On TB seals and an early peopling of America

My post of August 7, 2014 on Tuberculosis and the early peopling of America, was in a certain way, prophetic.

In that post, I suggested that there was a "unique" TB strain carried by Native Americans, an it arose because it was taken to the New World by Homo erectus or Neanderthals. My post wrapped up with the following remarks:

"The sequencing of the genome of the native American strain of TB will surely show that it does not share its root with the European clade. however the Amerindian strain is rare and surely overlooked in the samplings that have been carried out. Perhaps recovery of M. tuberculosis from ancient remains may provide evidence of its singular origin. We will have to wait for additional studies to prove or discard the possibility that Homo erectus or Neanderthals reached America with the ancient TB bacteria which evolved there into the Amerindian strain and back-migrated to Asia to continue evolving there."

My conjecture was confirmed by a paper published less than two weeks later (Kirsten I. Bos, et al., Aug. 20, 2014) [1] which reports that a group of scientists did just that, they sequenced the TB genome from the remains of three ancient Peruvian mummies that were over 1,000 years old, and found that the American Natives carried a very unique strain of tuberculosis, completely different to all of the Old World strains.

Of course, being mainstream scientists, they interpreted the results within the constraints of orthodoxy, and came up with some rather odd conclusions and even stranger dates. Today's post looks into this paper and its implications on the possibility of an early peopling of America by Homo erectus or Neanderthals.

Some background on Tuberculosis

Until recently, tuberculosis was believed to have been introduced to America by the Europeans after Columbus discovered the New World in 1492. The heavy death toll caused among the natives by discovery and conquest was mainly due to the devastating effects of disease (including TB).

However, evidence of Pre-Columbian tuberculosis among the natives of the New World has been suggested in several papers. A hypothesis which has been finally confirmed by this recent study [1] which sequenced DNA from Tuberculosis bacterium taken from the remains of Peruvian mummies that predate the arrival of the Europeans to America by some 500 years.

Of course, the current TB strains found in the New World are of a recent origin and were brought to America by the European influx post-1492. This European strain proved more virulent to the local natives and contibuted to their death toll, it also replaced virtually 100% of the Pre-Columbian lineages.

The Recent paper on Amerindian TB

The team which authored this paper [1] found that the Mycobacterium tuberculosis bacteria detected in the ancient Amerindian remains was not of the typical European strain, furthermore it was different to the current African and Asians strains. It is in fact very different to all other human TB clades.

The following image whcin I adapted from [1], displays the phylogenetic tree of various TB bacterial strains (both human and animal):

I added the geographical distribution of the strains [3] which in the original paper [1] are not mentioned. I did so because it provides an interesting perspective to the tree:

• There are two distinct West African lineages L5 and L6, known as M. africanum (Shown in brown and green, respectively).
• L1 (violet) is the strain that is predominant in South East Asia, central and southern India, and the Indian Ocean rim areas.
• L7 (yellow) is found exclusively in the Horn of Africa (Somalia, Eritrea, Ethiopia).
• L4 (red) is the Europan strain also found in America, where it was taken by European migrants after the discovery of the New World.
• L2 (blue) is the East Asian "Beijing clade".
• L3 (purple) is the Central Asian and northern India or "Delhi" strain.

The animal strains are shown in black. And the novel Peruvian "archaic Amerindian" strains in orange, on the right side of the tree.

The novelty here is the location of the Peruvian lineage, close to th TB strain found in seals, and apparently rising from the "animal" TB branches.

Animals and humans

This proximity between animal and human strains is not new, all mycobacterium lineages are classed together as the MTBC (Mycobacterium tuberculosis complex). Boritsch et al. (2014) [3] have also pointed it out: the West African strains are very close to the strains found in wild chimpanzees, to M. microti (detected in voles in the 1930s), M. pinnipedii (seals and sea lions) as well as the other strains found in cows (M. bovis), goats (M. caprae) and oryx (M. orygis). [3]

Taking a closer look at the image above, you will notice two main clusters or "clades":

1. One composed by the animal strains plus the West African human L5 and L6 strains (and also as the new paper shows, the novel Peruvian strain [1]). They are found on the upper part of the image.
2. The other clade comprises all the remaining human strains in Africa, Eurasia as well as the "recent" American strain of European origin (post-1492). They are found on the bottom part of the image.

This split between these two clades is defined by the "RD9-deletion" which marks the branching point between both lineages of MTBC: (i) The African-Animal (which now includes Ancient Amerindian) and (ii)the other Old World "human" strains which lack this deletion. A peculiar variety of Mycobacterium, the ancestral M. canettii marks the splitting point.

It seems that the ancestor of MTBC evolved into two separate groups, one with the RD9 deletion and the other strains, which we may call M. tuberculosis senso stricto without this deletion. This split took place during the early period of MTBC evolution (below we will see how long ago this split took place).

There is yet another split between the MTBC lines: on one side we have the African-animal-Peruvian plus L1 and L7 (East African and Indian Ocean Rim) clades while on the other we have the rest of the Eurasian strains. The split is caused by the presence or absence of a marker (Tuberculosis Deleted Region 1) or "TbD1".

This split can be seen above and also in another paper (W. Hildebrand, et al., 2008) [4], which studies the expansion of TB "Out of Africa" (O.o.A) and from which I took the following image.

The image below shows a phylogenetic tree split into two sections: on the bottom part is the "ancestral" clade or TbD1-intact (yellow) and on the top is the "modern" or TbD1-deleted strains (red).

EAI stands for East Africa and Indian Ocean Rim

These mutatons show that the "West African - animal - Peruvian strains" are the oldest, splitting earliest from the other MTBC clades. Then come the Horn of Africa and Indian Ocean Rim variants which together with the African - animal - Peruvian strains form yet another group, and finally the other younger clades found in Eastern and Central Asia, and Europe.

Both images are anchored in an ancestral group (M. canettii or M. prototuberculosis):

The ancestral TB strain - M. canettii

There are Mycobacterium strains that live in the environment, like other microbes, in the soil or in the oceans (M. marinum or M. ulcerans), and the MTBC which live inside the cells of mammals. How did they manage to jump from one to the other? It seems that another variety of Mycobacterium, known as M. canettii is the link between environmental and human mycobacteria.

The M. canettii strain was reported in 1970 by G. Canetti in Djibouti, in the Horn of Africa. It is a peculiar strain with unusual smooth features. It is believed that it "might represent a pool of strains from which the last common ancestor of the MTBC has emerged" [3]. M. canettii and the MTBC both arose from an archaic progenitor: "M. prototuberculosis". [3]

The M. canettii genome is 20% larger than that of MTBC (+900 genes), so it shows that MTBC shed genes as it specialized in its mammal hosts.

But when did TB appear?

Dating TB

As can be expected due to the molecular clocks and dating techniques used by scientists, the published dates for the origin of TB diverge greatly: from 15 to 70 kya. [3] Furthermore, since all papers assume that TB left Africa with the "O.o.A" event with modern H. sapiens some 40 - 50 kya, this assumption constrains the dating of the different TB strains to a rather "recent" date.

But, what if it left Africa inside infected Homo erectus or the ancestors of Neanderthals, or even earlier? The ancestral home of our predecessors is in the Horn of Africa, precisely where M. canettii is currently found.

The hypothesis of an East African origin is accepted by Boritsch et al., [3] (but the dispersal vector used in the paper is H. sapiens not Neanderthal or H. erectus). Furthermore, the spread from humans to animals is also believed to have taken place in Africa, where our ancestors managed to pass TB on to chimps, dassies, oryx, cows, voles and seals: "...the animal-associated strain lineages of the MTBC seem to have evolved from RD9-deleted M. africanum-like ancestor strains that may well have been adapted to humans already".[3]

I agree with that statement, I firmly believe that these RD9-deleted M. africanum-like strains adapted early to humans, so early that we were not yet H. sapiens, but probably H. erectus or the predecessors of Neanderthals.

The trees depicted further up show very close links between the roots of the animal and West African human TB branches.

But, there is an interesting peculiarity: the oldest African strains are found in West Africa and not in East Africa the cradle of mankind. And this has to be accounted for. Below I outline a hypothesis:

The origin of TB in Africa

Let's assume that M. canettii evolved into two strains the progenitors of modern MTBC, one with the RD9-deletion which infected a "hominin". This hominin lived in Africa yet, part of that population moved out of Africa and, via Asia, reached America, where it originated the "Peruvian strain" with the RD9-deletion. Those that remained in Africa infected animal species with TB.

This explains why Peruvians, West Africans and animals share the RD9-deletion lineages. Perhaps a know extinct Asian population also had this strain.

The second strain of MTBC was not RD9-deleted, and it infected other "hominins" these lived in East Africa from where they split into a group that moved out along the coast of the Indian Ocean. This migration may represent either the Homo erectus (1.8 Mya) or the H. sapiens (100 kya) moves Out of Africa.

Finally the TbD1 deletion appeared outside of Africa and the people carrying it colonzed Europe, Central Asia and Eastern Asia, some also back-migrated to Africa. Did it appear in Neanderthals or modern H. sapiens?

The ancient African "hominin" RD9-deleted variant survived among a relict archaic population in Western Africa, long after its relatives became extinct, and only recently admixed into Modern humans in West Africa. We have already posted on the possibility of archaic admixture as the source of the extremely ancient A00 Y chromosome haplogroup, which may have originated from the introgression of "an archaic form into the ancestors of AMHs...", remains combining "both archaic and modern features" were found at Iwo Eleru in Nigeria and are quite recent: around 13 kya.

The issue is who do we assign to each migratory event.

RD9-deleted hominin could well be Homo habilis which left Africa (H. Georgicus in the Caucasus). Did they reach America? If so, the Peruvian variant could have been carried by them. The RD9-intact group could be H. erectus who remained in Africa and also peopled the coasts of the Indian Ocean. Modern H. sapiens later mutated (TbD1 deleted) and dispersed the other strains around the globe. They could also be the ancestors of Neanderthals or even modern H. sapiens.

The dating as per orthodox science is based on an average mutation rate of roughly 0.5 substitutiones &frasl genome per year. But, the issue is that TB is caused by a clonal bacteria with a very small amount of SNPs (clonal microbes tend to keep stable since they are specialized to infect specific hosts and have discarded unnecessary genes). So, can we be sure that the model used to calculate its age is really valid?

On the other hand, M. canettii has a larger genome than MTBC and around 25 times more SNPs than the MTBC lineages. So if we assign 15 - 70 ky to MTBC, does this mean that the archaic M. canettii 25 times older? That is, 125 to 1,750 ky old? [3]

It is not such a far fetched notion, in fact Gutierrez et al., 2005 [5] suggested a 3 million year date for TB.

So, we have a very wide range of dates from around 15 kya to 3 Mya. So there is the chance that an ancient hominin carried the Peruvian mutation.

Having said this, let's the conclusions of the authors of the paper on pre-Columbian TB in Peruvian mummies:

Going back to the Peruvian mummies

I mentioned above that the paper has some odd findings:

(1). "Two independent dating approaches suggest a most recent common ancestor for the M. tuberculosis complex less than 6,000 years ago, which supports a Holocene dispersal of the disease" [1]. In other words, TB evolved only 6,000 years ago.

(2). Since by that time Beringia had flooded, they had to find a non-human vector that could take TB from its Source in Africa, to America. They sequenced the TB strains of different animals and found that seal TB shared similar traits with the Amerindian strain. So they concluded that seals were the source of Amerindian TB.

We can see in the images above that seal TB is one of the animal groups closest to human L5 and L6 strains. The gene sequencing conducted by Bos et al., [1] showed that the TB strains present in the Peruvian skeletons was very similar to strains of TB that are found in modern seals and sea lions.

The researchers concluded that seals somehow picked up TB from African humans and then carried it across the ocean to the New World where it spread among the native seal-hunters. [1]

I find the 6 kya date for the origin of TB too recent, and the theory of an "African to seal to Amerindian" infection route too complicated. Allow me to explain:

TB in seals

Tuberculosis in seals was studied in 2003 (D. Cousins et al.) [2], they compared genes from six species of seals taken in the UK, New Zealand, Argentina, Australia and Uruguay. They found that pinnipeds share their own strain of MTBC (Mycobacterium pinnipedii) which can also infect other creatures: "guinea pigs, rabbits, humans, Brazilian tapir (Tapirus terrestris) and, possibly, cattle" [2].

How does it spread in nature? "As with other members of the M. tuberculosis complex, aerosols are the most likely route of transmission" [2]. Seal TB is quite contagious, in fact, 6 out of 25 animal keepers at a zoo in The Netherlands became infected with M. pinnipedii when 13 out of 29 sea lions contracted the disease. [6] so no eating was necessary, just close contact with living seals was enough to pass on TB from seal to man.

But, does it work the other way round? Did Africans infect seals? or did they eat a dead Dassie or vole and became infected?

Note that African L5 and L6 strains are similar but not identical to seal TB. So it would have had to mutate in Seals and then jump again to infect Amerindians and mutate again... By the way, why didn't seals infect other seal hunting populations around the world? Only Amerindians?

Now, the interesting point is that "There seems to be a common ancestral source of M. pinnipedii across geographic locations, which raises questions about the original distribution of the seal bacillus in animals of different continents and the potential role of marine mammals in the spread and transmission of tubercle bacilli across oceans". [3]

Bos et al., assume that Africans infected seals with TB. The microbe then mutated among seals and led to a seal-specific lineage which they spread across the globe among seals and sea lions. It later infected some Peruvian seal hunters, and mutated into the Peruvian TB variety which spread among Amerindians. The image below shows this process:

An equally valid alternative is that, as outlined above, some archaic hominin took TB to America and the seals became infected in the New World, spreading the disease among seals around the globe. The Old World variants evolved separately for a very long period of time, and this explains why Amerindian and Old World TB lineages among humans are so different:

Taking a look at the trees above, it is clear that Seal TB branched from Peruvian TB and not the other way around. Actually the scarcity of mummy samples may have rooted Peruvians after voles instead of rooting it on the main branch, where all animal strains converge with African human ones. Perhaps futuer samples will modify the branching sequence.

Why would Amerindians, of all humans be the only ones to have evolved their strain of TB from animals. Are Peruvians the only seal hunters in the world? or the only ones that got infected?

Then there is the issue of the Dates. The researchers calculated that all modern MTBC strains date back to only 6,000 years ago.

If so, how did it spread globally so fast? By that time we were well established across the whole world!

So this means that it arrived to America between 1 and 6 kya. So, how do they account for a well known case ot TB in a 17,000 year old bison found in North America?

The bison, dated to 17,870 +⁄-230 BP had "ancient DNA characteristic of the Mycobacterium tuberculosis complex, confirming the oldest proven case of tuberculosis" [7], This bison case indicates that this disease evolved as a zoonosis in America long before the 6 kya limit established by Bos et al. Perhaps bisons (like oryx, goats, voles and seals) caught their TB from pre-Columbian humans which were already living in America 17 kya.

Sources

[1] Kirsten I. Bos, et al., (2014). Pre-Columbian mycobacterial genomes reveal seals as a source of New World human tuberculosis, Nature, doi:10.1038/nature13591, Published online 20 August 2014
[2] Debby V. Cousins, et. al., (2003). Tuberculosis in seals caused by a novel member of the Mycobacterium tuberculosis complex: Mycobacterium pinnipedii sp. nov.. International Journal of Systematic and Evolutionary Microbiology doi: 10.1099/ijs.0.02401-0 IJSEM September 2003 vol. 53 no. 5 1305-1314
[3] Boritsch, E. C., Supply, P., Honoré, N., Seeman, T., Stinear, T. P. and Brosch, R., (2014). A glimpse into the past and predictions for the future: the molecular evolution of the tuberculosis agent. Molecular Microbiology. doi: 10.1111/mmi.12720
[4] Wirth T, Hildebrand F, Allix-Béguec C, Wölbeling F, Kubica T, et al. (2008). Origin, Spread and Demography of the Mycobacterium tuberculosis Complex. PLoS Pathog 4(9): e1000160. doi:10.1371/journal.ppat.1000160
[5] Gutierrez, M.C., Brisse, S., Brosch, R., Fabre, M., et?al., (2005). Ancient origin and gene mosaicism of the progenitor of Mycobacterium tuberculosis. PLoS Pathog 1: e5.
[6] Kiers A, Klarenbeek A, Mendelts B, Van Soolingen D, Koeter G., (2008). Transmission of Mycobacterium pinnipedii to humans in a zoo with marine mammals. Int J Tuberc Lung Dis. 2008 Dec;12(12):1469-73.
[7] Lee OY-C, Wu HHT, Donoghue HD, Spigelman M, Greenblatt CL, et al., (2012) Mycobacterium tuberculosis Complex Lipid Virulence Factors Preserved in the 17,000-Year-Old Skeleton of an Extinct Bison, Bison antiquus. PLoS ONE 7(7): e41923. doi:10.1371/journal.pone.0041923

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