Likewise, a phylogenetic study of Papaver based on DNA sequences was unable to distinguish these two taxa The opium poppy P. Papaver setigerum is an annual plant, 60 cm high, a field weed occurring in disturbed grounds 14 and native to the western Mediterranean 6 , 14 in Algeria, France, Italy, Morocco, Portugal, Spain, Tunisia The history and mechanisms of opium poppy domestication remain unclear despite the abundance of archaeological seeds in sites dated to the Neolithic period, particularly in the Alpine Foreland 5.
Archaeobotanical remains are usually broadly identified as P. The domestication syndrome of opium poppy encompasses the increase in the size of the capsule and seeds, as well as capsule indehiscence 6. Previous studies attempted to distinguish wild from domestic opium poppy seeds based on the size, comparing archaeological seeds to modern species 17 , 18 , 19 , 20 , 21 , However, the size of the seed alone has not proven to be a good discriminating criterium 20 since it overlaps between the two species.
This paper addresses two questions: i can we distinguish between modern seeds of the wild Papaver setigerum , domestic P. If so, ii can we distinguish Papaver species in archaeological assemblages previously identified as P.
Two additional sets of 30 seeds of P. We first established new identification criteria between Papaver species, and chiefly between P. We applied traditional and geometric morphometrics on seeds, considering the number of cells, size measurements and shape using outline analysis.
Outline analysis has been successfully used to identify archaeobotanical remains of an array of species such as grape pips 23 , olive stones 24 cereals 25 , 26 , 27 , dates 28 and cherry stones The technical challenge for Papaver seed lies in the millimetric size of the seeds and their globoid shape. Prior to any morphometric analysis, repeatability tests were performed to establish the effects of taking the photos, cleaning and landmarking in the observed seed morphometric variation. This site is an ideal starting point since the Swiss Plateau is outside of the natural area of spread of P.
Furthermore, the waterlogging conditions maintained the original seed shape and size, unlike what is known to occur to charred remains The error measurements was quantified by acquiring data 3 times on 5 seeds independently for the three species Table 1 , which allow to test for the different steps of the protocol: positioning, image cleaning and landmarking.
These three steps yield contrasting results. This originates from the difficulty to orientate the seed consistently under the stereomicroscope, due to the small size and globoid shape of poppy seeds.
On the other hand, cleaning and landmarking errors are much lower. Despite the existence of a certain positioning error, this does not prevent taxonomic identification see below and the protocol can therefore be used for the purpose of this study.
Length and width show considerable variation with significant differences between the various Papaver species Fig. The two domestic species P. The seeds of P. For both species, the two investigated samples are close, yet some dimensions appear different Wilcoxon rank tests, P. The number of cells also present differences between species Fig. P somniferum and P. Boxplot of measurements number of cells, length, width, made on the modern seeds.
Length and width are natural logged. Archaeological seeds are presented in red. The first two PCs Fig. Asymmetry mostly represents intraspecific variability. It is higher for the species with the most rounded seeds P. Principal component analysis performed on shape coefficients. Archaeological seeds red dots are added as supplementary individuals, i. The species with the most elongated seeds P.
Shape overlapping is particularly important between P. The hierarchical clustering performed on the euclidean distance matrix computed on the coefficients averaged per taxa confirmed the shape proximity between P. The slight differences in shape between the species of the P. The unrooted tree obtained with hierarchical clustering on the Euclidean distance matrix between Fourier coefficients averaged per taxa.
Mean shapes pairwise comparisons for all Papaver taxa studied here. Orange colour corresponds to the taxon of the rows and the blue colour to the taxon of the columns. The linear discriminant analyses LDA on modern material allowed a good identification at the species level. Benchmarking of linear discriminant analyses on all species and using different proxies. Accuracy per classes and their variability were obtained using permutations on classes-balanced dataset with the error bars.
Although the performance of individual variables length, width, shape and cell number provided relatively good discrimination, the best percentages were obtained when all traditional and shape parameters were combined Fig.
The results were similar if we considered only the species from the P. Length and width showed considerable variation between modern seeds of Papaver and archaeological seeds Fig. The LDAs using various sets of descriptors were used to infer species on the archaeological material. Some seeds were identified as P. When all criteria are combined, no seed is allocated to P. In every case, and more especially when all criteria are combined, about half of the seeds are attributed to P.
Assignation of the 33 archaeological seeds: Benchmarking of linear discriminant analyses on A three taxa P. Here we show that the combined application of morphometric descriptors, number of cells and shape analysis outline elliptic Fourier transforms EFT allows the discrimination of seven modern species of Papaver genus.
In spite of a high positioning error, due to the small size of this material, morphometrics can be done. The various species and sub-species are well discriminated, which validates the methodology used Table 1. Indeed, the most interesting result was that by using this method, with all descriptors, the LDA gave optimal results when distinguishing between P. The second finding of this study was that allow for the first time the application of this method to archaeological seeds.
The first question that this study sought to answer was if it is possible to discriminate between the different taxa. The LDA results Fig. According to our results, adding the cell number to the size descriptors gives a better prediction for P. These are also the taxa where the shape yielded the lowest additional accuracy.
A possible explanation for this confusion might be that their shape is identically reniform as seen in Fig. Instead, shape is a powerful descriptor in the case of P. The results of the unrooted distance network Fig. On the one hand, Carolan et al. On the other hand, P. Surprisingly, it is a critical discriminant variable in the identification of P.
The importance of cell number, as well as cell size, were also the previous observed 18 , 20 , 21 , however, according to our results the total number of cells from one of the seed face gives better discrimination results than solely count the cells by rows or measuring the cells 18 , 20 , The results show that it is possible not only to distinguish P.
Previous studies on the Papaver genus encountered problems distinguishing mainly P. Based mainly on size, other authors 16 , 20 stated that the high variability within the species P. Future research may include a higher number of varieties and accessions to better assess the intra sub specific variability in terms of cell number, size and shape, including in terms of environmental conditions In this case, it is essential to combine all descriptors and not only the shape. Nevertheless, one should consider the caveats of using modern material to classify archaeological specimens and of the preservation of archaeological remains.
In order to develop an understanding of the possible effects of taphonomic factors in the future, two tasks should be implemented. One would be to increase the number of samples, and the other one should be to perform experiments to replicate the state of the archaeological seeds. Nevertheless, the archaeological material results suggest that well-preserved waterlogged seeds of Papaver species can be used for this type of analysis.
In our results, 18 seeds were attributed to P. This opens several scenarios to explain the process of domestication of poppy.
There is evidence of the use and potential cultivation of opium poppy in the Western Mediterranean since ca. The authors of both archaeobotanical studies actually suggest that opium poppy was cultivated, based on the number of finds and their ubiquity 3 , Nevertheless, it is possible that the plant was still morphologically wild since these sites fall within the native area of P.
Thus, it is unclear if opium poppy spread northwards as morphologically wild, not fully domesticated, or as a domesticated plant. It is actually possible that opium poppy arrived at the Alpine foreland as a domesticated form along with some wild P. The domestication process could have been accelerated with the beginning of cultivation of opium poppy outside of the area of the natural distribution of the wild subspecies such as in the Alpine Foreland around BC After ca.
This may be interpreted as an indication of a protracted domestication process 36 , as observed with other plants domesticated in the Neolithic period. In order to test this hypothesis, similar analyses should be performed on opium poppy seeds from archaeological sites located in the Western Mediterranean region. Another critical factor is that some of the seemingly early finds of opium poppy seeds outside of P.
Early deposits found in Israel 37 but also in central Europe 38 , 39 lack radiocarbon dates on the seeds or on direct contexts where the seeds were found. Future studies on poppy seeds should integrate the morphometric as well as the direct dating approaches Likewise, it is foreseen to attempt to obtain aDNA from the archaeological seeds and so confirm, if possible, their status as domestic or cultivated.
The present paper provides the first results of geometric morphometrics for Papaver taxa. The combination of descriptors such as the number of cells, size and shape of different modern species of Papaver allows to classify the seeds with good accuracy despite the methodological challenge due to the small size and globoid shape of poppy seeds.
The seeds were actually distributed within these two subspecies in equal parts, which might suggest that the plant has not yet acquired the morphometric characteristics of modern domestic seed. Further studies should be done in order to test the classification model. Future research should consider the study of opium poppy seeds from historical periods to confirm their assignation to the domesticated subspecies, as well as the study of earlier Neolithic finds in the Western Mediterranean in order to trace the pace of the domestication process.
A total of 33 whole and well-preserved uncharred waterlogged seeds with visible cells identified as P. All seeds were obtained from the sample Located in the northern shore of the Lake Zurich, eight settlement phases were identified and dendro-dated to — BC.
In this late Neolithic site, archaeological deposits related to pile-dwelling houses are preserved in a waterlogged state where thousands of plant remains are present in charred and uncharred states, especially in layer 13, large quantities of opium poppy seeds were found concentrated mostly within building limits These analyses were non-destructive and therefore no special permissions were required.
Niels Bleicher Office for Urbanism Zurich. All Papaver seeds were photographed from a lateral view, with the hilum to the right. In this angle, it shows the cells, including those close to the hilum Fig. The broader part of the seed at the bottom. Table Comparison of alkaloids identified on harvested and thermally processed poppy seeds.
This update related to the detection of morphine, codeine, oripavine, noscapine, and papaverine in poppy seed samples whereas, the previous report related only to the levels of morphine entering the food chain. Codeine values were given in relation to morphine equivalents, using a conversion factor of 0.
Noscapine and papaverine were considered in the most recent publication however, the data that was available to the EFSA did not allow for a hazard characterization but they did conclude that the presence of these compounds would not present a health concern.
In relation to the presence of thebaine and oripavine not included in the work of this paper , it was concluded that there was insufficient data to make any assessment. Based on these updated EFSA findings, the presence of the morphine and codeine in the poppy seeds analyzed in this work, would still would still fall below the recommendations outlined.
There is also little or no information on packaging of poppy seeds regarding what, if any, treatment has taken place prior to packaging. Since the ingestion of poppy seeds has been used as reasons for failure of workplace drug testing and roadside drug testing, more should be done to ensure as much information as is possible is available on the preparation methods of the seeds. The datasets generated for this study are available on request to the corresponding author.
This work was approved by the Northumbria University Ethics committee. The university holds a UK Home Office Drug License for the storage and use of controlled drug standards and for the extraction of alkaloids from poppy seeds. The laboratory work, analysis of data and writing was carried out predominantly by MC. JD and JA helped in the design and review of data and interpretation and all parties contributed to the writing and review of the article. All authors contributed to the article and approved the submitted version.
This work was funded with the support from the Department of Applied Sciences, Northumbria University. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Plants 8, 41— Straightforward analytical method to determine opium alkaloids in poppy seeds and bakery products. Coloured zincograph, c.
Burnett, M. Available online. Licence Public Domain Mark. You can use this work for any purpose without restriction under copyright law. Credit: Opium poppy Papaver somniferum : white flowers, seed capsule and seeds. Public Domain Mark. Selected images from this work. View 1 image. About this work. Description Opium poppy seed capsules yield opium and 25 different alkaloids used in medicine. Physical description 1 print : zincograph, with watercolour.
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