Wednesday, 30 August 2017

Broadening the mind regarding aurochs colour schemes

The “standard aurochs colour scheme” is as it follows: bulls being more or less completely black except a dorsal stripe and a muzzle ring of a light colour (whether there were also bulls with lightly coloured forelocks is unknown). For cows, various shades from completely reddish-brown, dark brown or black with a reddish-brown back/colour saddle and also “bull colour” are supported by evidence.
There is not one good reason to assume that European aurochs bulls had a colour saddle, but at least some North African aurochs (I tend to think it was universal in this subspecies) had one, as outlined in my post on Bos primigenius africanus. For the Indian aurochs, nothing is known of its life appearance except for what goes beyond osteologic information, but we can speculate (emphasis on speculate) that it might have had a very similar colour to that of the European aurochs, but it could also had some differences as I outlined in the posts on Bos primigenius namadicus (the most recent and most comprehensive one here).

But apart from what is either proven or at least not implausible based on the evidence, could there have been some more colour variants? The aurochs’ original range was quite large, and genetic drift and habitat differences could have produced some local variants, or some that were limited in time, or perhaps such that were so rare and unremarkable that they were not noticed. I made some thoughts on that, and also illustrated them and would like to share them with you in this post. I am going to start with the hypothetical colour variants that I consider most likely and end with the least likely one.

1: Completely black aurochs

The degree to which the muzzle ring and the dorsal stripe were expressed may have varied. Usually, as wildtype coloured cattle age, the muzzle ring gets reduced and may even almost disappear except for the chin (mostly in bulls). I would not be surprised if some or many wild aurochs bulls also showed only reduced muzzle rings or none at all, which might be the reason why there are no contemporaneous literature references to this trait. In Bantengs and Gaurs, we have the same situation – some individuals show them, others do not. Some aurochs bulls may have even lacked a dorsal stripe. But that is not what I am thinking about here. I am talking about the possibility that the Ed allele was actually present in wild aurochs populations before domestication and is actually a second wild type allele of the Extension locus. In this case, some aurochs would have been completely black in both sexes without any light markings, as we see it in many breeds (f.e. Angus, many fighting bulls et cetera). Usually, wild animal populations are quite uniform regarding coat colours, but the phenotypic difference between E+ and Ed, especially in bulls, would be so marginal that I hardly believe natural selection would have purged it out again in a few millennia after aurochs spread to Europe from the Middle East.
We cannot say there is direct evidence for this colour variant to have been present in Aurochs. Surely a lot of sources just describe the aurochs simply as “black” (f.e. Plinius), but that would also be the case if someone would describe an E+ bull without making the effort of making an extra remark on the light markings.
This is what aurochs with an Ephenotype would look like: 
The only way to test if there were Ed aurochs would be to do a genetic test for coat colour alleles in aDNA of fossil and subfossil aurochs material, as it has been done for wild horses in recent years.
I think that would be worth examining it, as I consider the possibility of aurochs with the Ed allele absolutely plausible. If it was found in Holocene or historic aurochs only, it may also be possible that the black allele would still be of domestic origin and found its way into the wild population by domestic introgression. Domestic animals escape all the time all over the world and can leave a mark in wild populations, especially in the form of colour variants as they are most neutral to selection. This is evident in some wolf and wild boar populations, and the same happened in wild horses in Europe, where the e mutation (sorrel) got into wild populations in historical times (Pruvost et al. 2011). So it might have happened in Holocene aurochs as well, and the black mutation would maybe not have been discernable for eyewitnesses.

2: Red aurochs in far Eastern Europe
 
Casta-Navarra bull showing the red colour of a cow
Many of you know this chart showing the maximum range of the aurochs. The way I understand it, it shows the sum of all ranges the aurochs originally had, and not where it once ranged all at the same time. It seems that the aurochs was not an animal of the steppes, it would probably not do well in the cold and dry Eurasian steppe of the Baikal area. There are bone findings from this area, but from a time when climate was warmer and allowed Bos primigenius primigenius, adapted to the temperate European climate, to live there. That is why the Holocene range of the aurochs ended in the west of Russia in the transition zone from the European temperate biome to the Eurasian steppe biome. What is interesting is that van Vuure notes that Russian and Romanian tales tell of “red aurochs”, while most Central and European literature refers to “black aurochs” or mention sexual dimorphism. Could that mean that the aurochs of far eastern Europe in the semi-steppe lacked sexual dimorphism and that their bulls were of a red colour, perhaps caused by genetic drift? This would add another colour variant to the list, and quite frankly, it would be very interesting.
But I consider the evidence for that too weak. First of all, folk tales are not all too precise. Furthermore, cattle usually form herds of cows with calves and young bulls, where most individuals would be of a red colour, and bulls form either small groups of youngsters or wander around solitarily. So the chance is good that when people thought of big herds of aurochs most individuals would have been red because they consisted of cows, calves and young bulls. Again, genetics could resolve this question, but examining the amount of sexual dichromatism would probably go less quick than just a test for colour alleles.

3: Aurochs with the White Park pattern

The cave paintings at Lascaux are from the Paleolithic and about 17.000 years old. It includes black bulls, red cows, and line drawings showing bulls. What is peculiar about these line drawings is that they show small, irregular black spots on the neck, face and shoulder area, distributed in the same kind of pattern we find in the British breeds White Park and Chillingham cattle. This colour variant is caused by the homozygous presence of the Colour sided allele Cs. Could it be that some, perhaps only Pleistocene Southern European aurochs showed the so-called White Park pattern? This is not entirely implausible, and I revealed this idea in 2013 already. Cave paintings also show spotted horses long before the emergence of domestic horses, and a study by Pruvost et al. 2011 showed that such spotted wild horses probably did exist and where no invention of Pleistocene artists. So why should not be the same possible here?

It seems that these line drawings at Lascaux would be the only evidence supportive of this idea. There are no artistic or literary references that ever mention white or very faintly coloured aurochs, not even mystery tales. But this colour variant must have survived in the aurochs population until at least 8.500 years ago when the first aurochs where domesticated, otherwise it would not be found among domestic cattle. Interestingly, the heterozygous state Cs/cs+ results in a spotted colour called “colour sided”, found in many Texas Longhorn, for example. So if aurochs with the White Park pattern would have mated with typically coloured aurochs, “colour sided” aurochs would be the result – such a piebald colour is rather untypical for wild animals and probably of selective disadvantage (camouflage, especially for calves). However, a disadvantageous heterozygous state is not impossible for a wildtype allele. An aurochs with a “colour sided” pattern would have probably been considered a hybrid by eyewitnesses, but there are no contemporaneous notions of (alleged) aurochs-cattle hybrids running around in the wild that I am aware of.
In any case, the line drawings from Lascaux are the only evidence that would support a White Park pattern in aurochs, but the small black spots can also be interpreted differently.  Perhaps the artist wanted to indicate curly hair. Many domestic bulls, especially those of populations/breeds where the bulls fight on regular basis (Chillingham, Betizu, Eringer, Lidia, some Heck cattle at Oostvaardersplassen), often have rather curly hair on head, neck, face and shoulder area what – I hypothesize – might protect their skin in a fight. Some, or even all, wild aurochs bulls might have had this trait as well (the curly hair between the horns is proven in any case). See this post.
So the evidence for this colour variant is very weak, but it could be worth to test aDNA from the suspected population (Southern European aurochs of the Pleistocene) for the Cs allele.

Note that I am neither saying that I “believe” these colours were present in wild aurochs, nor do I say that I consider that likely. I am just speaking of possibilities. To test these ideas, genetic tests of historic and prehistoric aurochs aDNA would be necessary. The same work has been done with wild horses, and revealed surprises. It is likely that testing aurochs would result in a confirmation of status quo, but it would be worth examining. However, the problem is that there is way fewer interest in cattle as animals than in horses and many geneticists might consider genetically examining the colour of wild aurochs too trivial.



Saturday, 26 August 2017

Genetic proximity: what is it, and how much is needed?

This is a topic that has been barely covered here before, but is surely of interest for many of my readers especially since the 2015 article on the genetic studies involved in the Tauros Project was published by Rewilding Europe. The reason why I have covered neither the article nor the issue of genetic proximity here as such is that it is a lot to write and explain, and I haven’t had the time previously. However, this summer I have the time and inspiration to finally cover this topic appropriately.

By genetic proximity, we of course refer to the genetic proximity of living domestic cattle to its wild progenitor, the Eurasian aurochs Bos primigenius primigenius. But there are several ways in which domestic cattle can be “close to their ancestor”, several ways how genetic proximity is defined or measured, and what it implicates for “breeding-back”. However, as I always admit, I myself am a layman in the field of genetics so please point me to mistakes if I made some.

What was especially striking when Rewilding Europe published their article on genetics was the chart of Nei genetic distance of a number of cattle breeds to the aurochs. The most obvious result is that there is seemingly no clear correlation between a less-derived phenotype and “genetic proximity” (see down below), which led some people to conclude that “genetics don’t matter”. But genetics do matter of course: if you insert an aurochs’ genome into a Holstein zygote, you get an aurochs. But why this discrepancy? To solve this question and to find out what it means for “breeding-back”, we have to look at what “genetic proximity” means in this case.
Nei distance analysis published here
How genetic proximity is usually measured

Of course it would be best and most comprehensive to compare the full length of two genomes against each other. However, as genomes are huge molecules with a lot of information (in the case of cattle, the genome includes 3 billion base pairs and 22.000 genes), this would be very effortful. And apart from that, only a very small fraction of the whole genome codes for the defining differences between closely related species, and an even smaller portion codes for individual variation. So in order to make it easier, geneticists often rely on easily identifiable, short DNA sequences such as marker genes, and haplotypes of the mitochondrial genome or Y chromosomes. The advantage of molecular marker genes, such as cytochrome c and others, is that there is a constant average mutation rate per generation and that they are barely effected by selection as they have minor influence on the individual phenotypic variation, especially because many variations are neutral. This also makes them useful for determining the time of splitting up between evolutionary lines (“molecular clock”, the markers being used are called molecular chronometers). While variations on the mitochondrial genome, Y chromosome and their haplogroups as well as other marker genes are often used to determine the time of divergence or degree of genetic proximity under the assumption of a constant mutation rate and that they are barely affected by phenotypic selection, their influence on the actual phenotype and thus the nature of the animals is comparably minor. Mitochondrial genes mostly serve functions in the mitochondria themselves, and there are only a few genes on the Y chromosome that are actually relevant (lying on the sex-determining region of the chromosome). Haplotypes (variations passed on by only one parental lineage) are often used as an indicator of relatedness, but they actually are just an accumulation of more or less neutral variation on haplotypes and their influence on the organism as a whole is really minor. That is why the variation on those markers usually studied are barely affected by selection, what makes them in turn good markers. But their influence on the genetic architecture of the animals is meagre. They can indicate relatedness, but do not guarantee that the rest of the genome, especially those regions coding for the particular differences between the taxa compared, will be similar too.

As an example: let us assume that we take the genome of an ordinary Holstein-Frisian and exchange all sequences of its marker genes, haplotypes and even the complete mitochondrial genome with those sequences of an original aurochs and let that embryo develop. The result will still be a Holstein-Frisian, because the genetic material exchanged are mostly more or less neutral variations on genes that have a minor influence on the individual variation within a species or even between related species and not the regions that define the differences between a Holstein-Frisian and an aurochs. A Holstein cow with aurochs mitochondria would still be a Holstein cow in our perception, because the influence of mitochondrial DNA of the organism as a whole is comparably small. Furthermore, haplotypic variation does neither determine the identity of an individual or a species.

Most of the identity of an organism is defined by the nuclear genome. If you want to determine truly influential differences between aurochs and cattle, you would have to look there. Which is why the geneticists cited in the Rewilding Europe article have had a look at nuclear autosomal SNPs (Single nucleotid polymorphisms). SNPs, as long as they lie on coding regions, do have an influence on the phenotype. Many mutations causing cancer in humans, for example, are SNPs. For the study, 770.000 SNPs have been investigated and compared between one aurochs individual and 35 cattle breeds. They calculated the Nei genetic distance and presented the results (shown above).

The genetic difference between wild aurochs and domestic cattle

As far as my understanding goes, the last word is surely not spoken with that. It is of course state of the art to measure genetic distance using SNPS, haplotypes, marker genes et cetera, but we have again to look at the relevant genetic differences between aurochs and cattle. Just as the aurochs was neither defined via its mitochondria or haplogroups, it also was not defined by a couple of thousand SNPs. I imagine that the truly vital genetic differences between an aurochs and domestic cattle concern the following biological aspects:

- neurology
- endocrinology
- developmental regulation: timing et cetera
- sexual dimorphism
- metabolism
- morphologic aspects
- immunology

As outlined in a number of previous posts where you also find relevant literature (here and here), a lot of the differences between domestic and wild morphology are probably caused by pleiotropic effects and developmental cascades and therefore concern the upper five points; probably only a few novel morphological mutations appeared (alleles such as those causing scurred horns, new colour variants, extreme cases of dachshund-leggedness etc.).
I think those seven aspects are where we should look for the defining genetic differences between cattle and Eurasian aurochs that have a large biological impact since they are probably those regions that determine whether we have to deal with an aurochs or domestic cattle. Presenting the full genome sequencing of a British aurochs, Park et al. 2015 noted that “important questions remain unanswered, including […] which genomic regions were subject to selection processes during and after domestication. […] Finally, the functions of genes showing evidence for positive selection in B. taurus are enriched for neurobiology, growth, metabolism and immunobiology, suggesting that these biological processes have been important in the domestication of cattle1, what fully supports my view.

It is therefore my suspicion that we really have to identify the regions coding the neurobiological, developmental, endocrinologic, morphologic, metabolic and immunologic differences between aurochs and domestic cattle if we want to make genetic comparisons that truly matter on a wider biological basis concerning the nature of the animals and not coincidental variations that have been accumulated on regions that are barely effected by selection.
It is therefore not surprising that standard genetic methods that are usually used to determine simple “relatedness” that look at marker sequences, be it haplotypes, mitochondrial genes, SNPs or molecular chronometers that are not deeply effected by phenotypical selection do not show a clear correlation between a less derived and a strongly derived phenotype, although the latter clearly implicates a lot of strong directive selection and mutations.
There are multiple angles to look at a genome, and if we would look at those regions named above that are probably where we should look for matches with the aurochs, we would probably receive a different picture because those regions are directly and highly involved in the shape, form and function of the organism and directly and highly affected by phenotypic selection.

Furthermore, and to come back to the chart presented by Rewilding Europe, I am not sure if the Nei distance is the right tool to compare aurochs and cattle. The Nei distance was developed to look at the divergence of populations via mutation and genetic drift. In the case of cattle we do not have nice clean cladogenesis, but at first we have a dramatic bottleneck, then strong selective pressure during domestication, then very likely also local introgression on multiple regions by different regional variants of aurochs, and, not to forget, very unequal selective pressure on the different populations/lines/breeds of domestic cattle we see today.

Implications for “breeding-back”

So we would actually have to clearly determine those particular regions determining the defining differences between aurochs and cattle and get an overview over the allelic differences there. I do not think that it is a problem that we have only one full aurochs genome yet, because those traits defining the aurochs will, unsurprisingly, be universal among aurochs. However, I do not think that the results will be that enchanting or provide an additional directive for “breeding-back” projects, because of two reasons: 1) the fundamental differences between aurochs and domestic cattle concerning the seven factors above will probably be more or less universal among domestic cattle because all of them show the typical traits of domestication to a more or less clear extent. It would surprise me if we could still find the genetic make-up for all defining wild aurochs traits split up and distributed among the cattle of this world. This could be expected if domestication was an uncoordinated process where each of the factors was modified separately, which was certainly not the case as an organism functions as a whole and domestication was a coordinated, conscious process with a clear objective 2) certainly some breeds will be closer to the aurochs than others, but the question is how much and if the extent is relevant at all. For example, if the phenotypically most primitive cattle on this world show a 1,01% match to the aurochs on those seven crucial factors (just a symbolic number) while the most domesticated cattle show a 1,009% match, then the difference has to be considered negligible despite the primitive cattle having a number of alleles for a primitive morphology. I don’t think the difference would be that small, but I also do not expect any miraculous surprises. I think that all domestic cattle on this world are pretty similar in being domestic, with the derived examples of course carrying it to the extreme concerning a few morphological and other traits.

The claim that “no aurochs genes were lost, but just split up and distributed among domestic cattle” that has been floating around in the web for a while not only has no empirical support but is also to be considered unlikely for the reasons outlined above. The dramatic genetic effect of domestication – a narrow genetic bottleneck at first, strong selection on a large number of genes that have a dramatic influence on fundamental aspects of the organism – probably irreversibly and universally purged off a lot of defining wildtype alleles and therefore aurochs alleles from the domestic cattle gene pool, creating modern domestic cattle. The occasional local introgression of aurochs may have left traces in the modern domestic cattle pool (especially in the form of immunologic adaptions etc.) but certainly it did not alter their domestic nature. And even if all the defining aurochs genes were indeed still present in the modern domestic gene pool, and even if we had already identified and tracked them down in the populations, one should not make illusions over uniting them by conventional breeding in an anywhere near future because we are probably talking about at least hundreds of gene loci here. Body size alone is determined by over 50 loci (in humans)2, and you already see how longsome it is to achieve the right colour setting although we are dealing only with a couple of genes there. Therefore, truly genetically reconstructing the aurochs by selective breeding would be a centuries-long project, and many of the key genes are probably lost anyway. It would probably be way faster, cheaper and endlessly more effective to genetically reconstruct an aurochs via either cloning or CRISPR-Cas9.

Therefore, studies on genetic markers and SNPs are nice, but we are certainly not looking at the key genes that are relevant for true genetic identity of the animal that the aurochs originally was. Furthermore, there are unfortunately good reasons to assume that many of those defining key genes are lost due to the dramatic process of domestication that all of our modern day cattle went through. That is why you read that few on “genetic proximity” on this blog. We don’t know what truly matters yet, and it is likely that we also don’t have it anymore. Thus, I fear that I have to say that claiming “we are genetically breeding-back the aurochs” is the same kind of simplicity once practised by the Heck brothers, just carried onto the next level – even if you back it up with marker or SNP analyses. Please do not get me wrong, this is just the personal opinion of a biology student sitting in front of its laptop, therefore I am open for any critique.


Literature

1 Park et al. 2015: Genome sequencing of the extinct Eurasian wild aurochs, Bos primigenius, illuminates the phylogeography and evolution of cattle.

2 Visscher 2008: Sizing up human height variation.

Wednesday, 23 August 2017

The Indian aurochs III

In 2013 and 2015 respectively, I did posts on the Indian aurochs, Bos primigenius namadicus, the wild predecessor of zebuine cattle, each time supported by an artistic reconstruction I made for the subspecies.
However, in this post I want to start from a new and go systematically over what we know about this divergent subspecies and what it might have been like. Bos primigenius namadicus is very enigmatic – based on current evidence, survived until 8.000 years ago at maximum, and there are no unambiguous artistic references and of course no literary references to the wildtype of the zebu.

Before we dive medias in res, I want to define some expressions: when I use the term “taurine clade”, I do not refer to taurine cattle exclusively, but all animals that are on the branch of taurine cattle since the branches of taurine and zebuine cattle diverged, so also B. p. africanus and B. p. primigenius of course. The same goes with “zebuine clade”, it includes also B. p. namadicus. The namadicus-clade is therefore synonymous with the zebuine clade, and the primigenius-africanus clade with the taurine clade (note that I am talking about clades).

Phylogenetic evidence

While B. p. primigenius and B. p. africanus were pretty much alike, seems as if the Indian aurochs was a kind of the “weirdo” of the species, and its descendants, zebus, are still the weirdoes among cattle. Part of the reason might be that the namadicus-clade split up from the primigenius-africanus clade rather early about 1,7-2 million years ago [1]. That is more than one million years earlier than the oldest remains that have been assigned to Bos primigenius [2]. That is ten times the time distance between the domestic horse and Przewalski’s horse [3]. One might ask why listing them as part of one species then, and indeed the use of zebus as a species separate from that of taurine cattle is widespread. However, taurine cattle and zebuine cattle still hybridize readily without any problems, what supports classifying them and of course all their ancestors back to their point of divergence 1,7-2mya as one species. It is my preference to refer to this species as Bos primigenius. Nevertheless, meiotic chromosome pairing abnormalities in zebuine x taurine hybrids suggest that postzygotic isolation mechanisms were starting to develop, what suggests a beginning state of speciation [1]. But subspecies are always “incipient species”, as already noted by Darwin more than 150 years ago. As taxonomy is often subjective and species definition is a very tricky issue (I have been planning to do a post on that topic for a while), the differences might be enough for some to regard the members of the taurine clade (and therefore also B.p. primigenius and B. p. africanus) and the zebuine clade (and therefore also B. p. namadicus) as separate species. In this case, the Indian aurochs would be no aurochs, but you could still call the species like this, since it is its Indian sister species. An alternative suggestion that I have would be wild zebu for the wildtype. You can still use the term wild zebu if you consider namadicus an aurochs subspecies.

So it seems that the ancestors of Bos primigenius namadicus migrated pretty early from Africa to India, while the common ancestors of the primigenius-africanus clade stayed in Africa for another while. This early divergence alone probably might explain a part of the differences we see between namadicus and other aurochs populations.

Ecology

If the Indian aurochs was ecologically similar to other wild members of the species, it would have preferred semi-open landscapes near rivers and floodplains. In India, it was sympatric with gaurs and water buffalo, and their respective ecological niches supports this idea: gaurs prefer to live in more forested areas, and water buffalo prefer more wet habitats. Therefore, we would see niche partitioning between the three bovine species if the Indian aurochs was ecologically comparable to the other two aurochs subspecies. Modern zebus often are adapted to very arid habitats, but that is probably a consequence of the fact that they are the cattle of people living in very arid regions. So this does not necessarily indicate that namadicus was particularly adapted to arid habitats.

Morphology and external appearance

The morphology and life appearance of Bos primigenius namadicus is not very well known. In contrast to B. p. primigenius, where we have plenty of well preserved, more or less complete material, we only have fragmentary postcranial material and a few skulls for the Indian subspecies (van Vuure, 2005). In order to resolve what namadicus’ life appearance was like, we have three sources of evidence and clues:
- Direct evidence
- Parsimony based on phylogenetic bracketing
- Zebuine traits that might be wildtype traits of namadicus
Now let us have a look at what we can deduce for the life appearance of B. primigenius namadicus, the Indian aurochs or wild zebu if you will.

Direct evidence:
Direct evidence is what the bone material tells us. We have no unambiguous artistic references to namadicus (this rock art might or might not show an Indian aurochs; the horn tip might be tufted to it could also be a Kouprey). So we have to rely solely on the scarce bone material.
It seems that the Indian aurochs was smaller than the European subspecies. I was unable to find measurement data in the literature. I remember that Cis van Vuure mentioned that the size of one Indian cranium was comparable to that of an European aurochs cow. So perhaps 150-160cm is reasonable for bulls of the Indian subspecies, which was the lower size end for European aurochs bulls.

This is the only illustration of bone material from namadicus that I was able to find. I will refer to it as the Lydekker skull. It is not clear which sex it is; its eye sockets are not nearly as expressed as in European aurochs bulls, and it is narrow in build. This does not necessarily imply it is a female skull. Perhaps bulls of namadicus had a narrower skull with less prominent eye sockets. We cannot say yet (at least I cannot, having seen only this one skull). The drawing also does not enable to tell the exact orientation of the horns relative to the skull, but it is apparent that they face more upwards and less inwards than in many European skulls. The literature says that the horns of the Indian subspecies were larger in proportion and more wide-ranging than in European aurochs. Considering that basal aurochs and basal members of Bos (such as B. acutifrons, B. buiaensis) had very large horns, it would perhaps be more correct to say the horns of the Indian aurochs never shrank down, while some single Northern European aurochs had horns that were not necessarily what I understand as “large” (see here, for example). What definitely changed as namadicus diverged was the orientation of the horns. Basal aurochs had horns at a rather sharp angle (45° in the case of the oldest cranium), inherited from its ancestors. The horns of the Indian aurochs were definitely more upright. This might have been a result of genetic drift. So being proportionally large, wide-ranging and comparably upright, the horns of the Indian subspecies might have resembled those of Heck cattle of the Wörth lineage (see here, for example) and a number of Watussi albeit not that huge. This analogue also fits the Lydekker skull.
This is all that we can say based on osteological evidence.

- Parsimony based on phylogenetic bracketing
This is a way of reasoning that enables us to deduce traits that namadicus must have had even if we have no direct evidence for it. When both its direct descendants (zebus) and closest relatives (taurine cattle, the other two aurochs subspecies, other Bos, Bison or buffaloes) show a specific trait, it is almost certain that namadicus had it as well because it is the most parsimonious and therefore most likely assumption. For example, almost all wild bovines show a more or less slanted pelvis that we also find in zebus. So it is very likely that the Indian aurochs also had a slanted pelvis creating a rounded rear (perhaps not that extreme as in some zebus). A more horizontal pelvis seems to be an autapomorphy of the taurine clade that might have originated in Bos primigenius primigenius or the common ancestor of  africanus and primigenius already, and probably was also transferred to the wisent by hybridization (the wisent seems to be a hybrid species of Eurasian aurochs and Bison priscus [4]).
Very likely the Indian aurochs was a long-legged animal with a square-like build. It is a common trait in wild bovines and zebus also often are comparably long-legged (or actually short-trunked compared to derived taurine cattle). Most likely the Indian aurochs also possessed the high processus spinosi in the shoulder region for large muscles to attach – a universal, functional trait for wild bovines and evident in each single skeleton of Eurasian aurochs. The size of this hump (not to be confused with the zebuine hump, I’ll come back to that later) might have been different in namadicus, it might have been larger or smaller. One would have to look at the relative length of those processes if some are preserved.
Zebuine cattle have the same E+ allele that is also found in taurine cattle causing wildtype colour, so it very likely was the base colour for the Indian aurochs as well: lightly coloured muzzle ring and dorsal stripe, degree of Eumelanin dependent on testosterone level. Probably the colour was very similar to the European aurochs, but not totally. The allele(?s) that regulate the distribution of the pigment in the coat seem to have diverged a bit in zebus. For example, in taurine cattle the snout is strongly melanised all the way down to the muzzle ring, creating a sharp edge for the muzzle ring. In zebus there is more like a smoothtransition. In the less eumelanised individuals you do not see a saddle like in taurine cattle, but a light area on the lateral side of the trunk that looks similar to the saddle though. I call it the lateral zebu saddle. So there are subtle differences that might have been inherited from namadicus as a result of the long time of divergence. Furthermore, there are some zebuine colour modifiers as well, but we are going to look at these in the next section.
Very likely the Indian aurochs also showed a strongly marked sexual dimorphism. Sexual dimorphism is universal to all other members of Bos (at least in the species whose life appearance is known), although the dichromatism is quite reduced in most gaur populations. It is the result of the social and mating system of large bovines (harem system), and I have not heard of any differences in zebus on this aspect. So it is very likely that namadicus also had a strongly marked sexual dimorphism.

- Zebuine traits that might be wildtype traits of namadicus
Zebuine cattle have a lot of quite characteristic traits, and it is possible that some of them might be in fact inherited from their wild ancestors. This suspicion is especially strong if these traits are universal among zebus or at least very common. These traits include:
- the fleshy zebuine hump
- hanging ears
- zebuine colour modifiers
- the large dewlap

However, prevalence in the domestic population of the modern zebu alone is not an argument for its presence in the Indian aurochs. The abundance of these traits can also be explained if it appeared directly after domestication, was preferred by artificial selection or became fixated by genetic drift. But if those traits apparently serve a functional purpose, especially if this purpose would have been advantageous for the Indian aurochs, we can speculate that they are in fact wildtype traits.
The fleshy zebuine hump, however, seems to serve no purpose for the animals. It is formed by a hypertrophied Musculus rhomboideous and does not influence food storage or thermoregulation [5]. It might actually be of disadvantage for the animals because male zebus lack the big muscular neck bulge that is typical of taurine bulls and many other large bovines as a consequence of the altered neck musculature. Farmers use the hump of zebus to attach the plough on their shoulders, so the zebuine hump might have been a mutation that was preferred by farmers and is thus prevalent in zebuine cattle now.
Many zebus have large, hanging ears. Hanging ears are, in various shapes, a typical trait of domestic mammals of any species, so it was probably not a feature of the Indian aurochs. We cannot rule out that its ears were a little bit lowered compared to other bovines, but if so probably only to a shallow extent like in this individual but not huge hanging ears like this cow has.

As for the zebuine colour modifiers, it becomes a little bit more complicated. The Agouti allele that causes red pigmentation to disappear and creates a grey colour is very widespread among zebuine cattle, and I think it originated in the zebuine clade. Zebuine cattle introgressed a lot into Steppe cattle which originated in the west of the Eurasian steppe6, and the vestiges of zebuine introgression are very apparent in their looks (upright horns, large dewlap). Probably the Agouti mutation found its way into Steppe cattle by introgression from zebus. Steppe cattle further influenced many other cattle such as those on the Balkans, Italy and the Alps. The Central European breed Grauvieh shows the same Agouti mutation, and it even found its way until the primitive Iberian breed Tudanca. The Agouti mutation is likely a zebuine allele, but is it a wildtype legacy of Bos primigenius namadicus?
It is not entirely impossible. Colour does not have such a great impact on evolutionary fitness in large animals as in smaller animals, and genetic drift often play a role as well, so it could well be plausible that the greyish colour of zebus (i.e. the lack of red pigment caused by the respective Agouti allele) originated in namadicus. But considering that it was a tropical bovine, I think it is less likely; tropical animals often tend to be more melanised than those of temperate regions (Gloger’s rule; however, one has to be careful with the so-called ecogeographical rules; I am planning to do a post on that as well), and other tropical bovines and bovids show very vivid colours as well. So I tend to think the Agouti dilution allele is a mutation that occurred after domestication. One would have to test this locus on aDNA of B. p. namadicus (which would be very difficult to acquire because of the preservation circumstances in India).

Authors also describe the so-called “zebu tipping gene” that causes a light colour on the ventral side of the body (van Vuure, 2005). But I wonder if this is truly confirmed to be an additional locus in zebuine cattle (and since Bantengs also show very light areas between the legs, it could also be a basal gene that was lost again in the taurine branch), or if the light ventral hairs we see in zebuine cattle are just caused by an allele on a locus that is shared with taurine cattle, since wildtype coloured taurine cattle also often have light areas between the legs. It is actually part of this colour scheme, but to a varying extent. In this case, we can only guess whether it was present in namadicus or not. The same goes for the lateral zebu saddle for bulls.
Another trait that is often displayed by zebus are white ocular rings. Ocular rings in the adult stage are widespread among bovids, and in taurine cattle it is found in many calves but often lost in adulthood. It is very common in wildtype coloured zebu cows (also in taurine cows having the Agouti dilution; it is a stable trait in Tudanca cows), but rarely also found in bulls, such as in this miniature zebu bull from Germany (photo by Markus Bühler):
The bull at the right has a plausible colour scheme for Indian aurochs
(photo: Markus Bühler)
However, miniature zebus might be prone to display juvenile traits, what might be the reason for this bull having ocular rings. Here once again we can only speculate if adult Indian aurochs had ocular rings, and if only the cows had them or even the bulls as well. A colour scheme like that one shown by the bull encountered by Markus Bühler looks really aesthetic to me, it also has the lateral saddle and the lightly coloured ventral side, and also looks very credible for a tropical bovid. But we cannot know.

The large dewlap is another typical, universal zebuine trait. Eurasian aurochs evidently had a short dewlap, and according to stone engravings that of African aurochs was short as well. Nevertheless, I do not rule out that the Indian aurochs was the subspecies with the largest dewlap, at least for bulls. It is a general rule that bovids/bovines in tropical climates tend to have large dewlaps for display and perhaps also thermoregulation, while it is counteracting in temperate climates (heat loss), which is why bovids/bovines in temperate climates tend to have furry ornaments like beards or manes. So the dewlap of the Indian aurochs might have been actually a little longer, perhaps comparable to that of gaurs, bantengs and koupreys and was later exaggerated in zebus. It could have also been as long as in zebus. We cannot know because do not have unambiguous art that could give us a clue. I like to restore B. p. namadicus with a dewlap that is comparable to the related bovines I just named.

Interestingly, while the European aurochs reportedly had frizzy forelocks which are found in many taurine breeds, no zebu has this trait. This matches with the assumption that furry ornaments are a characteristic of bovids in temperate climates (additionally to that, I have the little suspicion that the curly forelocks might be a consequence of bison introgression into European/Eurasian aurochs; we know that aurochs and bison hybridized, it must not have happened exclusively in one way).

All in all, the picture we have of the Indian aurochs is not that frustratingly incomplete as the bone material we have because we can infer some things; we know that it was smaller in overall size but had large and wide-ranging horns that were a bit more upright, it probably had a typical morphology of a wild bovine and thus was comparable to the other two aurochs subspecies, it probably had a similar base colour and sexual dimorphism but we have to be unsure about some details of its colour or the length of the dewlap.
Taking all that into account, I created this new reconstruction of a bull and a cow (I was, surprisingly, inspired by this photo of two English longhorns for perspective and stance). I decided to do two versions: one showing a bull with the zebuine lateral saddle and one without.

Using zebuine cattle in breeding-back

In the previous posts on the Indian aurochs, I suggested doing a “breeding-back” project with zebuine cattle. Looking at modern zebus, it is apparent that there are seemingly no truly primitive zebus left that resemble their ancestor to a large degree. This could be the result of genetic bottlenecks or very strong artificial selection. But it should be possible to select on traits that we know or can infer with a high degree of certainty. I suggest a mix of Watussi (for the horns), miniature zebus with taurine influence as few as possible (for the colour) and autochthonous Indian zebus like Guzerat/Krankeji, Kenkatha and Javari. It should be possible to fully reconstruct the horns of namadicus especially with the aid of Watussi, to get the right colour scheme also with some degree of sexual dichromatism, and a square-shaped build. Obvious domestic traits like overly hanging ears or a hanging spine should be avoided.
I did a photo manipulation by copying suitable Watussi horns on a photo manipulation by Jochen Ackermann on Wikimedia commons he did using a Taurus bull and a zebu:

That is what I envision such a “breeding-back zebu” to look like. For some aspects, such as the presence of the lateral saddle, no strict standards should be set, because we do not know about their presence in the wildtype. The funny thing is, I came up with the idea of a zebu “breeding-back” project in a dream were I saw zebus that looked exactly like the photo manipulation above back in 2011. Such a project should best be carried out in India so that the animals are automatically suited ecologically. Releasing them on a sufficiently large area size and leaving them exposed to natural selection will certainly alter their morphology in a way that could provide us some clues on the wildtype. Their morphology would become more like those of wild bovines, their horn shape might become refined, and perhaps even the zebuine hump might disappear after some generations if it really serves no purpose to the animals. It would be very interesting to watch that.

But this section is also about using cattle with zebuine influence in existing “breeding-back” projects focusing on European aurochs. It is controversial and especially focusing on the use of Watussi. I see that in my comment sections and also forum discussions. People often have objections against cattle with zebuine influence because the zebuine clade diverged a very long time ago, zebus have a pretty divergent morphology, and are adapted to arid and hot climate – not what is needed in Central or Northern Europe. Those are good reasons to be averse to cattle with zebuine influence in “breeding-back” projects for the European aurochs, but now I am going to explain my point of view to this subject very clearly.
First of all, and most important to note, the fact that they belong to a clade that diverged long time ago per se does not imply cattle from this lineage have to be destructive. Actually, if the Indian aurochs was still extant but the European one extinct, I would use it in “breeding-back” projects at any time in order to get beneficial wildtype traits.
Zebuine cattle have a very derived morphology, that is true. But for “breeding-back” projects, I do not care if an undesired allele is of zebuine or taurine domestic origin, because it is undesired in any case. The recessive Agouti allele, that is widespread in any “breeding-back” project/breed, is not desired for the European aurochs, no matter if it originated in taurine cattle, zebuine cattle, or even the Indian aurochs – so it makes no difference. The zebuine hump is not desired for the European aurochs, in the same way the overly long hair of Highland cattle or the virtual lack of sexual dichromatism in Sayaguesa is not desired for the European aurochs – again, it makes no difference on which clade this mutation originated because it is not desired in the population anyway.
The next and very important point is that whatever undesired trait you introduce to the population, you can breed it out again. The fact that zebuine cattle are adapted to hot and arid climate, and therefore have a short and less dense coat and less subcutaneous fat does not mean that using zebuine cattle will result in all crossed cattle being less well adapted to cold and wet environments but the traits are going to split up in the second cross generation and you have to pick the right individuals – just as with any undesired and desired traits. Steppe cattle, for example, are heavily influenced by zebuine cattle yet they are among the cattle that are best-suited to cold habitats and have a supreme winter coat, which is why they are used in all “breeding-back” projects. It not only depends on what you crossbreed, but also on what you select for.

The amount of desired vs. undesired traits of course implicates how to use a breed. To give an example, I take the Watussi crossbreeds in the National Park Hortobagy. I was told that second-generation crosses with Watussi influence often turn out unsatisfying. This is not surprising: Watussi have a number of undesired traits (zebuine hump, zebuine body, large dewlap) versus one desirable trait (horn size). So the likelihood for a second-generation cross to end up disappointing is larger than for it ending up pleasant. But that does not mean that Watussi is not a good choice for breeding, not at all. It means that the breed has to be used a bit more cautiously than breeds with a lot of desired traits and that one has to be a bit more patient and lucky to get really good second-generation crosses.

It is rare that Heck cattle can serve as an example for efficient selection, but the Wildgehege Neandertal, a German zoo, did a very good job in creating and spreading very large-horned Heck cattle without maintaining any of the undesired Watussi traits. They did this by using one Heck x Watussi cow in the 1950s, and using her quarter Watussi son as a breeding bull. While the other Watussi traits got reduced to almost nil after decades, they always kept those individuals with large horns. Nowadays there are a lot of Heck cattle that look totally taurine but have impressively large horns like aurochs. Only in a few you can see the Watussi descent (like this Bavarian Heck bull), but as I said, I regard an undesired domestic zebuine trait not anymore worse than undesired domestic taurine traits. The Neandertal lineage left a big mark on the German Heck cattle population and it did not have any negative effects on the cold tolerance of the cattle at all.

So I see no problem with using a breed that is either zebuine or zebuine influenced if it contributes precious traits. One just has to know how to use it wisely and breed out the negative traits, just with any other breed.

Literature

1 Hiendleder, Lewalski, Janke: Complete mitochondrial genomes of Bos taurus and Bos indicus provide new insights into intraspecies variation, taxonomy and domestication. 2008.
2 Martinez-Navarro et al.: The Early Middle Pleistocene archaeopaleontological site of Wadi Sarrat (Tunisia) and the earliest record of Bos primigenius. 2014
3 Ryder et al.: A massively parallel sequencing approach uncovers ancient origins and high genetic variability of endangered Przewalski’s horses. 2011.
4 Soubrier et al.: Early cave art and ancient DNA record the origin of the European bison. 2016.
5 McDowell, McDaniel, Hooven: Relation of the rhomboideus (hump) muscle in zebu and European type cattle. 1958. 
6 Decker et al.: Worldwide patterns of ancestry, divergence and admixture in domesticated cattle. 2014.

Cis van Vuure: Retracing the Aurochs - History, Morphology and Ecology of an extinct wild Ox. 2005.