” has therefore taken on negative connotations associated with this history.

**Marshall C, Tench PA, Cook, DC, Kaestle FA. 2011. Conjoined twins at Angel Mounds? An ancient DNA perspective. American Journal of Physical Anthropology 146: 138-142.

***Della Collins Cook (Indiana U), Charla Marshall (Southern
Illinois U Carbondale), Cheryl Ann Munson (Indiana U), and Frederika A Kaestle (Indiana U). 2012. If Angel Twins Aren’t Twins, What DO They Represent? Paper presented at Midwestern Archaeological Conference, East Lansing Michigan, Oct 17-21, 2012

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Reading and understanding scientific literature can be incredibly frustrating for most people. You may want to understand some cutting-edge finding, but find you can’t wade through the technical jargon and obtuse figures, so you give up and read some crappy summary in the news. This doesn’t mean you’re not smart! I’m want to assure you that this is a learned skill–we actually have to explicitly teach our students how to do it.

I feel very strongly about making science accessible to everyone. One of the ways I’m going to do it here is to walk people through recent and exciting scientific papers. Here’s my first attempt. Please feel free to give me feedback!

Summary of Brotherton et al: Neolithic mitochondrial haplogroup H genomes and the genetic origins of Europeans. 2013. Nature Communications 4:1764.

I talked recently about how you can use genetics to test the idea that cultural changes in the past were the result of migration. A few days ago, this study was published, doing just that. I want to go through their findings, because they’re exciting and important.

Europe has a very complex prehistory, characterized by lots of migrations of different ethnic groups. Understanding this prehistory genetically is a tricky endeavor, requiring the sequencing genetic lineages of both modern and ancient populations in order to try to link them in time and space. Remember how I said that the majority of ancient DNA research targets the mitochondrial (maternal) genome? By comparing the frequency of different groups of closely related lineages (called haplogroups) in different populations, we can see how closely they are related. More distantly related populations will have different proportions of haplogroups. This is pretty intuitive when you think about the story behind the science; women living in these populations were passing down their mitochondrial lineages through their daughters and grand-daughters. When a woman moved into a new place, she would have brought her lineage with her. Populations that shared greater proportions of related women would have similar haplogroup frequencies, and would differ from more distant populations.

In modern European populations the most common haplogroup is H; it comprises something like 40% of the population. In fact, my own mitochondrial genome belongs to H, reflecting my mother’s family’s Celtic origins*. It’s therefore crucial to understand how different lineages of haplogroup H are related to each other, or what their phylogeny is. Think of a phylogeny as being analogous to a family tree, with individual mitochondrial lineages being sisters, cousins, second cousins, etc. differing by the mutations they possess. You need to work out how they’re related to each other in order to start understanding their shared histories.

Now, the phylogeny of ancient haplogroup H lineages was worked out previously, but that was done using only the hypervariable regions of the mitochondrial genome. (Again, see this post for an explanation of what the hypervariable regions are, and why they’re the targets of ancient DNA research). It turns out that there’s a whole bunch of genetic variation in the rest of the genome, and without incorporating it, the phylogeny is inaccurate.

The mitochondrial genome, showing the hypervariable control region

The control region, containing two hypervariable segments, makes up only a small proportion of the mitochondrial genome, but is the most frequent target of ancient DNA research. (Image modified from an original source which I’ve unfortunately lost.)

So we (finally!) get to the paper itself! What Brotherton et al. (the authors**) did was first observe that haplogroup H was much less frequent among ancient populations than in modern Europeans; Early Neolithic (~5450 BC) farmers had only a 19% frequency of H, and the older Mesolithic hunter-gatherers basically didn’t have any H. The authors decided to completely sequence the mitochondrial genomes of a sampling of ancient people who were already (through previous research) known to belong to haplogroup H. By expanding sequencing past the hypervariable regions to get at the entire genome, they would be able to “capture” all of the genetic variation, and create more high-resolution phylogenies. This would lead to a better understanding of how individual maternal lineages within H moved into the region.

They chose to sequence DNA from 37 skeletons that spanned ~ 3,500 years of the European Neolithic period (roughly 5450-1575 BC) in the Mittelelbe-Saale region of Saxony-Anhalt (Germany). Without going into the chemistry details, trust me when I say that this is a technically impressive feat!

So what did they find? I’m going to focus only on one of their main results. I’ve excerpted Figure 1A from their paper to show you:

Modified Figure 1a from Brotherton et al., 2013

Modified Figure 1a from Brotherton et al., 2013

I realize this looks like something created by a demented spider. Bear with me, and I’ll explain.

This picture is a network diagram, showing the phylogenetic relationships of all the lineages they obtained from the ancient individuals. The circles are the individuals themselves, colored to represent the different cultures they come from (see the key at top left). The lines are the mutational steps between them, with longer lines indicating more mutations (and thus greater genetic distances). The mutations are listed alongside the lines. Unfilled circles are lineages which aren’t actually present at the sites, but are known about from other places. For fun, I’ve indicated with a purple arrow where I fit in on this network. (Have you ever had your mitochondrial DNA sequenced by one of the commercial genome services? If you belong to haplogroup H, see if you can find yourself on this network, too!)

How do the authors interpret this phylogeny? First, look at the position of the red circles. These are the oldest samples in the study, dating to 5450-4775 BC. Do you see how they’re on shorter lines, closer to the central node? That means they have fewer mutations away from the “basal” H type, and are therefore the oldest lineages! (Remember that lineages accumulate mutations over time, so younger, “more derived” lineages are going to have more mutations). And indeed, we see that the youngest lineages (the ones with the most mutations) tend to correspond to the more recent archaeological sites. It’s a cool pattern, that reinforces the validity of this approach.

This also shows something more subtle, but very important. We’re looking at genetic lineages present throughout time within a single region, remember? So…if that region was continuously occupied by the same group of people and their descendents, we would expect to find the oldest lineages on the same branches as the later lineages. Specifically, we’d expect to see the Early Neolithic individuals (red, orange, yellow, green) to be on the same lines (but closer to the central H node) as the Late Neolithic (light blue and blue), and the Bronze and Iron Age (brown and black) individuals. Instead, they’re all on different lines. This means they’re distinct lineages (not-very-closely-related female ancestors).

And this means that, most likely, there was considerable migration of women (and probably men, though we can’t tell from these data) into Central Europe over time, beginning around 4000 BC. The authors suggest (for various reasons which I won’t get into here) that they were likely immigrants from the West, who interacted with the early Neolithic farmers, and ultimately “superseded” their genetic diversity to shape the patterns of genetic diversity seen in present-day Europeans (including myself!). How cool is that?

Does this explanation make sense? Do you have any questions? Let me know in the comments!

*Specifically, H 5
** We have a convention for referring to a study as “So-and-so et al.” that recognizes the first author (who did most of the work). “Et al.” is short for “Et alii” which means “and the others”. It’s a cool/ pretentious bit of science tradition that reflects the discipline’s historic usage of Latin.