That Nei distance chart

In 2015, Rewilding Europe published a PDF with a Nei distance chart showing the purported distance of 34 European cattle breeds to the one resolved aurochs genome. Many thought that we finally know which cattle breeds are genetically closest to the aurochs. But interpreting the chart that way would be a huge oversimplification and, in my opinion, inacurrate. In fact, I think we do not know anything more now than we knew before the Nei distance chart. With this post, I want to give my reasons for why I think that. Mind that I am not a geneticist, so this is completely my own interpretation. Critique is much appreciated.  

The Nei distance chart published by Rewilding Europe

1. The Nei distance analysis looked at only a very small fraction of the genome 

 

The cattle genome has 3 billion base pairs, the Nei distance analysis looked at 700.000 base pairs. Precisely, the analysis studied single nucleotid polymorphisms (SNPs). 700.000 SNPs is certainly a lot, but only a very small fraction of the total genome, thus it does not tell us much about the genetic closeness of the cattle breeds to the aurochs. With another 700.000 SNPs the results could be completely different. 

 

 

3. Only one aurochs genome was used for the analysis 

 

We have only one resolved complete aurochs genome for now, which is a problem for trying to analyse the genetic closeness of cattle breeds to their wildtype. It is unlikely (or actually impossible) that one individual comprised the full genetic diversity found in the wildtype populations, thus there must have been wildtype alleles found in other aurochs but not found in this one particular individual. The problem is that these wildtype alleles would be considered domestic alleles if found in a modern cattle individual and not found in the one aurochs individual that had its genome resolved. It could be the case that Caldela, a breed scoring very low on the chart, actually has a lot of wildtype alleles that just happen to be from other aurochs individuals than that of the one genome that we have, and so it results scoring low in the chart. That relativizes the relevance of the Nei distance analysis considerably. 

 

4. Nei distance might not be the ideal tool for analysing the situation of aurochs and cattle 

 

The Nei distance was developed for populations that diverged by mutation and genetic drift in isolated populations. But this is not the scenario that happened in the case of aurochs and cattle. In the domestication of cattle, we have at first drastic genetic bottleneck (since modern domestic cattle go back to only about 80 female founders), then massive directive selection (selection on tameness and economic value) during which many wildtype alleles might have been lost and mutated (= domestic) alleles became fixed, then we have local introgression from different types of aurochs in different regions of the world into the cattle genome (in Africa and Europe at least). Not to forget the not uncommon intermixing between taurine and indicine cattle, which descend from two rather divergent aurochs subspecies. 

 

That is why I have covered the Nei distance chart in only one post on my blog till now. I think it does not tell us much about the actual genetic distance between the aurochs and domestic cattle breeds. That is why the score in the chart seems to be rather coincidental and there is no correlation between a less-derived phenotype and the purported genetic closeness to that one aurochs individual analysed in the chart. For example, Fleckvieh scores higher than the Spanish fighting bull. Of course, it can be possible that a breed that has a rather derived morphology shares more alleles with the aurochs than one with a less-derived morphology, since many of the differences between aurochs and cattle might be non-visible, for example immunology, development, endocrinology, neurology, metabolism, or physiological aspects. But I consider it highly unlikely to be the case with Lidia and Fleckvieh, because Fleckvieh has experienced far more intense selective breeding than the Spanish fighting bull. Their score in the Nei distance chart is not evidence for that either, as outlined above. 

Furthermore, which aurochs are “the aurochs”? Even if we only care about the primigenius subspecies it is complicated to give an answer to that question. British cattle landraces have been found to share nuclear alleles with the British aurochs, likely due to introgression, which we cannot expect for Iberian, Italian or Near Eastern breeds and vice versa. 

Also, the total genetic closeness to the aurochs does not tell us which alleles are present in which breed, which is crucial information if one wants to unite aurochs alleles in one population with selective breeding. A breed scoring low in the overall genetic closeness to the aurochs might have alleles which all the other breeds do not have, and this is exactly the case in this Nei chart: Nelore, as an incidine breed, will share alleles with the British aurochs which taurine cattle have lost, because this is what was found by Orlando et al. 2015 (by the way, if the Tauros Programme is indeed aiming for breeding for “aurochs alleles”, why aren’t they breeding with zebus? There is no other way to get these alleles into the population. The answer is: Because they are not breeding on a genetic level, contrary to what they claim in press releases…). 

Thus, I think that this Nei distance chart does not tell us anything of value for “breeding-back”. Why did the Tauros Programme conduct this analysis, then? I think that’s because they needed to publish something “genetic”, in order to back up their claims that they are breeding for aurochs-like genetics. The project was very content with the results of the chart. They claimed that the breeds used in their project were particularly high-ranking. As you can see in the chart (the Tauros Programme breeds are those written in bold), this is not necessarily the case. Their breeds seem to be rather evenly distributed along the chart. However, this is not relevant for “breeding-back” anyway, at least in my opinion, for the reasons outlined in this post. 

 

Literature

 

Orlando, L.: The first aurochs genome reveals the breeding history of British and European cattle. 2015. 

 

Heck cattle at Oostvaardersplassen develop aurochs-like horns

I have made a couple of posts about the possible changes in horn shape in the Heck cattle in the Oostvaardersplassen reserve. Recently, I found some new photos of a group of young bulls in the reserve that all show interesting horns from March this year. You can see the animals here. 

Not only are the horns interesting, the bulls are also much more long-legged and less massive than Heck cattle found in zoos (which is were they descended from). Morphology can be influenced by phenotypic plasticity, so these changes in body shape and proportions do not necessarily indicate a genetic change in these cattle. In the case of the horns, however, I see no way how phenotypic plasticity can influence the horn curvature and orientation relative to the snout, thus I think we see genetic changes here. A change in allele frequency as a result of natural selection is the population genetic definition of evolution. Thus, we might see evolution at work here. 

In how far are the horns of these young bulls different? For once, they face clearly forwards in a 70 to 60° angle relative to the snout, which is identical with the horn orientation of the European aurochs. Earlier Heck cattle from the reserve (which can be seen on older photos easily available on the internet) never have forwards-facing horns, at least not on the photos I have seen so far, which are quite a lot. Furthermore, while the horns of early Oostvaardersplassen Heck cattle are curved rather straight, the horns in these bulls are curved more clearly. They do not curve inwards as strongly in the aurochs by far, but a tendency of the horn tips to grow a curve is there. This can also be seen, even more clearly, in some of the cows in the reserve (the photos aren't online anymore, unfortunately). To me, this suggests that the horn shape of the Heck cattle in the Oostvaardersplassen reserve is evolving. Precisely, evolving to a more aurochs-like horn shape. 

This is not surprising, as the horn shape of the aurochs probably had a purpose. Their strong inwards-curve enabled the bovine to push and pull the opponent during a fight, and the fact that they faced forwards and not upwards or downwards was likely functionally advantageous as well. If the horn shape of the Heck cattle in the reserve is indeed evolving, which can only be proven by gathering photos of individuals from the early years till today, it would endorse the following thoughts: 

1. the horn shape in cattle/aurochs has a function and the horn shape of the aurochs was not a coincidence

2. This has an impact on the evolutive fitness of the individuals (the more functionally advantageous the horn shape the higher the likelihood to win an intraspecific combat for breeding rights, feeding grounds etc.)

3. Eventually, the wildtype horn shape will develop in a cattle population that is exposed to natural selection, especially intraspecific selection 

The more genetically diverse the population, the faster the changes evolve (Fisher's fundamental theorem). Since Heck cattle is a mosaic breed based on many different cattle breeds, the wide range of phenotypes displayed by the individuals might have enabled the adaption process to become visible quite fast. 

I postulated that natural selection will make a variable cattle population in the wild more aurochs-like because wildtype traits are functionally advantageous multiple times on my blog, and I think these recent photos of Heck bulls at the Oostvaardersplassen reserve endorse this idea when you compare them with earlier individuals of the same population. In my opinion, the Heck cattle at this reserve are a valuable example for dedomestication. 

It is no secret that the original founding population of the Heck cattle at the Oostvaardersplassen, being ordinary Heck cattle from various locations, were not prime examples for aurochs-likeness. Yet, natural selection produced at least partly aurochs-like horn phenotypes after 40 years of intraspecific competition. This fits my idea that natural selection will "refine" any "breeding-back" product once released into the wild - if it worked on the mediocrely aurochs-like founding population of Oostvaardersplassen, imagine what kind of phenotypes might be produced by 40 years of natural selection with more aurochs-like cattle. 

Which horses should be used for a reintroduction in Europe's nature?

The discussion which horse breed or type is closest to the European wild horse and which horses should be used for a reintroduction of the species into European nature systems is sometimes carried out rather controversially and is sometimes needlessly emotionalized. In recent years, a variety of horse landraces have been used for “rewilding”, including the Konik pony, the Exmoor pony, the Garrano pony, the Hucul, Retuerta, Sorraia and the Bosnian Mountain horse. Some of them have popular background stories that claim they are, respectively, the most recent descendants of the European wild horse – none of these popular background stories are scientifically tenable. That does not make those breeds any more, or any less, suitable for natural grazing projects or even establishing truly feral populations. The Konik pony and the strongly Konik-influenced Heck horse are most frequently used in natural grazing and “rewilding” projects, probably due to their scientifically untenable reputation of being wild horses, recent wild horse descendants or phenotypic copies of the European wild horse. However, the range of pony or horse breeds used in “rewilding” is slowly diversifying. Currently, the only place in Europe where horses live completely free of human influence (except for poaching, unfortunately) is the Chernobyl exclusion zone, where a population of about 100-200 Przewalski’s horses thrives. They also happen to be true wild horses instead of domesticates. But which type of horse should be used for a reintroduction of Equus caballus into European wilderness?

One of the problems we face when trying to resolve that question is, apart from all the confusion that the mythologized breed origin stories of certain landraces have created, that we do not know how the wild horses in Europe exactly looked like. Not a single complete skeleton of a Holocene, predomestic European wild horse has been described so far. It is likely that it had the robust pony morphology with a thick head, as this morphotype is found in the closely related Przewalski’s horse and Pleistocene wild horse skeletons from Europe. But we do not know the morphology for sure. What is much more certain is the colour phenotypes, as the colour loci of ancient DNA samples from European wild horses have been tested for the respective alleles. It turns out that during the early and middle Holocene, both bay dun (the colour of the Przewalski’s horse) and black dun (the colour found in Koniks, Hucule and Sorraias) were found in European wild horses. During the later Holocene, however, black dun became the prevalent phenotype as a//a is the prevalent genotype found in the ancient samples [1]. It is also possible that non-dun wild horses existed in Europe, but the Dun locus has not yet been tested in European wild horse samples. I believe that it is likely that dun was prevalent (go here). A tricky question is the mane of Holocene European wild horses. All wild equines today have a standing mane, while hanging manes are found exclusively in domestic horses and donkeys. Nowadays I think it is much likelier that European wild horses had a standing mane as all other living wild equines do (go here for a post). 

It is also important to note that there was not one European wild horse during the Holocene, but at least two subtypes: Iberian wild horses and wild horses on the rest of Europe. It turns out that, genetically, Iberian wild horses are less closely related to the ancestors of domestic horses than the Przewalski’s horse [2]. Furthermore, it is important to note that the range of wild horses was likely continuous from Europe to Asia and that there was a continuum between European wild horses and the Asiatic Przewalski’s horse, as introgression from the latter subspecies has been found in a European wild horse stallion’s DNA sample [3]. 

 

Combining these facts, there are two concepts for horse reintroduction in Europe that I prefer. I cannot decide which one of the two concepts I favour as both have pretty strong pro-arguments. I see that there are diverse options for “rewilding” horses on this continent and that each project is free to pick those types of horses they prefer, but I think there should be a somewhat consistent baseline for why choosing a particular breed for a true reintroduction into European nature, at least in my opinion. By true reintroduction I mean the establishment of self-sustaining, unregulated horse populations in a nature system or wilderness area. 

 

1. Using pure Przewalski’s horses 

 

There are two very obvious arguments for choosing Przewalski’s horses exclusively for the reintroduction of horses into Europe’s nature. For once, they are the only wild horses left. It is true that the Przewalski’s horse is the Asiatic subspecies, and therefore not the native subspecies, but it has to be considered that it is closer to European wild horse subspecies outside Iberia than the Iberian wild horse was, and that there apparently was a continuum between both subspecies. Furthermore, it is very likely that the Przewalski’s horse would have recolonized the European continent after the man-made extinction of wild horses in Europe if it had not been for human influence. It is true that the habitat of the Przewalski’s horse was restricted to the steppe in historic times, but we do not know the natural plasticity of the ecotype as it might have also inhabited other biomes previously. Przewalski’s horses do very well in grazing projects in Central and Western Europe and also wild in the European wilderness as the Chernobyl population demonstrates. The second argument for using Przewalski’s horses exclusively is that it would be a very valuable contribution to the preservation of this endangered last remaining wild horse subspecies. The Konik population in Oostvaardersplassen numbers around 1000 individuals – image Przewalski’s horses would have been chosen for that initiative. It would have grown to the largest single Przewalski’s horse herd on the continent (or perhaps even the entire world). There are dozens of grazing projects in Germany alone, if all of them chose Przewalski’s horses instead of domestic horses it would not take long until the last wild horse on this world is not endangered anymore. 

The question then is, why are not all “rewilding” and grazing projects using the Przewalski’s horse? This has two very practical reasons. First of all, the Przewalski’s horse is not just another domestic breed, but a genuine wild animal with the behaviour of a wild animal. They can be very aggressive, particularly the stallions, and may attack humans. Przewalski’s horses are very difficult to handle. The other reason is that Przewalski’s horses are not as easily available as domestic breeds are. 

 

2. Using hybrids of Przewalski’s horses and robust landraces 

 

The second concept is using hybrids of Przewalski’s horses and robust landraces that are adapted to the climate and vegetation of the respective region. The reason for that is that European domestic horse breeds are the descendants of the European wild horse, and thus there is a chance that they preserve at least some of the wildtype alleles that were unique for this wild horse type. The Przewalski’s horse should be in the mix because it is a wild horse with a wildtype morphology, genetics and behaviour. This would also create a greater genetic diversity than using Przewalski’s horses only, as they have a limited genetic diversity due to their genetic bottleneck during the 20^thcentury. As a continuous range of free-roaming horses from Iberia to Asia is illusional in modern times, there is no danger of intermixing between pure Przewalski’s horses that have been released back into the wild and the hybrids in isolated European reserves. 

Creating hybrid populations of Przewalski’s horses and robust domestic landraces is also the chance to mimic the phenotype of the European wild horse. As mentioned above, the exterieur of that wild horse type during the later Holocene likely was the pony morphotype coupled with a standing mane and the black dun coat colour. The Przewalski-Konik hybrids living in the Lippeaue (go here or here) bear great potential for achieving that with selective breeding. The recessive standing mane and the recessive black dun coat colour can be fixed rather easily by breeding. I do not necessarily suggest that the combination Przewalski-Konik is the way to go for entire Europe. Many local landraces could be crossbred with Przewalski’s horses for “rewilding”. For example, the already established populations of Garranos, Sorraias, Exmoor ponies, Hucule and Bosnian mountain horses could be supplemented by single Przewalski’s horses, most ideally stallions. Surplus animals from zoos could be used so that the population of Przewalski’s horses that is used to preserve the subspecies is not depleted. And as the Lippeaue horses have shown, the introgression of one single animal can have a great impact on the phenotype of the entire herd without affecting Przewalski’s horses preservation efforts. I would pay attention that the genes for a black dun coat colour are always in the mix, as this was the original colour of the late Holocene European wild horse. In the case of the Sorraia, Hucule and Bosnian mountain pony, these alleles would be in the mix. In the other breeds, it might be best to introduce black dun stallions with a standing mane from another location to produce the desired phenotype. 

 

I think Przewalski’s horses should always be in the mix because they represent the last wild horse and the populations in Chernobyl and grazing projects have shown that they do well in the European biome. Alas, most projects will likely pick domestic horses only, because they are easier to handle, easily available, cheaper and there are no legal restrictions. 

 

Literature

 

[1] Sandoval-Castellanos et al.: Coat colour adaptation of post-glacial horses to increasing forest vegetation. 2017.

[2] Fages et al.: Tracking five millennia of horse managment with extensive ancient genome time series. 2019. 

[3] Wutke et al.: Decline of genetic diversity in ancient domestic stallions in Europe. 2018.