Alentoft 2015: more ancient DNA from beyond the Rhine

I've got some "friendly spam" insisting that I write something on this paper:

Morten E. Alentoft et al., Population genomics of Bronze Age Eurasia. Nature 2015. Pay per view (supp. materials are freely accessible though) → LINK [doi:10.1038/nature14507]

On the positive side, I think the study is much better quality than Haak's, with much more extensive and good quality supplementary materials, many of which are interesting. On the negative side, it totally lacks of any Western European samples, excepted the already known Epipaleolithic ones, being therefore pretty much useless to the understanding of the formation of the modern European genetic pool, except in a negative sense (stuff that is still clearly missing with the Central and Eastern references we have).

This huge blank in Western Europe, who nobody seems willing to fill up, would be quite apparent in the locator map except that it is poorly dissimulated by cutting a good chunk of Europe out of it:

from fig.1

What do we get in this paper then? Lots of info about Central Europe, notably the Carpatho-Danubian region, also about the Greater Caucasus, Siberia and historical Denmark. Add to that a couple of novel sequences from Bronze Age Montenegro and one from Estonia. 

Lack-tase

It's interesting in its own way but I'm still demanding Western European ancient DNA. In fact this study (piling up with others) clearly evidences that the Western European, particularly Atlantic aDNA must be key to the understanding of the formation of European peoples. For example:

Something is amiss, right? Well, that is the LCT allele. Bell Beaker Blogger spotted it very well but he could not explain it. So I told him: There are no samples from Western Europe. That's why! 

Later I added:

Actually Chalcolithic Basques from the Ebro banks already had more rs4988235-T than any of the samples shown here: 27% overall with as much as 31% in San Juan Ante Porta Latinam.

Notice anyhow that T allele frequency is not equal to "lactase persistance" because, on this allele alone, it may be distributed unequally through individuals (and that's precisely what we see in the Chalcolithic Basque samples, suggesting two different populations). Also there may be other alleles producing the same effect, just that they are not that famous or even known at all.

I must say that the recent finding of similar mtDNA pools to those found in ancient Basques and resembling modern ones in the Seine basin, does reinforce the notion of Western Europe being key, not just for this allele but in general for the conformation of modern genetic pools in much of Europe. 

It would seem (Mälstrom 2007) that the Gokhem people (Megalithic SW Swedes) also had some notable LCT frequencies, which are missing everywhere else (in Central-East and Mediterranean Europe, that is) before modernity. Hence the European LCT allele must have expanded from the Atlantic.

Y-DNA

It's not the only this particular trait, mind you. For example, another detail that Bell Beaker Blogger spotted in this paper's data, is that the first known modern S116-derived is in a Bell Beaker individual. Not in Yamnaya, not in Corded Ware, not in Unetice... but in the Bell Beaker carriers that (culturally at least) came from the Southwest.

Specifically it is R1b-U152 (alias S28), which has sometimes been dubbed the "Celtic" subclade because of its distribution across the Alps, being particularly important in Switzerland and North Italy (including non-Celtic regions like Tuscany, Piamonte and Liguria, as well as Corsica). It has also some notable presence in much of France, SW Germany and Belgium. 

I already argued back in 2010 that R1b-S116 must have expanded from Southwestern Europe, possibly Southern France. No need to extend myself on this matter because there has been nothing new in all these five years (sadly enough). Just attach some old maps here for your convenience:

Relative prevalence of R1b-M269 subclades
note: M529 is wrongly listed as M259

Frequencies of the main European R1b subclades: S116 (red) and U106 (blue)

ADMIXTURE

Hidden deep in the supplemental materials there is sup. fig. 6, which is an ADMIXTURE analysis of ancient and modern sequences. I simplified it by removing modern Asian and African samples as well as low K scores, all them pretty much irrelevant, adding more clear labels and rotating it:

The most interesting aspect is that this analysis pretty much gets rid of the exaggerated Yamna influence attributed by Haak, bringing it down to much more acceptable levels. Even Corded Ware peoples were, it seems now, only weakly related to Yamnaya and much more strongly related to Paleo-European hunter-gatherers (tan) and Mediterranean early farmers (yellow). Some of the Caucasus (teal) component was anyhow already present in pre-Indoeuropean farmers from Hungary and it is only the minor Siberian element (blue shades) what really marks the distinctiveness of Indoeuropeans. 

Notice that in this analysis, early European farmers like Stuttgart are not single color but dual: they appear as almost exactly a 50-50 mixture of Paleo-Europeans (tan) and Mediterranean farmers (yellow). The only (almost) true yellow reference are Naqab Bedouins, pointing again to a PPNA origin of the migrant farmers, who, after admixture with European aborigines, surely in Thessaly, spread the Neolithic through the subcontinent. 

When, at K=19, the Naqab Bedouins form their own distinctive component (pale yellow) the yellow component suddenly expands in all other samples at the expense of the rest. It changes meaning at that point, beware: it has become the Sardinian component, although one can well argue that this component is very much dominant among early European farmers (but always mixed with some Paleo-European, some Caucasus, some Bedouin even), there is not any single sample that is clearly dominated by it at >80% frequencies (visual estimate), only Sardinians score that high. Admittedly it'd be interesting to re-run this without Sardinians and see what happens. 

From the Basque origins viewpoint, I find notable that (again) the Basque and Swedish farmer sequences are similar all along. The latter have more yellow component however, implying that Basques are even more Paleo-European than they were. 

In contrast, in extended data fig. 6, Basques appear as less related to WHG than our immediate neighbors (Spanish and French), what is probably an artifact of Indo-European admixture in the latter, as Indoeuropeans were no doubt largely Paleo-European and the Siberian elements they carried may also weight in that same direction. This is also visible in the ADMIXTURE graph above: French clearly carry some more tan Paleo-European, in addition to the Caucasian teal (and in some K-values also minor Siberian blue shades).

In general we see an apparent increase of the Paleo-European component as we move away from the Neolithic but while in the French case this can be partly attributed to Indoeuropean flows from, ultimately, Eastern Europe, in the Basque case that is not the case. There must be another source of that excess Paleo-European element and that source must necessarily in Atlantic Europe. 

Time to do proper research, time to sample the ancient Far West.

Lactase persistence genetics in Africa

This month we get to know a bit more about the ability of humans to digest milk sugars (lactose) in adulthood, with particular emphasis on Africa:

Alessia Ranciaro et al., Genetic Origins of Lactase Persistence and the Spread of Pastoralism in Africa. AJHG 2014. Pay per view (free access after 6-month embargo) → LINK [doi:10.1016/j.ajhg.2014.02.009]

Abstract

In humans, the ability to digest lactose, the sugar in milk, declines after weaning because of decreasing levels of the enzyme lactase-phlorizin hydrolase, encoded by LCT. However, some individuals maintain high enzyme amounts and are able to digest lactose into adulthood (i.e., they have the lactase-persistence [LP] trait). It is thought that selection has played a major role in maintaining this genetically determined phenotypic trait in different human populations that practice pastoralism. To identify variants associated with the LP trait and to study its evolutionary history in Africa, we sequenced MCM6 introns 9 and 13 and ∼2 kb of the LCT promoter region in 819 individuals from 63 African populations and in 154 non-Africans from nine populations. We also genotyped four microsatellites in an ∼198 kb region in a subset of 252 individuals to reconstruct the origin and spread of LP-associated variants in Africa. Additionally, we examined the association between LP and genetic variability at candidate regulatory regions in 513 individuals from eastern Africa. Our analyses confirmed the association between the LP trait and three common variants in intron 13 (C-14010, G-13907, and G-13915). Furthermore, we identified two additional LP-associated SNPs in intron 13 and the promoter region (G-12962 and T-956, respectively). Using neutrality tests based on the allele frequency spectrum and long-range linkage disequilibrium, we detected strong signatures of recent positive selection in eastern African populations and the Fulani from central Africa. In addition, haplotype analysis supported an eastern African origin of the C-14010 LP-associated mutation in southern Africa.

The study detects in essence four alleles that explain lactase persistence in Africa: one (Fulani and other Central Africans) is the same as in Europe, another (Afroasiatics and some Nilotics from NE Africa) is related to Arabia but at least two other variants are African-specific:

Figure 3. Contour Maps of Africa Show the Allele Frequency Distribution for the Four Primary SNPs Associated with the LP Trait in the Current Study

 

from Fig. 4 (haplotype network)

Interestingly, three of the alleles are related in the haplotype network (to the right), suggesting a common remote origin, maybe in the shared Neolithic origins of West Asia. The fact that they mostly affect peoples of Afroasiatic language (and to lesser extent some other groups of the Sahel) rather suggests a common origin in either West Asia or the Nile (which most likely had some sort of impact in the Western Neolithic, judging from the distribution of the lineage E1b1b1 in the present as in the past and the minor African affinity of early European farmers).

Instead the C-14010 allele, present in East Africa around Lake Victoria, and to some extent also in SW Africa, seems very much unrelated.

Alleles and their variants 

The European T-13910 allele as three main haplotype variants in Africa: 

• The Mozabite one is related to West Asia
• The main Fulani and Bulala one is related to Europe
• A third variant includes only Fulani and Arabic Baggara (strictly African therefore)

Judging on the haplotype tree to the right, the Mozabite/West Asian variant probably derives from Europe but the case for the Central African ones is much less clear.

The authors estimate the allele to have an age between 12,000 and 5,000 years, i.e. approximately early Neolithic. This would allow it to have arrived to Africa and Europe separately before booming. 

The G-13915 allele is attributed by the authors to Arabian origins c. 4000 years ago, based on previous studies and its presence only in Afroasiatic and Nilo-Saharan speakers. However I can't but notice that the haplotype structure above is centered in East Africa, with Arabian and Middle Eastern sequences being found only at the branches, what makes me cast a prudential question mark on that claim.

The G-13907 variant is clearly NE African in origin and distribution, with the greatest frequency being among the Beja (Afroasiatic pastoralists of coastal Sudan). This one is restricted to Afroasiatic speakers only, mostly of the Cushitic branch. The authors did not produce an age estimate for this branch.

The C-14010 allele could be as old as 23,000 years or as recent as only 1200 using the 95% C.I. Again it is essentially present among Nilo-Saharan and Afroasiatic (Southern Cushitic) populations, rather than among Bantu speakers, suggesting again association with pastoralism. 

Notably this allele has reached Southern Africa but it seems more directly related to the Khoe-Khoe pastoralist history and maybe other populations of possible East African origins (Himba, Herero) than to anything Bantu. 

Lactase persistence in hunter-gatherers and other roles of the enzyme

To the surprise of the authors the Hadza hunter-gatherers have high frequencies (47%) of the lactase persistence phenotype (not associated to any of the previously mentioned alleles), something they try to explain on the grounds that the same enzymes that allow lactose digestion, also works as the enzyme phlorizin-hydrolase, which uses the substance phlorizin, found in many Rosaceae (the main group of fruit-producing plants). Besides its anti-diabetic properties, phlorizin has also been used to fight against malaria, what may well be another pathway of positive selection, alternative to usefulness of lactose digestion.

This is a particularly interesting note because it could potentially feed also alternative explanations for the strong selection of lactase persistence alleles in other populations, particularly Europeans. Just tentatively, it might have helped to fend off diabetes (type II) in high carbohydrate diets typical of the Neolithic onwards. The fact that people of West Eurasian and African ancestry are somewhat less prone to this type of diabetes than people of other Asian and Native American ancestry may support this function of lactase persistence.

However in the case of the Hadza a role against malaria (same enzyme) seems more likely. 

Still plenty of room for further research

There are still way too many pockets of lactase persistence phenotype which remain unexplained and more rarely (Afghan Tajiks particularly) also pockets of lactose intolerance lacking explanation, as I discussed in 2010. Map of worldwide actual LP phenotype from Yuval Itan 2010:

In the case of Africa the Western Sahel pocket is most intriguing. Also the alleged 88% LP phenotype in the Sudanese (not apparent in the map but data from the same study - maybe they meant Western Sudanese from the Sudan region, i.e. the Sahel and not the Republic of Sudan?), which is only explained in half. Even Germans and Italians have much greater ability to digest milk than what their known alleles can explain so far. 

Neolithic peoples from Britain and Ireland ate a lot of dairies and nearly no fish

I just discussed again the genetic sweep that apparently has happened in Europe after the Neolithic strongly favoring the selection of alleles that allow the digestion of lactose (the sugar present in milk and often in other dairies) by adults. However our knowledge of ancient European genetics is probably not sufficient (nor that of lactose tolerance genetics) and in any case the question remains, where did those lactase persistence (LP) alleles come from if all ancient Neolithic remains test negative?

An interesting possibility is opened by another recent study, not at all genetic in nature but rather bio-archaeological:

Lucy J. E. Cramp et al., Immediate replacement of fishing with dairying by the earliest farmers of the northeast Atlantic archipelagos. Proceedings of the Royal Society B 2014. Open access → LINK [doi:10.1098/rspb.2013.2372]

Abstract

The appearance of farming, from its inception in the Near East around 12 000 years ago, finally reached the northwestern extremes of Europe by the fourth millennium BC or shortly thereafter. Various models have been invoked to explain the Neolithization of northern Europe; however, resolving these different scenarios has proved problematic due to poor faunal preservation and the lack of specificity achievable for commonly applied proxies. Here, we present new multi-proxy evidence, which qualitatively and quantitatively maps subsistence change in the northeast Atlantic archipelagos from the Late Mesolithic into the Neolithic and beyond. A model involving significant retention of hunter–gatherer–fisher influences was tested against one of the dominant adoptions of farming using a novel suite of lipid biomarkers, including dihydroxy fatty acids, ω-(o-alkylphenyl)alkanoic acids and stable carbon isotope signatures of individual fatty acids preserved in cooking vessels. These new findings, together with archaeozoological and human skeletal collagen bulk stable carbon isotope proxies, unequivocally confirm rejection of marine resources by early farmers coinciding with the adoption of intensive dairy farming. This pattern of Neolithization contrasts markedly to that occurring contemporaneously in the Baltic, suggesting that geographically distinct ecological and cultural influences dictated the evolution of subsistence practices at this critical phase of European prehistory.

Not only fish consumption was pretty much abandoned in Britain and Ireland with the arrival of Neolithic (only recovered under Viking influence many millennia later) but the most striking fact is that it was replaced by milk as main source of proteins. 

This fact, considering that farmers studied in Central Europe and Iberia have systematically tested negative for lactase persistence, really opens an avenue for the possible origins of this nutritional adaptation because it is most unlikely that they were such notable dairy consumers without the corresponding digestive ability (even cheese may be harmful to lactose intolerant people unless it is aged, while yogurt was almost certainly not known yet in Europe). 

While the evidence comes from the Atlantic Islands, it is worth to notice that their chronologically late Neolithic has its origins in the much older agricultural cultures of NW France, another blank spot in the ancient DNA map of Europe. Nowadays NW France is high but not particularly high in this phenotype but SW France and Basques have among the highest LP scores (both phenotype and rs4988235(T) genotype) in Europe, together with the Atlantic Islands and Scandinavia. 

Then again it is worth recalling that one of the first areas where the rs4988235(T) allele is found is in the southern areas of the Basque Country, with clear signs of two different populations (one lactose tolerant and the other lactose intolerant) being still in the first stages of contact and mostly unmixed.

This leads us to the issue of Atlantic Megalithism (tightly associated to Atlantic Neolithic) and its still unsolved, but likely important, role in the conformation of the modern populations of Europe. 

Whatever the case the first farmers of the islands were heavy dairy consumers, although in Britain (but not in Ireland and Man) they eventually derived into heavy meat eaters later on:

Figure 1.

Prevalence of marine and dairy fats in prehistoric pottery determined from lipid residues. (a–f) Scatter plots show δ¹³C values determined from C_16:0 and C_18:0 fatty acids preserved in pottery from northern Britain (red circles), the Outer Hebrides (yellow circles) and the Northern Isles of Scotland (blue circles), dating to (a) Early Neolithic, (b) Mid/Secondary expansion Neolithic, (c) Late Neolithic, (d) Bronze Age, (e) Iron Age and (f) Viking/Norse. Star symbol indicates where aquatic biomarkers were also detected. Ellipses show 1 s.d. confidence ellipses from modern reference terrestrial species from the UK [19] and aquatic species from North Atlantic waters [13]. (g–i) Maps show the frequency of dairy fats in residues from Neolithic pottery from (g) Early Neolithic, (h) the Middle Neolithic/Secondary expansion and (i) Late Neolithic. Additional data from isotopic analysis of residues from Neolithic southern Britain (n = 152) and Scotland (n = 104) are included [19,20].

The data of this study also suggests that the so much hyped high-meat "Paleolithic diet" is more of a Late Neolithic (Chalcolithic) thing, with the real hunter-gatherers of Europe being more into fish in fact.


Correction: I wrongly reported the main European lactase persistence SNP as rs13910*T, when it is in fact rs4988235(T) (already corrected in the text above) This was caused by the nomenclature used in the Sverrisdóttir paper, where it refers to it as -13910*T, which must be some other sort of naming convention. Thanks to Can for noticing.

New lactase persistence study rejects "calcium absorption" hypothesis

The "calcium absorption" hypothesis has been proposed as hypothetical mechanism to explain the apparent genetic sweep of lactose persistence alleles in Europe. According to this hypothesis, the possible role of milk in improved calcium absorption would counter the poor vitamin D synthesis in Northern Europe, preventing rickets.

However this hypothesis seems very weak, as I explained recently, notably because bone formation is only one of the various roles of vitamin D and it is probably much more crucial in correct brain development in childhood. Also there is another clear adaptation that actually solves the problem very well: whiter skin able to much more efficiently produce vitamin D in our bodies surfaces by mere exposition to sunlight, a trait that seems to have been increasingly favored after the Neolithic drop in fish consumption (the only actual nutritional source of vitamin D at relevant doses).

This new paper confirms my skepticism.

Oddný Ósk Sverrisdóttir et al., Direct estimates of natural selection in Iberia indicate calcium absorption was not the only driver of lactase persistence in Europe. MBE 2014. Pay per view → LINK  

[doi:10.1093/molbev/msu049]

Abstract

Lactase persistence (LP) is a genetically determined trait whereby the enzyme lactase is expressed throughout adult life. Lactase is necessary for the digestion of lactose – the main carbohydrate in milk – and its production is down-regulated after the weaning period in most humans and all other mammals studied. Several sources of evidence indicate that LP has evolved independently, in different parts of the world over the last 10,000 years, and has been subject to strong natural selection in dairying populations. In Europeans LP is strongly associated with, and probably caused by, a single C to T mutation 13,910bp upstream of the lactase (LCT) gene (-13,910*T). Despite a considerable body of research, the reasons why LP should provide such a strong selective advantage remains poorly understood. In this study we examine one of the most widely cited hypotheses for selection on LP – that fresh milk consumption supplements the poor vitamin D and calcium status of northern Europe's early farmers (the calcium assimilation hypothesis). We do this by testing for natural selection on -13,910*T using ancient DNA data from the skeletal remains of eight late Neolithic Iberian individuals, whom we would not expect to have poor vitamin D and calcium status because of relatively high incident UVB-light levels. None of the 8 samples successfully typed in the study had the derived T-allele. In addition, we reanalyse published data from French Neolithic remains to both test for population continuity and further examine the evolution of LP in the region. Using simulations that accommodate genetic drift, natural selection, uncertainty in calibrated radiocarbon dates, and sampling error, we find that natural selection is still required to explain the observed increase in allele frequency. We conclude that the calcium assimilation hypothesis is insufficient to explain the spread of lactase persistence in Europe.

The study finds most likely that, most likely, there is population continuity between Neolithic farmers and modern local peoples in Northern Iberia and SE France. Technically they could only not reject this population continuity for all population parameters, but, considering that the same tests strongly reject it for Central Europe and Scandinavia, the most parsimonious conclusion is that some important population continuity does exist in SW Europe since Neolithic. In the words of the researchers:

It thus seems likely that population turnover since or shortly after the Neolithic transition has been less severe in southwestern Europe than in central or northern Europe.

However these ancient populations were lactose intolerant (rs4988235(C)) while modern ones in Northern Iberia are massively able to digest lactose (rs4988235(T)). This supports the theory of adaptive sweep for this allele. 

They suspect that the real reason behind the lactose persistence sweep is caused by basic nutritional reasons (calories and proteins) because milk may have been less subject to fluctuations in crops (traditionally cattle ate grass and not cereals, as happens in modern industrial production, while goats have even more varied natural food sources). In such circumstances episodic famines would have strongly favored lactose tolerant phenotypes, more so if lactose intolerant people would have drank milk or ate high-lactose dairies in desperation, causing them potentially deadly diarrhea.

This is not the same but fits well with my class structure hypothesis, outlined recently. The main reason why I favor this hypothesis is that this generalizing pattern should have affected farmers since very early in the Neolithic, even when they were still living in Asia or Greece, so it is very strange that the genetic sweep only appears since or after the Chalcolithic period, when a hierarchical class society is formed everywhere.


Correction: I wrongly reported the main European lactase persistence SNP as rs13910*T, when it is in fact rs4988235(T) (already corrected in the text above) This was caused by the nomenclature used in the Sverrisdóttir paper, where it refers to it as -13910*T, which must be some other sort of naming convention. Thanks to Can for noticing.

See also:

• Actual lactase persistence more common than genes predict
• Childhood and death in Neolithic and Bronze Age Iberia
• Medieval Germans, Hungarians and the spread of lactose tolerance
• Caught in the act: lactose intolerant and lactose tolerant populations together but mostly unmixed in Chalcolithic Upper Ebro
• And in general category: lactose intolerance

Medieval Germans, Hungarians and the spread of lactose tolerance

A new ancient DNA study found that 800 years ago in Dalheim (Western Germany) lactase persistence was already similar to modern day frequencies (h/t to Chad):

Annina Krüttli et al., Ancient DNA Analysis Reveals High Frequency of European Lactase Persistence Allele (T-13910) in Medieval Central Europe. PLoS ONE 2014. Open access → LINK 

[doi: 10.1371/journal.pone.0086251]

Abstract

Ruminant milk and dairy products are important food resources in many European, African, and Middle Eastern societies. These regions are also associated with derived genetic variants for lactase persistence. In mammals, lactase, the enzyme that hydrolyzes the milk sugar lactose, is normally down-regulated after weaning, but at least five human populations around the world have independently evolved mutations regulating the expression of the lactase-phlorizin-hydrolase gene. These mutations result in a dominant lactase persistence phenotype and continued lactase tolerance in adulthood. A single nucleotide polymorphism (SNP) at C/T-13910 is responsible for most lactase persistence in European populations, but when and where the T-13910 polymorphism originated and the evolutionary processes by which it rose to high frequency in Europe have been the subject of strong debate. A history of dairying is presumed to be a prerequisite, but archaeological evidence is lacking. In this study, DNA was extracted from the dentine of 36 individuals excavated at a medieval cemetery in Dalheim, Germany. Eighteen individuals were successfully genotyped for the C/T-13910 SNP by molecular cloning and sequencing, of which 13 (72%) exhibited a European lactase persistence genotype: 44% CT, 28% TT. Previous ancient DNA-based studies found that lactase persistence genotypes fall below detection levels in most regions of Neolithic Europe. Our research shows that by AD 1200, lactase persistence frequency had risen to over 70% in this community in western Central Europe. Given that lactase persistence genotype frequency in present-day Germany and Austria is estimated at 71–80%, our results suggest that genetic lactase persistence likely reached modern levels before the historic population declines associated with the Black Death, thus excluding plague-associated evolutionary forces in the rise of lactase persistence in this region. This new evidence sheds light on the dynamic evolutionary history of the European lactase persistence trait and its global cultural implications.

Table 2. Results of genetic sex and LP allele genotyping.

So lactase persistence was already highly prevalent in West-Central Germany 800 years ago, much as it is today.

Very interesting also is their mention of a previous study in Medieval Hungarians (Nagy 2011, PPV):

A study of medieval Hungary found moderate levels of LP in local commoners (33%) ca. AD 900–1100, but extrapolating from these results is complicated by the region's history of conquest by lactase non-persistent Asian invaders.

While these frequencies are clearly much higher than Neolithic ones (zero), they were still much lower than present day (c. 60%). 

They also mention the oldest know lactase persistence alleles in Europe, which correspond to Chalcolithic findings in Götland and the Basque Country, albeit still at low frequencies and, in the Basque case, showing strong linkage disequilibrium pointing to an initial admixture episode between two different populations: one lactose-tolerant and the other intolerant. See this previous entry for more details.

As I see it, these two data points help us to better understand the still very wide window when lactose tolerance spread among Europeans, which begins in the Chalcolithic and, at least in the case of Germany, seems closed by the Middle Ages. Although in the Hungarian case remained still half-way in that period. 

It is quite possible that instead of a single selective swap affecting this trait, the process took place in several bouts, each one with their own geography and timeline. 

Still, the reasons behind this apparent positive selection for milk-digesting genes, remain ill-explained at academic level. Recently I tried to articulate a consistent theory on it, based on the fact that the Metal Ages, when this sweep happened almost certainly, were characterized by the accumulation of agricultural resources, wealth and power in few hands, producing a class-structured society in which the vast majority were poor and lived precarious lives, in which the general availability of, particularly, goat milk may have been an important nutritional relief (calories and proteins). See: Is the ability to digest milk in Europeans caused by ancient social inequality?

Is the ability to digest milk in Europeans caused by ancient social inequality?

I've got involved these days in a discussion at Dienekes' Anthropology Blog on the causes of lactase persistance (LP), i.e. the ability to digest milk as adults, in Europe. 

The discussion orbits around a recent pay-per-view study by O.O. Sverrisdóttir, which claims, with some soundness for what I can discern, that LP in Europeans must have gone through positive selection. 

Actually the study, as most of its kind, deals only with one LP marker, the well known SNP rs4988235, whose T variant allows adults (in dominant fashion) to digest milk, an ability often lost after weaning. 

As I discussed back in 2010, there must be other such SNPs because actual LP phenotype only partly corresponds with the known LP alleles. But for whatever is worth, this is the (2010) "known allele" LP map:

In Europe at least, it essentially corresponds with the T variant of rs49235, which concentrates in Scandinavia, Atlantic Islands and the Basque/SW French area. 

At first I boarded the discussion with perplexity, because, even if the positive selection argument seems sound, it seems hard to find a reason for it: milk is not such a "great" source of food, excepting the issue of calcium and a high content of protein and fat, and the occasional claims that it is related to vitamin D deficiency seem extremely feeble because this vitamin is present at extremely low frequencies in natural milk, being rickets (where milk's extra calcium could play some role) only a "less important" side effect of vitamin D deficiency, because its main harmful effect is to impair early brain development, a most serious problem for which calcium seems quite meaningless. So why would the ability to digest milk would have become such a matter of life or death to be actively selected for generation after generation until near-fixation?

A key piece of information is that not a single sequenced Neolithic farmer has ever been found to carry the relevant LP allele (being all CC) and only since Chalcolithic we begin to find some TT and CT individuals. These are found in Sweden and in the southern areas of the Basque Country (see here for a lengthier discussion):

• In Götland (Pitted Ware culture) only 1/20 alleles was T (i.e. 1/10 persons had the CT combo, all the rest being CC and therefore likely lactose intolerants). 
• In Longar (Navarre, dated to c. 4500 BP) 1/7 individuals was TT, while the other six were CC (intolerant). There were no CT cases.
• In San Juan Ante Porta Latinam (SJAPL, Araba, dated to c. 5000 BP), 4/19 were TT, 2/19 were CT, while the remaining 13 were CC.

In the Basque cases we can appreciate that there must have already been two different populations regarding this SNP, because the CT cases are rare, implying that the two groups were only beginning to mix. It is worth mentioning that the Basque sites are odd in several aspects: on one side they seem to be military cemeteries (mostly males, arrow injuries and arrow points) and, on the other, they are rather exceptional in the Basque historical sequence of mtDNA pools (a lot more K and some other lineages than usual, less H and U).

But a key finding in this study is that a Neolithic sequence from Atapuerca (near Burgos city, historical Basque SW border) was again CC for the relevant SNP (and therefore likely lactose intolerant). So it is very possible that proto-Basques did not have the T allele in notable frequencies either (although I keep some reservations for lack of larger samples).

Whatever the case, if the T allele was selected positively as it seems, there must be a powerful reason for it. Was it cows, as some have claimed a bit too vehemently? I doubt it. 

Why? Because for all we know from the Middle Ages, a period very similar in many aspects to the Metal Ages, it were goats and not cows the main providers of milk. This makes total sense because the hardy goats are rather inexpensive to rear, while cows are more costly and were often reserved for traction jobs. In most cases, cow produce, be it milk or meat, was an expensive luxury apt only for the upper echelons of a society that was becoming more and more hierarchical and unequal since precisely the Chalcolithic period. 

Some oral accounts I have heard tell that not so long ago "acorn bread and goat milk" were often staple for the poor. In other areas maybe it was not acorn bread but, say, oat meal (or whatever else), but almost certainly the milk came almost invariably from goat udders, which very efficiently transform leaves and almost any vegetable, even thorny ones, into milk (and meat) for our consumption.

It is crucial to understand that only if milk was a key survival staple, LP would have become fixated. Otherwise people would have preferred alternative foods and survived in similar shape, so positive selection would never have happened at this locus (non-LP individuals would have survived easily, selection would never have happened or would have been mild enough to retain much greater diversity). 

It is also crucial to understand that, for all we know, this positive selection only happened since the Chalcolithic, i.e. when social stratification, inequality and private aristocratic property became common. Obviously the upper classes (or castes) had no problems accessing high quality foods, including meat, but the masses probably had growing problems in this aspect as the land and cattle became more and more concentrated in few hands. 

Even where a wide class of free peasants existed, as was probably the case in much of Atlantic Europe, these were surely often not well-off enough to afford dairy cows. Instead goats would have been available for almost everybody, even the poorest of farmers. And very likely they were the only steady supply of proteins and fat, mostly via milk.

Plausibly this need of extra nutrients of animal origin was more intense in the Atlantic areas of Europe because cereals do not perform so well in the prevalent humid conditions. Also before the medieval development of the heavy plough, the deep Atlantic soils were not at all as productive as they are now (and that's why NW Europe only got its economic prominence in the last millennium, being before a peripheral area to the much more productive Mediterranean climate). 

But climatic and agricultural issues aside, I strongly suspect that the main driver of LP positive selection, were goats, because these and their dairy produce were almost certainly available for almost everyone and, in the Metal Ages, the vast majority of people were farmers, often rather poor peasants who had to rely on their goats for survival, very especially in the bad times.

I really do not see any other explanation that fits the data.


PS- This social inequality & goats argument makes sense assuming that the positive selection theory is correct. However before I fully embrace it, I would need a half-decent sample of aDNA sequences from the Atlantic areas of Europe, notably Britain & Ireland, the Basque Country & SW France and mainland Scandinavia, where the T allele peaks. I say because what we find in some Chalcolithic sites, notably in the Basque Country, rather strongly suggests that there was already a TT population somewhere and we have not yet found it. So maybe some of the premises of the positive selection theory are not as sound as I said above - but we do not know yet.

Was the first ever cheese from Poland?

Modern oscypki cheese from Poland
(CC by Pawel Swiegoda)

While today it is maybe France the most famed cheese-making and cheese-eating region on Earth*, we knew very little about cheese-making origins... until now.

Melanie Salque et al., Earliest evidence for cheese making in the sixth millennium bc in northern Europe. Nature 2012. Pay per view → LINK [doi:10.1038/nature11698]

Abstract

The introduction of dairying was a critical step in early agriculture, with milk products being rapidly adopted as a major component of the diets of prehistoric farmers and pottery-using late hunter-gatherers^{1, 2, 3, 4, 5}. The processing of milk, particularly the production of cheese, would have been a critical development because it not only allowed the preservation of milk products in a non-perishable and transportable form, but also it made milk a more digestible commodity for early prehistoric farmers^{6, 7, 8, 9, 10}. The finding of abundant milk residues in pottery vessels from seventh millennium sites from north-western Anatolia provided the earliest evidence of milk processing, although the exact practice could not be explicitly defined¹. Notably, the discovery of potsherds pierced with small holes appear at early Neolithic sites in temperate Europe in the sixth millennium bc and have been interpreted typologically as ‘cheese-strainers’¹⁰, although a direct association with milk processing has not yet been demonstrated. Organic residues preserved in pottery vessels have provided direct evidence for early milk use in the Neolithic period in the Near East and south-eastern Europe, north Africa, Denmark and the British Isles, based on the δ¹³C and Δ¹³C values of the major fatty acids in milk^{1, 2, 3, 4}. Here we apply the same approach to investigate the function of sieves/strainer vessels, providing direct chemical evidence for their use in milk processing. The presence of abundant milk fat in these specialized vessels, comparable in form to modern cheese strainers¹¹, provides compelling evidence for the vessels having being used to separate fat-rich milk curds from the lactose-containing whey. This new evidence emphasizes the importance of pottery vessels in processing dairy products, particularly in the manufacture of reduced-lactose milk products among lactose-intolerant prehistoric farming communities^{6, 7}.

See also the related article by Niddhi Subamaran at Nature News and the one by Hanna Briggs at BBC News.

The dating for the cheese-making artifacts (holed pots used to press curdled milk, discarding the whey, which is a preliminary step in cheese making) is of similar age as the arrival of Neolithic itself and the first evidences of dairying in Europe and Africa. So I guess that we can conclude that farming arrived to Europe, at least to Central and Northern Europe, together with cattle herding, dairying and cheese-making.

While the potsherds are known to exist elsewhere in Central Europe, the ones analyzed for this paper are specifically from Cuyavia, Poland, which used to be the most Northeastern offshoot of the Danubian Neolithic.

Cuajada or mamia
(GFDL+CC-BY-SA-2.5)

Just one weak caveat. While Richard Evershed (in the BBC article) asks rhetorically what other milk product could it be? This question is not as trivial as it may look on first sight because there is indeed another such milk product: the cuajada (Spanish name) or mamia (Basque name) which is nothing else but the preliminary product of cheese making (curdled milk) and a much coveted delicatessen when properly made.

But, well, I guess it can be considered a form of cheese... more or less. Also real fully-processed cheese achieves a further purpose: to preserve milk (or rather key parts of it) for delayed consumption and easier transport.

________________________________________

* Actually Greeks eat quite more cheese per capita than the French, but they are the only ones. 

The Maasai: rich ancestry, lactase persistance and low cholesterol

Maasai warriors jumping (cc by javic)

There is a new paper at the Public Library of Science on the genetics of the Maasai with emphasis in diet-related aspects:

Kshitij Wagh et al., Lactase Persistence and Lipid Pathway Selection in the Maasai. PLoS ONE, 2012. Open access ··> LINK [doi:10.1371/journal.pone.0044751]

Abstract

The Maasai are a pastoral people in Kenya and Tanzania, whose traditional diet of milk, blood and meat is rich in lactose, fat and cholesterol. In spite of this, they have low levels of blood cholesterol, and seldom suffer from gallstones or cardiac diseases. Field studies in the 1970s suggested that the Maasai have a genetic adaptation for cholesterol homeostasis. Analysis of HapMap 3 data using Fixation Index (Fst) and two metrics of haplotype diversity: the integrated Haplotype Score (iHS) and the Cross Population Extended Haplotype Homozygosity (XP-EHH), identified genomic regions and single nucleotide polymorphisms (SNPs) as strong candidates for recent selection for lactase persistence and cholesterol regulation in 143–156 founder individuals from the Maasai population in Kinyawa, Kenya (MKK). The non-synonmous SNP with the highest genome-wide Fst was the TC polymorphism at rs2241883 in Fatty Acid Binding Protein 1(FABP1), known to reduce low density lipoprotein and tri-glyceride levels in Europeans. The strongest signal identified by all three metrics was a 1.7 Mb region on Chr2q21. This region contains the genes LCT (Lactase) and MCM6 (Minichromosome Maintenance Complex Component) involved in lactase persistence, and the gene Rab3GAP1 (Rab3 GTPase-activating Protein Catalytic Subunit), which contains polymorphisms associated with total cholesterol levels in a genome-wide association study of >100,000 individuals of European ancestry. Sanger sequencing of DNA from six MKK samples showed that the GC-14010 polymorphism in the MCM6 gene, known to be associated with lactase persistence in Africans, is segregating in MKK at high frequency (~58%). The Cytochrome P450 Family 3 Subfamily A (CYP3A) cluster of genes, involved in cholesterol metabolism, was identified by Fst and iHS as candidate loci under selection. Overall, our study identified several specific genomic regions under selection in the Maasai which contain polymorphisms in genes associated with lactase persistence and cholesterol regulation.

The study actually has two elements of interest for my point of view, on one side it is the dietary aspects of Maasai genetics underlined in the title and the abstract but then there is a more generic element of understanding the relevant population genetics with the help of STRUCTURE,  reflected in fig. 1 but better addressed in the supplemental materials (Appendix S1).

The Maasai as product of 6 ancestral populations

K=6 is what STRUCTURE indicates as clear best fit (lowest log probability of fit) in a no-admixture model and all the six reconstructed ancestral populations are all present in them (unlike what happens for example with the CEU sample of European ancestry):

From Appendix S1 - click to expand
CEU: European-Americans (USA), ASW: African-Americans (USA)
LWK: Luhya (Kenya), MKK: Maasai (Kenya), YRI: Yoruba (Nigeria)

We can see that the influence of West Eurasian genetics (teal) appears to be almost nil in Africa, although the Maasai (MKK) have some (c. 2%), probably infiltrated via Ethiopia, Somalia or Sudan.

West African genetics (pink) are slightly more influential in the Maasai but only c. 10%.

Instead the Maasai display four different East African specific "ancestral populations" of which two are larger and the other two smaller in the sampled populations:

• The yellow component is almost exclusive of the Maasai (69%), with some very minor penetration in other populations.
• The green component is more outstanding among the Luhya (32%), reaching barely 5% among the Maasai.
• The brown component is again almost exclusive of the Maasai (12%), with some individuals apparently displaying it as very dominant.
• Finally, the purple one is almost exclusive of the Maasai but at much lower frequecies (3%). However it is also dominant in a few individuals.

The fact that the Maasai show obviously marked individual variance at levels not apparent in other populations, and that this variance includes some smaller "mystery" components, seems to mean that they are in ongoing or very recent admixture with other populations (maybe other Maasai clans of distinctive origin, maybe other pre-Bantu East African peoples). It indicates that there is still a lot to research in the Population genetics of East Africa, what requires of extensive new sampling among the many diverse small ethnicities of the Upper Nile and nearby regions.

Yes to milk, no to cholesterol

The other, more central aspect of this study is the localization of specific genomic regions or even alleles that guarantee that this pastoralist people can largely live off the milk produced by their cows and, quite interestingly, not to have circulatory problems because of the associated excess cholesterol.

The authors detected the following regions as very clearly under selection (several approaches being convergent in their detection):

Fig. 5

The first of these regions, in chromosome 2, seems particularly long and selected for both lactase persistence (the ability to digest lactose as adult) and low cholesterol, according to this paper. 

The region in chromosome 7 regulates the enzyme Cytochrome P450, related to oxidation processes in metabolism of lipids, steroids, drugs and toxins, probably also regulating cholesterol.

See also in this blog:

• Claim that cholesterol related gene was positively selected in Africa but not elsewhere
• Positive selection for arthritis and short-size alleles in Eurasia?
• Khoesan genetics helping to understand the evolutionary history of Humankind as a whole
• Khoesan autosomal genetics
• North African autosomal genetics (also considering the Maasai). 
• Chad basin mtDNA
• Sudanese autosomal genetics
• African diversity and the possible origins of Humankind
• Eastern Pygmies are not mtDNA L1

I also recommedn Ethio Helix blog, generally quite informative in all things African and genetic.

Lactose tolerance favors obesity

While the lactose tolerance allele may have some positive health effects, notably because milk is one of the few good dietary sources of calcium, it seems to correlate also with some negative effects, namely obesity.

Ricardo Almon et al., Association of the European Lactase Persistence Variant (LCT-13910 C>T Polymorphism) with Obesity in the Canary Islands. PLoS ONE 2012. Open access ··> LINK [doi:10.1371/journal.pone.0043978]

Canary Islands, in spite of its subtropical geography, is one of the regions of the European Union where milk is most consumed, at levels comparable to Scandinavia. 

Although there is a strong correlation between being lactose tolerant and milk consumption it is not fully clear yet if it is excess milk consumption what makes people obese or an unknown collateral effect of the European lactase persistence allele.

Interestingly the correlation, very strong, is only found for obesity and not for being overweight:

Fig. 1 - BMI classification by LCT genotypes (LP: n = 330; LNP: n = 221)

Dairying in Africa some 7000 years ago

At least.

Julie Dunne et al. First dairying in green Saharan Africa in the fifth millennium bc. Nature 2012. Pay per view (supplementary information is accessible).

Abstract

In the prehistoric green Sahara of Holocene North Africa—in contrast to the Neolithic of Europe and Eurasia—a reliance on cattle, sheep and goats emerged as a stable and widespread way of life, long before the first evidence for domesticated plants or settled village farming communities1, 2, 3. The remarkable rock art found widely across the region depicts cattle herding among early Saharan pastoral groups, and includes rare scenes of milking; however, these images can rarely be reliably dated4. Although the faunal evidence provides further confirmation of the importance of cattle and other domesticates5, the scarcity of cattle bones makes it impossible to ascertain herd structures via kill-off patterns, thereby precluding interpretations of whether dairying was practiced. Because pottery production begins early in northern Africa6 the potential exists to investigate diet and subsistence practices using molecular and isotopic analyses of absorbed food residues7. This approach has been successful in determining the chronology of dairying beginning in the ‘Fertile Crescent’ of the Near East and its spread across Europe8, 9, 10, 11. Here we report the first unequivocal chemical evidence, based on the δ13C and Δ13C values of the major alkanoic acids of milk fat, for the adoption of dairying practices by prehistoric Saharan African people in the fifth millennium bc. Interpretations are supported by a new database of modern ruminant animal fats collected from Africa. These findings confirm the importance of ‘lifetime products’, such as milk, in early Saharan pastoralism, and provide an evolutionary context for the emergence of lactase persistence in Africa.

While there are some related articles (different author) that propose yogurt instead of milk as being the actual consumed product, this seems mostly a molecular-clock-o-logic wild speculation. Otherwise, the invention of yogurt is generally attributed to the steppe peoples (or Indians maybe) and arrived to the West only in the Middle Ages. There is no classical source discussing it at all (cheese or butter however are mentioned, as is raw milk) and instead Medieval Arab sources consider it a typical Turkish product. 

No African tradition exists of yogurt, unlike the case of butter or diverse ways of drinking raw milk, be it alone or mixed with blood.

Most likely, as in Europe and elsewhere, the relevant alleles pre-dated Neolithic changes (after all there's no advantage in lactose intolerance, so no reason why it would have been fixated other than random drift) although they may have been somewhat favored by the development of Neolithic dairying, specially in areas where other foodstuffs were not easily available.

The archaeological site of Tadrar Acacus is at the in the Central Sahara, Fezzan region of Lybia, bordering the lands assigned to Algeria and Niger. Its chronology is illustrated in the supplemental figure 2:

click to expand

Equivalent evidence of dairying in Europe is from similar dates (or somewhat earlier in the Balcans and West Asia).

Caught in the act: lactose intolerant and lactose tolerant populations together but mostly unmixed in Chalcolithic Upper Ebro

There is a new paper on ancient DNA from the southernmost Basque Country that has found the oldest known lactose tolerance alleles in prehistoric Europe:

Theo S. Plantiga et al., Low prevalence of lactase persistence in Neolithic South-West Europe. European Journal of Human Genetics 2012. Pay per view.

Context

The samples studied are the same (in part) as those from Izagirre and de la Rúa 1999, specifically 19 individuals from San Juan Ante Porta Latinam (Araba) and 7 from Longar  (Navarre). In spite of both sites being inside Euskal Herria (Basque Country) as usually acknowledged, the are both very close to Logroño, capital of La Rioja autonomous region. I mention because this area of the Upper Ebro is at least somewhat distinct from the higher reaches of the core Basque Country and may not be representative of ancient (proto-)Basque genetics but rather of Ebro Valley populations.

It is easy to appreciate in genetic research as that of Adams 2008, that Aragon (I presume representative of the overall Ebro Valley) is markedly distinct from sub-Pyrenean neighbors like Basque Country or Catalonia, holding a much larger apportion of presumably Neolithic haplogroups of Transmediterranean origin (surely E, G, J and T, as well as maybe most of I), being more similar in this to Valencia, Ibiza or Western Iberia.

Paleo- and Epipaleolithic Basque Country

Similarly the Ebro Valley is a region of very early Neolithic settlement in which often no pre-Neolithic habitation is known to have existed. However the data I know for the Basque part of the Upper Ebro basin (X. Peñalver 1996), suggests an Epipaleolithic (epi-Magdalenian) settlement of most of Araba (but not the Ebro banks as such) and only the highland parts of Navarre (right). 

It is therefore only in the Neolithic and later on in the Chalcolithic (term used sometimes loosely to indicate the late part of the Neolithic, when social complexity increases, regardless of the presence of copper, gold or silver), when the Upper Ebro, including that within conventional Basque borders is colonized. 

These colonists may or not have been of aboriginal (proto-Basque) stock. In fact anthropometric estimates emphasize the presence of Gracile Mediterranean types in devastated La Hoya town, contrasting with the native Pyrenean type of apparent Paleolithic stock.

Chalcolithic and Early Bronze sites

Whatever the case, it is clear that the Upper Ebro valley is not the core of the Basque Country, located further north, but a border area which may have been colonized partly by people of exotic origins upon the arrival of Neolithic. Of course all this is open for discussion and further analysis of the available data. 

[See also: Wikipedia: Basque Prehistory].

Findings

Luckily several readers have sent me copies of the paper (you people are awesome, thanks), and even some ideas on what is most interesting (not always the most obvious).

The findings are detailed in table 3:

The most important finding is also the most obvious: the lactose tolerance allele (or more specifically rs4988235 in its T variant, as there are others less famous such alleles) is found at the oldest known site in Europe and that is (in spite of all my previous caveats) in the Basque Country. 

There is a similar aged site in Götland, Sweden (Mälstrom 2010), but the apportions are even lower (1/19 alleles or 1/10 persons had the CT combo). All other older of contemporary sites in Central Europe or nearby Occitania have yielded negative for what is nowadays a dominant allele in Western and Northern Europe. However there are many blanks: for example no Neolithic (never mind Paleolithic) British or Atlantic French or Portuguese samples have ever been tested for. In fact no Basque samples other than these either... so the question marks are still many.

The second finding is that the apportions are low, even if it is enough for lactose tolerance to kick in that a single T allele exists (classical Mendelian dominance), only 31% and 14% of the people sampled were lactose tolerant, meaning that (most probably) non-processed milk was rare in their diet. This is contradictory with the idea that the allele became common thanks to positive selection, after all these people had been herding livestock of various types for 2000-2500 years already (just in situ, i.e. not counting their likely ancestors in Italy, the Balcans or West Asia: add other 3000 or 4000 years maybe) and yet most were lactose intolerant (apparently). 

This is something that the authors also find surprising, pondering that maybe its frequency levels may have risen more recently just because of cultural pressure. What?! Drinking milk became fashionable and you were stoned to death if you found it nasty? Hard to believe, honestly, and if you did not die from not drinking milk, there is no (or extremely low) selective pressure we can take seriously.

Two populations?

But the most striking finding, not even mentioned by the authors is the following: there are almost no heterozygous individuals in these samples: with two exceptions in the San Juan APL  sample, people fall either to the CC (lactose intolerant) or the TT genotype. The percentage of heterozygous is much lower than that of the allele itself (11% vs. 26% in SJAPL and 0% vs. 14% in Longar).

I don't know how can you explain this but for me it is crying two populations that are still at the early stages of blending: one almost homogenously TT and the other almost homogenously CC. 

Otherwise, if the admixture process would be advanced, there would be more heterozygous (CT) individuals than TT homozygous ones. 

Which can be these two populations? Hard to say for sure but my natural intuition, knowing what I know about Basque Prehistory, is that the CC population would be made up of colonists of Mediterranean stock (possibly trans-Mediterranean: Balcanic, Italian or West Asian, as suggested by recent studies of mitochondrial DNA from Epicardial sites) who were quite obviously lacking the T allele altogether even if they were the ones to have the goats, sheep and cows initially.

These peoples left a legacy in the Ebro Valley and elsewhere in Iberia and Europe but they ultimately must have failed to become fully dominant and today their ancestry seems to be just a fraction of modern Western Europeans, seldom looking dominant even in the patrilineages. 

The TT population must therefore be some other. Which one? In this context I can only think of the local aborigines rooted in the Paleolithic cultures of the area and described by anthropometric usages as of Pyrenean stock (I think Heraus would say Dinaromorphic Atlanto-Mediterranean but that's too long - incidentally he just posted on the phenotypes of Arabako Errioxa). 

So this brings me to my fallback theory: the lactose tolerance allele is a mere fluke which became fixated randomly in pre-Neolithic populations just because (or maybe, speculatively, related to motherly lactation practices), eventually showing an unexpected use in drinking milk and eating desserts, what was nice for the development of Basque cuisine but mostly unrelated to survival. 

Being able to enjoy rice pudding (as I'm doing right now) is probably the most clear advantage of the T allele

See also: Leherensuge: Actual lactase persistance more common than genes predict.

Update (Jan 19): a scanned paper by Belén Márquez et al., describes in detail the burials of SJAPL and Longar, both looking rather militarized (mostly adult males, abundance of arrow points and arrow injuries...)