Phytoliths in selected broad-leaved trees in China

Broad-leaved trees are widely distributed from tropical to temperate zones in China, reference collections of phytoliths from these taxa are crucial for the precise reconstruction of paleoenvironments and the study of early plant resource exploitation. However, not much has been published on the phytoliths produced by modern broad-leaved trees. In this study, we collected samples of 110 species that cover the common species distributed in Northern and Southern China, and extracted phytoliths from leaves, twigs and fruits, in order to investigate the phytoliths types and production in these species. We found that only 58 species were phytoliths producers, and that 23 distinct phytoliths morphotypes could be recognized. The results showed that phytoliths types and production in Northern and Southern China could be similar in the two regions. Through analyzing previously published data and our data, Elongate brachiate geniculate, Polygonal tabular, Elongate facetate, Tracheary annulate/facetate geniculate and Tracheary annulate/facetate claviform have been proposed to be the potential diagnostic types for broad-leaved trees in general. This study provided a preliminary reference of phytoliths in modern broad-leaved trees, and could be used in the identification of phytoliths in sediments and archaeological contexts.


Results
Phytoliths types in the studied species. A total of 23 different types of phytoliths were observed in the studied species. Typical phytoliths types are shown in Figs. 1 and 2, and more detailed illustrations of phytoliths produced by each specimen can be found in the Supplementary Figures 1-13. Phytoliths types are described in Table 2.
Most phytoliths observed in this study were found in leaves, except for Elongate entire ( Fig. 1-10) which were also observed in the vine of Ficus tikoua, the twig of Pittosporum truncatum and Tilia mandshurica, and Irregular articulated granulate ( Fig. 1-15), which were only observed in the fruit husk of Aleurites moluccana. Because many phytolith types have the same anatomical origin, to simplify the further analysis, we further classify the phytoliths types into 4 categories or classes: • the stomata class, phytoliths that were formed in the stomata in the leaves, which includes the Stomate stellate; • the hair tissue class, phytoliths that were formed in the hair tissues in the leaves, which includes the Trichome irregular tubercule, Trichome bulbous irregular, Acute bulbosus, Acute uncinate, Acute, Acute acicular, Acute echinate, Hair base, Trichome spheroid plicate/cavate, Trichome fusiform cavate; • the tracheid/vascular tissue class, phytoliths that were formed in the tracheid/vascular tissues in the leaves, which included the Elongate facetate, Tracheary annulate/facetate geniculate, Tracheary annulate/facetate claviform, Tracheary annulate, Tracheary helical; • the silicified cell class, phytoliths that were formed in the cells of mesophyll or epidermis in leaves/branches/ fruit, which includes the Elongate brachiate geniculate, Irregular sinuate, Polygonal tabular, Spheroid favose, Elongate entire, Spheriod hollow, Ellipsoidal nodulate.
The total count of phytoliths in each specimen and the percentage of phytoliths in each category are reported in Table 3. We carried out a PCA analysis using this set of data, to find out the relationship between the phytoliths types and species. The result is reported in Fig. 3. We note that the spheres (the red spheres) that represent the four categories of phytolith types form a tetrahedron in the coordinate system Fig. 3, with each sphere occupying an apex of the tetrahedron, indicating that the four categories can be clearly separated. We further note that the spheres that represent the species are scattered throughout the coordinate system with their positions reflecting their relationship with the four phytolith type categories. This PCA closest relationship paradigm between  Table 1. Information of the studied specimens. a Phytolith production index refer to the result part, which NP non producer, A abundant, C common, U uncommon. Of 110 species we analyzed, 58 produced phytoliths and 52 were non phytolith producers Table 1. The production index for 58 phytolith producers was mostly recognized as abundant (A) and common (C), except for Berberis poiretii (which was rare), and Leptopus chinensis and Zelkova schneideriana (which are uncommon).

Scientific RepoRtS
Among the phytolith producers, 21 species were collected from Northern China (Changbai Mountain and Beijing) and 37 were from Southern China (Gongga Mountain). To compare phytolith production between the two regions, we applied an independent-samples Mann-Whitney U test using the data in Table 3. The results showed that phytolith production in the stomata class (Sig. = 0.147), the hair tissue class (Sig. = 0.792) and the  www.nature.com/scientificreports/ silicified cell class (Sig. = 0.226) showed no significant differences between the two regions, however, phytolith production in the tracheid/vascular tissue class (Sig. = 0.028) was significantly different between Northern and Southern China. Also, despite some differences in the taxa, the total count of phytolith showed no significant differences between the two regions (Sig. = 0.601). Such results indicated that although the tracheid/vascular tissue class differed between the two regions, the production of most other phytoliths types might not be influenced by regional differences. The differences in the production of the tracheid/vascular tissue class might reflect the different hydrothermal conditions in the two regions.

Discussion and conclusions
It is widely known that in general, woody plants produce fewer phytoliths than grasses 4,5 . The results of our study are consistent with the previous studies. Only 58 out of the 110 species we analyzed were phytoliths producers.
Most of the phytoliths we observed were extracted from leaves, the other plant parts, such as twigs and fruits typically showing a lack of silicification. Phytolith types belonging to the silicified cell class make up the largest portion of the phytoliths produced by the 58 phytolith producing taxa, followed by the stomata class, the tracheid/vascular class and the hair tissue class. Species belonging to the same genus usually produced the same types of phytoliths, and the phytolith production was typically similar. However, we found that phytolith types and production in species belonging to different genera of the same family can be very different. Such results suggest the possibility of identification of taxa on the genus level using phytolith analysis, which is in consist with the study of grasses 22 , however, studies that involve more species and more samples of species are needed to confirm such findings.
To date, no especially diagnostic types of phytoliths have been identified for broad-leaved trees in general or a certain family. After reviewing other phytolith studies of species belonging to the broad-leaved trees 12,15,[46][47][48][49][50][51][52]55,56 (also see Table 4, we here propose several phytolith types that have the potential to be diagnostic to broad-leaved trees: Elongate brachiate geniculate ( Fig. 1-3), Polygonal tabular ( Fig. 1-5), Elongate facetate ( Fig. 1-8), Tracheary annulate/facetate geniculate ( Fig. 1-9) and Tracheary annulate/facetate claviform ( Fig. 1-10). Because these types of phytoliths are rarely seen in grasses and have been extracted from broad-leaved tree taxa in other studies, we suggest that they might have the potential to be diagnostic types for broad-leaved trees. Although some types of phytoliths have distinct morphological differences with other              This type of phytolith was found in the fruit husk of Aleurites moluccana, has a twisted elongate morphology, can be highly variable, the surface has a granulate texture, found articulated forming a layer (single disarticulated phytoliths of this type could not be observed without breaking the layer) Aleurites moluccana, Silicified cell class  Originating from a not fully silicified hair cell, the tip is bent over to form a hook shape Smilax sp., Morus australis Hair tissue class www.nature.com/scientificreports/ types (such as Trichome irregular tubercule ( Fig. 1-6), Trichome spheroid plicate/cavate (Fig. 2-7), Ellipsoidal nodulate ( Fig. 2-8) and Trichome fusiform cavate (Fig. 2-9), considering the lack of crossexamination of these types, further studies were needed to evaluate their potential in being diagnostic types. The Acute acicular (Fig. 2-4) and Acute echinate (Fig. 2-5) were only observed in Moraceae plants 4,7,48 , combined with our results, they might be the potential diagnostic types for Moraceae, while observation of more specimens from Moraceae and other plants was needed to confirm this finding. Although Irregular sinuate phytoliths were observed in many broad-leaved trees, they were also observed in many ferns 4,45,54,65 , thus they were not proposed as the potential diagnostic types for broad-leaved trees. The Irregular articulated granulate ( Fig. 1-15) which we found in the fruit husk of Aleurites moluccana (which could be used as food or sauce in Malaysia and Indonesia), is also noteworthy as it has not been reported yet. Such silicification in fruit husks might be a protection strategy 22,66 , and the presence of this type may provide insight into ancient plant resource exploitation. In this study, we have provided an illustration of several distinct phytolith types we observed in the common broad-leaved trees in temperate China, and reported that there appears to be little difference in broad-leaved trees phytolith production between the northern and the southern regions. Although we have proposed several specific phytoliths types as potentially diagnostic (which we believe to be reliable), pending further confirming research involving more taxa and samples, researchers should not solely use our findings as identification criteria, but rather as a guidance and reference for the future studies.

Acute acicular
Originating from a not fully silicified hair cell, has the shape of a lance, sometimes a line could be observed along the axis of symmetry (it might be caused by the insufficient silicification)

Morus australis, Sorbus multijuga
Hair tissue class         This type of phytoliths have been frequently reported to be observed in broad-leaved trees, thus it might be of high potential to be a diagnostic type for broad-leaved trees Irregular sinuate (Fig. 1-4) Silicified epidermal cell 46  This type of phytoliths have been frequently reported to be observed in broad-leaved trees, however, they have also been reported to be observed in ferns, thus it might of low potential in being a diagnostic type for broad-leaved trees Polygonal tabula ( Fig. 1-5) Silicified epidermal cell 46  This type of phytoliths have been frequently reported to be observed in broad-leaved trees and have distinct difference with those from grasses (mostly rectangle-shaped), thus it might of high potential in being a diagnostic type for broad-leaved trees Trichome irregular tubercule ( Fig. 1-6 This type of phytoliths have been frequently reported to be observed in broad-leaved trees, thus it might of high potential in being a diagnostic type for broad-leaved trees Tracheary annulate ( Fig. 1-11 This type of phytoliths have been commonly observed in plants, thus it might have low potential in being a diagnostic type for broad-leaved trees; however, the Acute type of phytoliths in woody plants were commonly larger than in Poaceae plants, the morphometric approach might help to increase the potential of Acute type of phytoliths in being a diagnostic type for broad-leaved trees Acute uncinate (Fig. 2-2) Silicified epidermal hair 46 , thin, curved hair cell phytoliths 7 , long point 4,9 This type of phytoliths have been commonly observed in plants, thus it might have low potential in being a diagnostic type for broad-leaved trees; however, the Acute type of phytoliths in woody plants were commonly larger than in Poaceae plants, the morphometric approach might help to increase the potential of Acute type of phytoliths in being a diagnostic type for broad-leaved trees Acute ( Fig. 2-3) Silicified epidermal hair 46 , long point 4,9 ; long, threadlike nonsegmented hair phytolith 7 , square proximal hair cell 48

Data availability
The raw materials of studied species and slides of phytoliths involved this study can be found in the phytolith lab at the Institute of Geology and Geophysics, Chinese Academy of Sciences.
Received: 26 March 2020; Accepted: 25 August 2020 Table 4. Comparison of phytoliths nomenclature and evaluation of their potential in being diagnostic types for broad-leaved trees.

Potential of being diagnostic types for broad-leaved trees
Acute acicular (Fig. 2-4) Nonsegmented hair phytolith 7 , long point 4,9 This type of phytoliths have been reported only being observed in Moraceae plants, thus it might have high potential in being a diagnostic type for Moraceae Acute echinate (Fig. 2 -5) Hair phytolith with small spines 7 , armed hair 48 , long point 4,9 ; hair 12 , long acicular granulate segmented hair 49 This type of phytoliths have been reported only being observed in Moraceae plants, thus it might have high potential in being a diagnostic type for Moraceae; however a confuser from some grasses (typically Asteraceae) showed similar morphology, but the confusers were observed to be segmented, while in Moraceae they were all nonsegmented Hair base (Fig. 2-6) Silicified epidermal hair base 46 , hair base phytolith 7,48 , silicified hair base 4 , hair base 12,49,55,56 This type of phytoliths have been commonly observed in plants, thus it might have low potential in being a diagnostic type for broad-leaved trees Trichome spheroid plicate/cavate (Fig. 2-7) Decorated sphere 48 , hair base 12 , ovate striate 55 This type of phytoliths have been reported to be observed in some broad-leaved trees and have not been reported to be observed in grasses, thus it might have the potential in being a diagnostic type for broad-leaved trees; however, this type of phytoliths seemed to be thin-walled and might hardly be preserved in sediments Ellipsoidal nodulate (Fig. 2-8) Spherical nodular 45 This type of phytoliths have been rarely reported, unlike the common spherical phytolith observed in Palmaceae, this type of phytoliths were larger (over 20 microns in diameter) and mostly not spherical but ellipsoidal, thus it might have the potential in being a diagnostic type for broad-leaved trees or genera Populus Trichome fusiform cavate ( Fig. 2-9) First reported in this study This type of phytoliths belong to the hair/trichome class, and it has distinct morphology that differs from others, and it has not been observed in grasses, thus it might have the potential in being a diagnostic type for broad-leaved trees; however, this type of phytoliths seemed to be thin-walled and might hardly be preserved in sediments