Bulliform Phytolith Research in Wild and Domesticated Rice Paddy Soil in South China

Bulliform phytoliths play an important role in researching rice origins as they can be used to distinguish between wild and domesticated rice. Rice bulliform phytoliths are characterized by numerous small shallow fish-scale decorations on the lateral side. Previous studies have shown that domesticated rice has a larger number of these decorations than wild rice and that the number of decorations ≥9 is a useful feature for identifying domesticated rice. However, this standard was established based on limited samples of modern rice plants. In this study, we analyzed soil samples from both wild and domesticated rice paddies. Results showed that, in wild rice soil samples, the proportion of bulliform phytoliths with ≥9 decorations was 17.46% ± 8.29%, while in domesticated rice soil samples, the corresponding proportion was 63.70% ± 9.22%. This suggests that the proportion of phytoliths with ≥9 decorations can be adopted as a criterion for discriminating between wild and domesticated rice in prehistoric soil. This indicator will be of significance in improving the application of fish-scale decorations to research into rice origins and the rice domestication process.


Introduction
Rice (Oryza sativa) is among the world's most important and ancient domesticated crops [1]. There is still controversy regarding when rice cultivation and domestication started [2,3]. Some researchers believe that rice was domesticated 9,000-10,000 years ago, based on evidence from archaeological rice fossils and rice DNA [4][5][6][7][8][9][10], while others argue that the process of rice domestication is only known to have begun with certainty 8,000-7,700 years ago, based on the study of unearthed rice spikelet bases [11,12]. One cause of this disagreement is the lack of unified standards for distinguishing between wild and domesticated archaeological rice remains, with this remaining an urgent problem in the research of rice origins [13].
Traditionally, charred rice grains and spikelet bases from archaeological sites have been employed to identify wild/domesticated rice remains [4,11,12,14]. Past research on rice grain morphology has shown that a wild rice grain is thinner and longer than a domesticated grain, which indicates that it has a greater length-width ratio (with a ratio boundary of 3.5) [15]. The spikelet bases of domesticated rice can be identified by their uneven profile, dimpled appearance, and less symmetrical scars. In contrast, wild-type rice spikelet bases typically have a straight profile at their base, and shattering results in a smooth and round abscission scar, with a small, distinct vascular pore [11,16]. However, the application of these indictors to archaeological remains still leaves a degree of uncertainty [2]. Liu et al. [2] have proved that the size and shape of rice grains are not reliable identifiers for distinguishing domesticated grains from wild grains; in addition, charred remains and spikelet bases are heavily dependent on unique burial conditions and preservation processes.
To date, phytolith analysis has been a crucial method for identifying rice remains uncovered from archaeological sites and sediments. Rice plants produce three distinctive phytoliths: bilobate phytoliths from rice leaves and stems, double-peaked phytoliths from the rice husk, and bulliform phytoliths from the rice leaves [17]. The bilobate phytolith is typical of the Oryzoideae subfamily and contrasts with the characteristic features of Oryza plants [18]. However, measurement of bilobates does not enable discrimination of cultivated and wild Oryza species [19]. Double-peaked phytoliths can be used to distinguish domesticated rice from wild rice based on multivariate linear discriminant function analysis and three-dimensional measurements [19,20]. However, very few double-peaked phytoliths have been found in prehistoric rice soil or sediments [21,22].
Rice bulliform phytoliths are abundant and unique to the rice leaf, with the presence of fishscale decorations on fan edges (bulliform phytoliths) [18]. Bulliform phytolith measurement is a method that could potentially be used to distinguish domesticated from wild rice. However, previous studies on rice plants and their relatives have suggested that bulliform measurement alone is unable to distinguish wild Oryza species from domesticated ones [19,[23][24][25].
Another method for discriminating bulliform phytoliths of wild and domesticated rice is based on the number of scales on fan edges. Fujiwara [26] was the first to find that fish-scale decorations are different in wild and domesticated rice. The scales of domesticated rice bulliform phytoliths are larger and have irregular shapes, while those of wild rice bulliform phytoliths look like a tortoise shell. However, this kind of qualitative discrimination is hard to apply to research in practice and it is thus important to explore quantitative standards.
Lu et al. [22] studied the number of fish-scale decorations on the rim of rice bulliform phytoliths from seven species of wild rice and six species of domesticated rice, finding that bulliform phytoliths of domesticated rice species generally had 8-14 fish-scale decorations, while those belonging to varieties of wild rice commonly had <9. It would thus seem that bulliform phytoliths with 9 decorations would be a useful standard for identifying domesticated species.
In practice, however, because of the overlap in the number of scale decorations between wild and domesticated rice species, a single bulliform phytolith is not sufficient to enable a distinction to be made between domesticated and wild rice species. Moreover, in locations where wild and domesticated forms of rice are likely to have overlapped, determining the precise source of fossil phytoliths recovered from sedimentary records can be problematic [23]. For these reasons, more clear specification is needed.
This study on domesticated and wild rice paddy surface soil suggests that the proportion of bulliform phytoliths with 9 decorations can be adopted as a criterion to discriminate between wild and domesticated rice.

Materials and Methods
Seventy-one samples of surface rice soil from south China, including from Hainan, Hu'nan, and Jiangxi Provinces, were collected for phytolith analysis (Fig 1). These included 29 surface soil samples from 9 wild rice fields, 30 surface soil samples from 24 modern rice paddies, and 12 soil samples from 9 other fields. Details of all samples are given in Table 1. All necessary permits for the described samples from Jiangxi Province were obtained from the Jiangxi Academy of Agricultural Sciences. No permits for other samples were needed.
Extraction of phytoliths from soil samples followed procedures described by Zhang [27], with slight modifications. Initially, 5 g of soil were weighed. Subsequently, 30% hydrogen  peroxide (H 2 O 2 ) and cold 15% hydrochloric acid (HCl) were added to each sample to remove organic matter and carbonates. The samples were then subjected to heavy liquid flotation using zinc bromide (ZnBr 2 , density 2.35 g/cm 3 ) to separate phytoliths, with these mounted on a slide with Canada Balsam. Phytolith counting and identification were performed using a Leica microscope with phasecontrast at 400X magnification. The bulliform phytolith selection criteria were modified from Wang & Lu [28] (both symmetric and asymmetric ones). Phytoliths <10 um in size were excluded because of the inability to clearly count decorations. For each rice bulliform phytolith, the number of fish-scale decorations around the edge of fan-shaped phytoliths (Fig 2) was counted. Each sample was scanned until 50 rice bulliform phytoliths were encountered [29]. In each case, the proportion of rice bulliform phytoliths with 9 fish-scale decorations was calculated.

Results
Bulliform phytoliths in soil samples were mostly well preserved, enabling correct identification (Fig 3 and S1 Table). Abundant rice phytoliths (including bulliform phytoliths and bilobate phytoliths) were found, but these included almost no double-peaked phytoliths.
Bulliform phytoliths from other fields-Rice bulliform phytoliths were found in only five other field soil samples (Fig 4, right). Of these, the highest proportion of bulliform phytoliths with 9 fish-scale decorations was 62% (No. 63), while the lowest was 16% (No. 71). Although the five soil samples were neither from wild rice fields nor from domesticated rice paddies, their locations were very close to paddy or wild rice fields, so it is possible that these samples contained rice bulliform phytoliths because of soil tilling or other disturbance activities.
We also analyzed the proportion of bulliform phytoliths with 9 fish-scale decorations from different climatic regions. Jiangxi and Hunan Provinces are subtropical, while Hainan Province is located in the tropics. Table 2 shows the number of samples from different climatic regions. From Fig 5, we can see that in wild rice soil samples, the highest proportions of bulliform phytoliths with 9 fish-scale decorations were found in samples from the tropics, and the lowest proportions of bulliform phytoliths with 9 fish-scale decorations were found in samples from the subtropical region. Moreover, as shown from the analysis of the box height in Fig  5, the proportions from both wild and domesticated paddy soil samples from the tropics were more scattered than in the case of samples from the subtropical region.

Discussion
From our study, it is evident that the indicator-proportion of bulliform phytoliths with 9 fish-scale decorations-can be used to clearly discriminate wild and domesticated rice. We therefore believe that when the proportion of bulliform phytoliths with 9 fish-scale decorations is higher than 63.70% ± 9.22%, the sample can be regarded as domesticated rice; when the proportion of bulliform phytoliths with 9 fish-scale decorations is less than 17.46% ± 8.29%, the sample can be classified as wild rice.
Previous studies have often employed morphometric parameters (length, width and b/aratio of length of handle to fan) of bulliform phytoliths to analyze ancient rice remains [31][32][33][34][35][36][37][38]. Some researchers have established discriminant equations to differentiate between japonica and indica rice [39][40][41]. However, Wang et al. verified these discriminant equations and found that wild rice was always misjudged; for this reason, when using discriminant equations to determine the origin of archaeological samples, extreme caution is required [28]. Moreover, Gu et al. found that three-dimensional morphological features of bulliform phytoliths from Oryza sativa are scattered, with significant overlap of this species with its relatives. Due to this wide overlap, bulliform phytolith measurement alone cannot be used to distinguish wild Oryza species from domesticated ones [19].
The fish-scale decoration features in single bulliform phytolith have shown great potential in previous studies [22,27,[42][43][44][45]. However, use of these features in single bulliform phytoliths to distinguish wild/domesticated rice remains controversial. One important reason is that the number of fish-scale decorations overlaps across species, so that it is not possible to use a single bulliform phytolith to classify rice properties. In order to improve the validity of this method, in this study we examined at least 50 phytoliths from each sample to calculate the proportion of bulliform phytoliths with 9 fish-scale decorations. This method can help avoid the uncertainty inherent in single phytolith analysis. Besides, bulliform phytoliths are abundant in the genus Oryza and previous research has shown that the highest silica percentage is present in the leaf blade [46]. It is therefore feasible to discriminate wild and domesticated rice through the number of fish-scale decorations found around the bulliform phytolith.
Bulliform cells (motor cells), situated in the upper epidermis of leaf blades [47], are water storage mechanisms and play a role in mature leaf rolling and/or folding in case of water stress [48,49]. During periods of excessive water loss, the bulliform cells became flaccid and enable the leaf to roll in order to maintain water; under sufficient water conditions, bulliform cells are filled with water and expand, and the blade thus flattens [50]. Leaf rolling can affect light interception of the base and enhance the ability to resist water stress [50,51].  Bulliform cells are closely associated with adjacent colorless cells [52]. Their morphology, combined with that of enlarged colorless cells, has been used as a taxonomic characteristic [53]. The colorless cells are smaller than the bulliform cells, translucent, voluminous, highly vacuolized, and arranged in uniseriate columns connecting the abaxial epidermis and bulliform cells [52]. The colorless cells are variable in shape and size [53]. Fish-scale decorations on the bulliform phytolith are comprised of cavities squeezed by colorless cells [28] and leaf curling will increase the number of cavities. Wild rice usually grows in swampy conditions [54], where water is abundant (Fig 2, left), and so leaves curl less; on the contrary, domesticated rice leaves are erect and distant from water (Fig 2, right), and so the leaves need to curl repeatedly to hold water. This might explain why bulliform phytoliths of domesticated rice have more fish-scale decorations than those of wild rice.
In our study, the proportion of bulliform phytoliths with 9 fish-scale decorations in samples from different climatic regions was analyzed. We found that in subtropical and tropical regions, the proportion of bulliform phytoliths with 9 fish-scale decorations in wild rice field soil samples was notably different, while the difference was smaller in the case of domesticated rice paddy samples; it is unclear whether the difference in the former case is climate-related. All domesticated rice paddy samples collected in the study came from southern China; however, since northern China is also a main rice growing area, we hope to study more soil samples from northern China in future.
Admittedly, the discrimination of wild and domesticated bulliform phytoliths might be influenced by other factors, such as erosion and dissolution of bulliform phytoliths [55], making it more difficult to precisely count the number of fish-scale decorations. In addition, hybridization of wild rice and domesticated rice species would also affect the ability to discriminate between wild/domesticated rice. We, therefore, recommend further research on wild-domesticated hybridization, which should help understand the variation in decorations and document cultivation and domestication.

Conclusion
This study systematically analyzed differences in bulliform phytolith fish-scale decoration numbers between domesticated rice paddy soil and wild rice field soil in South China. Results showed that, in domesticated rice soil, the proportion of bulliform phytoliths with 9 fishscale decorations was higher than 63.70% ± 9.22%, while in wild rice soil, the proportion was less than 17.46% ± 8.29%. The study therefore indicates that the proportion of bulliform phytoliths with 9 fish-scale decorations can be used to successfully discriminate between wild and domesticated rice. This provides significant insights for research into rice origin and domestication.