How to discover ploidy levels of charred free-threshing wheat caryopses?

the caryopses using a digital reflected light microscope at 200× magnification to obtain an average cell width. The width values are based on a total of 478 aleurone cell measurements (Table 2). Mean aleurone cell width values were calculated from the cell measurements of the lines of neighbouring cells. The means of the cell widths of the caryopses can be grouped into three classes centred around 28.9 μm, 34.8 and 41.6 μm. During charring, caryopses shrink in length by about 10% or more, but increase in width (Hopf 1955; Braadbaart 2008). Accordingly, archaeobotanical aleurone cell width values should be more than corresponding recent ones. For uncharred recent material of eight provenances, Fritsch et al. (1977) report an aleurone cell width of 29.8 μm for tetraploid free-threshing wheats and 36.3 μm for hexaploid T. aestivum, which corresponds to an increase in cell width from tetraploid to hexaploid by about 22% (Table 1). This is close to the aleurone cell width difference between the size classes 34.8 μm (interpreted as tetraploid) and 41.6 μm (hexaploid) of about 19.6% in the archaeobotanical material (Table 2). As expected, the absolute width averages for both charred (assumed) ploidy levels are higher than for the corresponding recent width values and account for differences of 16.9 and 14.6% respectively (Table 3). Hence, the morphologically identified T. aestivum/durum/turgidum caryopses with aleurone cell widths of around 42 μm are here identified as T. aestivum, while widths around 35 μm may represent tetraploid wheats like T. durum and T. turgidum. The occurrence of both tetraploid and hexaploid freethreshing wheats together, as in the Vráble material, has already been reported for other Bronze Age sites based on rachis remains (Stika and Heiss 2013). The few identifiable Charred caryopses (grains) of tetraploid (Triticum durum, T. turgidum) and hexaploid (T. aestivum) free-threshing wheats cannot be distinguished by morphological characteristics. Consequently, they are mostly referred to as T. aestivum/ durum/turgidum in archaeobotanical contexts. Until now, only rachis fragments can be assigned to a particular ploidy group. For more details and archaeobotanical nomenclature used, see Stika and Heiss (2013). In this short contribution, we present a suggestion how to identify the ploidy level of morphologically identified free-threshing wheat caryopses, based on their aleurone cells. As already published by Fritsch et al. (1977), cells of the aleurone layer, the outermost layer of the endosperm, which lies beneath the seed coat and the pericarp, show a considerable increase of width with ploidy level (Table 1), as the sizes of the pollen grains also do (Beug 2004). This outer layer of the endosperm was exposed in places on some caryopses from the early Bronze Age settlement Fidvár near Vráble, Slovakia (Fig. 1; Schlütz and Bittmann 2015, 2016). It was possible to measure the aleurone cell width for eleven of them. For each caryopsis, the widths of several neighbouring aleurone cells were measured across the width of

the caryopses using a digital reflected light microscope at 200× magnification to obtain an average cell width.
The width values are based on a total of 478 aleurone cell measurements ( Table 2). Mean aleurone cell width values were calculated from the cell measurements of the lines of neighbouring cells. The means of the cell widths of the caryopses can be grouped into three classes centred around 28.9 μm, 34.8 and 41.6 μm. During charring, caryopses shrink in length by about 10% or more, but increase in width (Hopf 1955;Braadbaart 2008). Accordingly, archaeobotanical aleurone cell width values should be more than corresponding recent ones.
For uncharred recent material of eight provenances, Fritsch et al. (1977) report an aleurone cell width of 29.8 μm for tetraploid free-threshing wheats and 36.3 μm for hexaploid T. aestivum, which corresponds to an increase in cell width from tetraploid to hexaploid by about 22% (Table 1). This is close to the aleurone cell width difference between the size classes 34.8 μm (interpreted as tetraploid) and 41.6 μm (hexaploid) of about 19.6% in the archaeobotanical material ( Table 2). As expected, the absolute width averages for both charred (assumed) ploidy levels are higher than for the corresponding recent width values and account for differences of 16.9 and 14.6% respectively (Table 3). Hence, the morphologically identified T. aestivum/durum/turgidum caryopses with aleurone cell widths of around 42 μm are here identified as T. aestivum, while widths around 35 μm may represent tetraploid wheats like T. durum and T. turgidum. The occurrence of both tetraploid and hexaploid freethreshing wheats together, as in the Vráble material, has already been reported for other Bronze Age sites based on rachis remains (Stika and Heiss 2013). The few identifiable Charred caryopses (grains) of tetraploid (Triticum durum, T. turgidum) and hexaploid (T. aestivum) free-threshing wheats cannot be distinguished by morphological characteristics. Consequently, they are mostly referred to as T. aestivum/ durum/turgidum in archaeobotanical contexts. Until now, only rachis fragments can be assigned to a particular ploidy group. For more details and archaeobotanical nomenclature used, see Stika and Heiss (2013). In this short contribution, we present a suggestion how to identify the ploidy level of morphologically identified free-threshing wheat caryopses, based on their aleurone cells.
As already published by Fritsch et al. (1977), cells of the aleurone layer, the outermost layer of the endosperm, which lies beneath the seed coat and the pericarp, show a considerable increase of width with ploidy level (Table 1), as the sizes of the pollen grains also do (Beug 2004). This outer layer of the endosperm was exposed in places on some caryopses from the early Bronze Age settlement Fidvár near Vráble, Slovakia ( Fig. 1; Bittmann 2015, 2016). It was possible to measure the aleurone cell width for eleven of them. For each caryopsis, the widths of several neighbouring aleurone cells were measured across the width of The caryopses with the smallest aleurone cell widths ( Table 2, #028, #006), however, are not morphologically different from the tetraploid and hexaploid T. aestivum/ durum/turgidum caryopses investigated. In contrast, their aleurone cell widths fall within the range of diploid wheat species as represented by cultivated and wild glume wheats (Table 1, T. monococcum, T. urartu, T. boeoticum). Could this imply that free-threshing diploid wheats might have been present in the past, but the differences of their rachis fragments to other free-threshing wheats are small and not yet recognised? It would be worth noting that possibility, especially as recent variants of free-threshing diploid wheats do exist (Amagai et al. 2014;Sood et al. 2009).
The aleurone cell width measurement technique presented here seems to open a way to directly determine the ploidy level of wheat caryopses identified as freethreshing from archaeobotanical contexts and might also be applicable to other polyploid plant groups. Because the amount of data is still quite limited, further measurements and tests of the method are needed on material from other sites with well preserved rachis fragments as a control. Therefore, any feedback (especially confirmation) would be welcome.
The authors strongly appreciate the comments of two reviewers which have been very helpful and the financial support by the Deutsche Forschungsgemeinschaft (DFG) for the project BI 783/5 "The rise and decline of the central settlement of Fidvár near Vráble (Southwestern Slovakia) -Studies on the economy, social structure and political organization of a social unit within its wider surroundings".  Table 2 Widths of 478 aleurone cells of charred free-threshing wheat caryopses from early Bronze Age Fidvár, assigned by their cell widths to ploidy levels. In most cases only one out of several caryopses morphologically identified as free-threshing wheat had parts of the aleurone layer exposed, but two grains did, in samples #129 and #112 Sample ID Cells per line (n) Length of line ( Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Funding Open Access funding enabled and organized by Projekt DEAL.
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