Thursday 23 November 2017

The earliest wheats of Ukraine (5400 BC)

The eastern areas of Europe and their transition to the steppe that lead to Central Asia remains one of the less well-studied regions archaeobotanically. The sparseness of reliable evidence has meant that the region is sometime discussed in terms of an alternative eastern source of crops from Europe, in addition to the main thrust from Anatolia through Greece and the Balkans, and it is sometimes mooted as a region of some crop origins, such as spelt wheat. New data is always welcome, especially of a high empirical calibre, from systematic sampling and backed up by AMS dating.

New data from the Ratniv-2 site in Western Ukraine, near the eastern frontiers of the Linear Pottery (LBK) culture, has been published by Motuzaite Matuzeviciute and Telizhenko in Archaeologia Lituana. This is an important record of early crops, and as the authors point out, it clearly points to similarities to the West and Southwest in Neolithic  Europe and suggest a spread towards Ukraine from the West in the Neolithic, rather than the east. The assemblage consists of wheats, barley, flax, lentil and pea. Two direct AMS radiocarbon dates on emmer wheat grains place these assemblages between 5400 and 5200 cal.BC. Of particular interest is that the wheats here include not just einkorn and emmer but apparently at some of the socalled "new type glume wheat," which these and other authors sometimes equate with Triticum timopheevi, and 20th century relict wheat found north of the Caucasus (western Georgia). That the archaeological "new type" has the AAGG genome of timopheevi remains unproven-- although I agree it is likely. It is perhaps more accurate to regard T. timopheevi as the relict remnant of what was a once a much more diverse and widespread species of wheat, which in all likelihood originated in the Anatolia and spread through many part of Europe and east through northern Iran in the Neolithic. I have sometimes offered the name "striate emmeroid" as a descriptive alternative to  "new type", as it is hard to think of something that has been largely extinct since the Bronze Age as new, and this wheat type has been in discussion by archaeobotanists for around 20 years...

In any case, what is notable about this assemblage is that is corresponds to those crops that are most common in the Neolithic of southwest Europe, supporting the ceramic and settlement evidence that attributes to the origins of agriculture in western Ukraine to spread from the west.

The lost rice of South America

One of my pet interests is lost crops, or largely forgotten ones-- species that were important in the past which are either completely lost from cultivation today or very nearly so. They serve to remind us that the ethnographic present does not provide a full range of potential economic activities nor the full range of crops. They demonstrate that archaeobotanical evidence can provide important broadening of our list of potential crops to consider in future breeding and sustainability efforts. An endemic rice of South America can now be added to the list of lost crops.

This exciting find, that received quite a bit of media attention (e.g. in Science) was the recent report of a rice that was apparently undergoing morphological change, i.e. domestication. The archaeobotanical evidence, published in Nature Ecology and Evolution last month by Hilbert, Iriarte and colleagues as part of the ERC Pre-Colombian Amazon Scale Transformations project, comes 

from  phytolith anaylses through a stratigraphic sequence at the site of Monte Castelo in southwest Amazonia, dating to between 5300 BP and 700 BP, which includes rice husk phytoliths (the double-peaked cells) and bulliform throughout. The proportion of rice increased somewhat in the past 4000 years, but shifted especially towards a much higher ratio of husk types to bulliforms, suggesting the concerntration of husk phytoliths that one might expect from dehusking or harvested rice spikelets. It is at this stage that the shape of the husk phytoiths also starts to change, with phytoliths getting wider, taller and with more pronounced peaks. These are the kinds of changes that may be indicative of a domestication process and be a proxy for increasing grain size. The change takes places somewhere in the upper levels of the site, which are unfortunately not well constrained in dating, except being younger than 4000 BP and up to 700 BP or so. This suggests that the inferred domestication process took place or was even ongoing upto shortly before Colombian arrival from Europe and Amazonian population decimation.

One can quibble over whether changes in husk and inferred grain size increases must be human caused. The classic case of such a change in Chinese rice is decidedly NOT about domestication, as it takes place in sites South of Yangtze at the transition from the LGM to much warmer conditions, i.e. around 18,000-16,000 years ago. A full 10,000 years before the appearance on non-shattering rice spikelet bases appear-- domesticated by definition. When originally published by Zhao (1998 Antiquity) this was mistakenly dated to the Holocene, and thus inferred to represent domestication, but this falled a false equations (domestication because change is start of Holocene; start of Holocene because advent of ceramics could not possibly be any earlier). We now know the advent of ceramics transition in China took place around 18,000 years ago at Yuchanyan and possibly even earlier at Xianrendong, as already discussed for at least 7 years (e.g. Fuller et al. 2010). while domesticate rice, i.e. that was dependent on humans for dispersal, evolved during the middle Holocene, with the earliest large assemblage of non-shattering spikelet bases at Baligang by ca. 6500 BC and predominance in the Lower Yangtze as late as ca. 4000 BC (for a updated summary see here). It now appears most likley that the morphological change in husk phytoliths in South China was driven by the rapid climate change and especially the increase in carbondioxide which has major repercussions on plant productivity and morphology (see experimental work by Cunniff et al 2010), and thus the near doubling of carbondioxide that took place in the millennia just after the LGM (along with increasing temperature) ought to have hade major effects on rice productivity and aspects of morphology).

However, in the past 4000 years it seems unlikely that there were any climate or carbon dioxide shifts on quite the necessary scale, which makes the inference of a local rice domestication process much more likely.  A shift in grain size, however, would be expected to be accompanied by some selection for reduced shattering-- as this co-evolves in all of our better documented cereal domestications, most notably in Asian rice. Thus good flotation samples, with the required fine mesh of ca. 250 or 300 microns, ought to produce small charred rice spikelet bases. Recent experience suggests that everywhere we look, and do the requisite flotation, in tropical Asia, we find now that rice spikelet bases greatly outnumber charred grains and this tells us that they survive well and are archaeobotanically recoverable. This is also true to the major rice growing areas along the ancient Niger river. Some macro-remains would seem the obvious next step to pinning down more details about the evolution of this lost rice of South America. It would be highly unexpected if selection for larger grains did not take place alongside increases of indehiscent spikelet bases, as these co-evolve in other well documented cereals (as illustrated in a PNAS 2014 article).

It is also highly likely that increase in grain size implies management of soils, i.e. some sort of cultivation. This is contrary to the novel, but rather unconvincing, hypothesis of the authors that grains would have been encased in clay and dropped into the water. They cite as an ethnographic parallel systems of reseeding American wild rice (Zizania palustris) stands in the Great Lakes region of North America. But in that context there is no evidence for prehistoric grain size increase or domestication processes.The rice represented at Monte Castelo was likely a productive annual, as the authors note, and could have been encouraged by burning of competing vegetation after seeds are shed, in which case selection for seed size increase can be expected from the levelled playing field conditions of freshly cleaned fields which put a premium on rapid seedling establishment against competition from conspecific seedlings. 

South America boasts 4 indigenous wild rice species, Oryza alta, O. latifolia, O. grandiglumis, and O. glumaepatula, and only the last has annual ecotypes. South America's O. glumaepatula is also an AA genome, like domesticated Oryza sativa or Oryza glaberrima, and thus this suggests something inherently attractive for, or conducive to, domestication in the AA wild rices. Like Oryza species everywhere these are water-loving grasses, but there are still two ends of a spectrum from perennials in deeper water and annuals and places that are seasonally dry. Oryza alta, which can form mats along river margins, is a perennial (see, for example the photo at left lifted from Duncan Vaughan's 1994 monograph on the wild rices). Annual Oryza are prolific seasonal grain producers, and thus lent themselves easily to forager intensification, and it was such annuals that were ancestral of the early cultivars of Asian indica and aus rices, or African glaberimma (from wild annual O. barthii). By contrast perennial rices are less prolific grain producers due to investment in perennating stems, roots and more leaves. Thus Asian rices when available ought perhaps be expected to be used resources. The other continent with annual AA genome wild rices in Australia, where these are found in the northern parts. Lets see some archaeobotanical work carried out there, in the region of Oryza meridionalis, as one might expect parallel evolution for utilization and even management there.

Tuesday 21 November 2017

Using big machines to look at the finer aspects of seeds

This year has seen three studies on high resolution x-ray computed tomography applied to archaeobotany, one using ct-scanning to recovered chaff hidden in ceramics (see Finding Rice Domestication in Clay), and two using a synchrotron to peer inside seeds, including soybeans and horsegram. past summer, I published with colleague Charlene Murphy, a Scientific Reports article on domestication of the Indian crop horsegram. While this article represents an important contribution on the domestication history of a major crop in India, and evidence for evolution of morphological change during that crops domestication in South India (see also our GRCE paper, reviewing all that is known about horsegram origins), this is really more significant for the methodological contribution to the archaeobotanical documentation of domestication. We were able to put our small archaeological seeds in a very large machine, the Diamond Light synchrotron (shown at left). which allowed us to non-destructively capture the the internal structure of the entire seed (not as straightfoward as it sounds as it takes a lot of computing time). And from this we could measure seed coat thickness on any of the 1000s of cross-section slices through our seeds (like that below/right)
horsegramOne of the well-known domestication syndrome traits in pulses is the thinning of the seed coat, tied to loss of germination inhibition. But it has been difficult to document this archaeologically. Seed coats are often destroyed in charring, but even if preserved they study on charred seeds would require destructive breaking of seeds. And even if damaged, it might only be possible to document the seed coat thickness in one or two places with an SEM or high powered normal microscope. As a result this has been rarely documented, which has lead to a fair degree of speculation on the evolution of thin-seedcoat, readily germinating pulses, as the result of conscious selection of the readymade mutants in the wild (although none have been documented in the present day)-- the domestication before cultivation hypothesis applied to lentils-- or positing a rapid conscious selection by those who initiated cultivation-- lets call this the pea breeding before agriculture hypothesis. The truth appears to be, however, a gradual evolutionary process as seed coats thinned over time, much like the evolution of increasing seed size or the non-shattering in cereals-- at least in horsegram. This can be seen in the chart below showing the thinning seed coat along side a trend in seed size increase in horsegram. Further work is needed on additional pulses to see if this pans out as typical of the pulses domestication processes, or whether there was variation, or indeed any cases of plucking domesticated types from the wild-- of which I am doubtful. At least now we have a method for approaching this.
This is actually, quite logical: established stands of pulses could be maintained and wild-type dormant seeds would constitute an established seed, and would recurrently add new plants to the the stand over a series of years. But due to annual human harvests mutations that reduced dormancy would get selected, and would be particularly important for any new populations planted in areas without existing wild populations. In this context we can expect the gradual evolution for thinner coated, more easily germinating seeds through selection across what are presumably multiple loci, as is evident in our archaeological horsegram data (shown left).

Soybean oil content in charred seeds?
The claim for earliest use of a synchrotron to look at charred archaeological pulse seeds, however, goes to our colleagues in China, in collaboration with Prof. Gary Carwford, Shandong archaeobotanist Xuexiang Chen. They argue that soybean underwent selection for increased oil content in prehistory during domestication-- undoubtedly true-- and that this can be tracked archaeological through a change in the number and size of pores visible on the inside of charred soybeans viewed through the synchrotron and High-Resolution Computed Tomography. I remain unconvinced on this last point, and although the paper reports on examination of modern soybeans and, other oily crop seeds, and experimentally charred seeds none of these are illustrated or really described so as to support this interpretation. The authors infer that more small pore is a product of more oil whereas large pores represent burned out protein, but is this true. The differences look to me more like artefacts of carbonization processes, and not a good proxy for the internal anatomy of the original uncharred soybeans. As the few illustrated example suggest larger and irregular pore are present in seeds with more distorted external surface anatomy (e.g. c), whereas small pores are more evident in better preserved examples (e.g. f).

Unfortunately, the central claim in this paper does not really add up, or at least are not well justified and explained in the text. This makes me very nervous about accepting the main conclusion of the paper, i.e. that the authors have demonstrated an increase in oil content in soybean during domestication by measuring the quantity of bubbles (voids) of different sizes in charred archaeological soybeans. Small voids are attributed to oil content and large voids to protein—but this is never demonstrated (for example in modern and experimental charred examples) or backed up by citations on soybean anatomy, as to why these voids should differ between oil and protein. That soybeans are oily, in contrast to most pulses is clear, but this also has major implications for the nature of archaeological finds. Most carbonized archaeological soybean are poorly preserved, distorted, full of large voids and small voids and very shiny on their interior. This is contrast to pretty much every other pulse I have seen archaeobotanically, from Vigna spp. to lentils and peas to Lablab. Even in the Chinese samples, presumably subjected to similar formation processes Vigna angularis seed present typical features of carbonized pulses, including a dense charred matrix with distinct cotyledons. In Glycine cotyledons are rarely evident and their interiors are heavily distorted by voids and bubbles. The obvious deduction is that this state of things is the result of the oil content in soybeans, and of course many other oily seeds, from cotton to sesame, also tend to show similar levels of bubbling and porosity when charred. If large voids in soybean are due to protein burning up during carbonization then surely one would expect to see this in any pulse, all of which have at least 20% protein content. It is true that the oil in soybean is contained in fresh seeds in many small droplets/sacs but upon charring things are likely to end up being very different. Oils are going to burn to more readily to gas than carbohydrates or proteins and thus create more bubbles and explosions of expanding gas. As this progresses and cracks to the outside of the seed allow penetration of gas (and some oxygen) from the exterior, one would expect this to speed up. The persistence of small voids then might be predicted to be the result of less oxidation, less temperature and perhaps other variables of charring conditions of a given seed. Cracking and penetration of gases into the charring seed may indeed be affected by aspects of domestication—thinning of seed coat, increase in seed size. Indeed, larger seeds seem likey to leave larger parts of their interior cotyledons unexposed to exterior cracks and oxygen; and in this context would be expected to preserved more small oil bubbles as a side effect of seed volume increase: i.e. the difference over time would reflect preservation artefacts rather than selection for genetic change. It is hard to see how at this stage we can deduce difference in underlying phenotype and genetics from this sort of data—at least until we have much better grasp on who charring conditions affect the distribution of seed contents, and this calls for some systematic experiments.
Undoubtedly soybeans were selected for oil content, but when and how this took place in relation to other domestication traits remains sadly unclear. I find I have to reject to conclusions of Zong et al., although their paper doe illustrate the potential analytical power of using a synchrotron to peer inside archaeological seeds