X-ray computed tomography (CT) and ESEM-EDS investigations of unusual subfossilized juniper cones

Recent investigations of a Greco-Roman site at Sais have provided well-preserved archaeobotanical remains within a pile of metal fragments. The remains are compared with comparable modern taxa. The morphology and anatomy are studied using Light microscope (LM), Environmental scanning electron microscope (ESEM) and X-ray computed tomography (CT). To investigate the preservation mode, Energy dispersive spectroscopy (EDS) analysis and elemental mapping are conducted. Results revealed that the archaeobotanical remains are exhibiting close affinity with modern juniper cones. Although, the studied archaeobotanical remains are buried for more than 2 millenniums, they underwent early stages of silicification and copper mineralization. These results are discussed in relation to other excavated objects in the find and to our knowledge and understanding of daily life in the Greco-Roman period.


Scientific Reports
| (2021) 11:22308 | https://doi.org/10.1038/s41598-021-01789-z www.nature.com/scientificreports/ This study aims to identify the excavated archaeobotanical material, illuminate the environmental and archaeological context, to increase our knowledge about its importance and use. The study also aims to explain the specific preservation mode and to find out the impact of the presence of metal fragments on the preservation mode.

Material and Method
Sample collection. This study is complied with relevant institutional, national, and international guidelines and legislation. This study does not contain any studies with human participants or animals performed by any of the authors, where archaeobotanical material and the associated metal fragments have been registered as (nos. 19-97/43/11 A-Q) ( Fig. 1a-d), in Tanta Museum, Egypt.
Light microscopy examination and SEM-EDS micromorphology. The archaeobotanical specimens were observed and photographed using (Leica EZ4) Stereo Microscope and Canon EOS 4000D/Rebel T 100 Digital SLR Camera.
Modern dried juniper cones were collected from the best-selling herbal market in Egypt (Harraz for food Industry and Natural Products, Egypt). One of them was sectioned for comparison with the archaeobotanical specimen. The modern specimen was soaked overnight in warm water then cut with a scalpel to show the number and arrangement of seeds.
For transverse sectioning, one of the archaeobotanical specimens was embedded in a polyester resin block then cut into two halves using a jeweler saw then polished using a series of coarse-to fine-grit SiC polishing paper and ground down to 2000 grit. The same procedure was conducted for sectioning the modern juniper specimen for micromorphological examination and analysis.
The transverse section micromorphology of the archaeobotanical specimen was investigated using an environmental scanning electron microscope with energy dispersive spectroscope SEM-EDS (Quanta FEG250, with tungsten electron source, at 20 kV). Four analysis spots were analyzed. Mapping was undertaken through the scanned area to show the distribution and relative proportion (intensity) of the defined elements. The "ZAF correction method" was used for quantitative analysis of elements.
C-scanning examination. The archaeobotanical specimen was scanned using X-ray computed tomography (CT) Scanning at Majd El-Eslam Medical Centre, Egypt, using Toshiba Aquilion 16 CT Scanner, Japan. Datasets were visualized, and images and videos were captured, 3-D Imaging: image quality with surface shaded- renderings and volume-rendered 3-D images. Zooming and panning over the 3-D surface and performs distance measurements. High Image Quality: The Aquilion 16 features 896 channels in 40 rows of solid-state detectors; specialized, user-selectable, image-reconstruction algorithms; and a wide selection of slice thicknesses. The system provides low-contrast resolution of 2 mm at 0.3% and high-contrast resolution of 0.35 mm. The used parameters are as follows: voltage 120 kV, current150 mA, timing 15.819 s, no. of X-ray projection, thickness 0.5 X 16 mm.

Results
Morphological visualizing and CT examinations of archaeobotanical and modern specimens. Taphonomy; Visual and microscopic examination (Fig. 2a) revealed that the archaeobotanical material composed of five rounded to oval shaped, 0.8-1.3 cm diameter cone withdark brown/black surface colour with green hue. The surface was rough with scaly skin. The archaeobotanical cone specimen seemed exceptionally well-preserved when compared with modern taxa. The size, shape features and the transverse section of the archaeobotanical cone specimen show that it includes intact seeds. The transverse section found similar to modern Juniper cone specimen (Fig. 2b). At scale bar of 1 mm using SEM examination, the archaeobotanical specimen ( Fig. 2c) was compared with the modern juniper specimen (Fig. 2d). The morpho-taxonomic study revealed that they are having a close affinity with each other.
By using the CT scanner for the archaeobotanical cone specimen, the surface appearance, the rough skin ( Fig. 3a) and the scaled surface ( Fig. 3b) are clearly shown. Five fleshy fused scales, each with a single seed are shown with smooth layer (Fig. 3c-e). The longitudinal or side view of the cone is shown in Fig. 3f.
For the modern dried cone (Fig. 4), the top and inside views are clearly shown in Fig. 4a,b, respectively, with the arrangements of five fleshy scales. In the cone transverse sections (Fig. 4c,d) the cone's anatomical structure with five scales are clearly shown under CT scanning, each with one seed (Fig. 4e) and the longitudinal scanned view with the anatomical structure of one scale are illustrated in (Fig. 4e), while the whole shape of the cone is shown in Fig. 4f. Microchemical analysis of the archaeobotanical material. The microchemical analysis results of the archaeobotanical specimen are given in Table 1. The elemental mapping show that C and O are homogenously distributed with weight percentages of 55.76 and 41.16%, respectively, while Si, S, Cl, K and Cu are found distributed in cell walls and voids. The inorganic elements are distributed along the outer layer of cone (scaley skin) towards the ovules in the scanned area (Fig. 5).
The analysis results of the modern cone specimen are given in Table 2. They indicated the presence of C and O, as main elemental composition with weight percentages of 59.93 and 38.6%, respectively, other elements such as Si, Ca and Cu were also identified (Fig. 6). The elements in both specimens almost followed the same manner in their distribution. However the archaeological specimen contains chloride, sulphur and copper. These elements are of the common components of copper corrosion.

Discussion
Scanning electron microscopy proved to be efficient for studying small fossils, charcofied and lignified mesofossils 16 . Their assemblage shown well-preserved angiosperm seeds, flowers, fruits, leaf fragments, wood, shoots, cone scales, leaves, pollen cones, and seeds of conifers 16 . µ-computed tomography scanning (µCT) was also used to investigate fossil cones of Pinus sp. and Keteleeria sp. 17 . X-ray micro-computed or computed tomography (CT) is ideal for studying three-dimensional fossils, it can be a good tool for the identification and the documentation of seed cones and other part of plants [18][19][20][21][22][23] . It was used to study permineralized plant fossils 24 and to identify fruits and seeds in pyrite-permineralized specimens from the London Clay Formation 25 . Other methods such as diffuse X-ray methods have also been applied for investigating fossil fruits, e.g., Crepetocarpon 26 and Spirematospermum 27 .
By comparing the archaeobotanical material with the modern taxa using visual, CT and SEM investigations, it was found that the archaeobotanical specimen can be identified as seed cones. They show a close affinity with juniper sp. Juniper cones have been found in ancient Egyptian tombs in multiple locations and were studied by visualizing morphology as fleshy berry-like cones 28,29 .
Cones of Juniperus excelsa and J. oxycedrus were found also in the tomb of Tutankhamen (1341-1323 BC) 30 . Medicinal use of juniper cone goes back to ancient Egypt 1500 BC, it was mentioned in a prescription for treating a tapeworm infection and for mummification 31,32 and from ancient times it has been widely used as herbal medicine as antidiarrhoeal, anti-inflammatory, astringent, and antiseptic 33,34 . Volatile juniper seed oil was also used as a laxative 35 . The Romans used juniper for treatment of stomach diseases, as well as a cheap domesticallyproduced substitute for the expensive black pepper [36][37][38] . The Greeks also used juniper as medicine and more interestingly, on many of their Olympic Games occasions due to their belief that juniper cones increase the physical endurance of athletes 39 .
J. phoenicea was reported and conserved in northern mountains of Sinai 40,41 , although, Juniperus sp. are not known to grow in Egypt 30 . They were most probably imported from Greece 42 . Earlier studies showed that J. oxycedrussubsp. oxycedrus 43 populated in East-and West-Mediterranean origins.
Based on morphological data, J. excelsa is divided into two subspecies 44,45 : J. excels subsp. excelsa, covering mountain and sub-mountain areas from the Balkan Peninsula in the west, through Anatolia, Syria and Lebanon to Crimea in the north and Iran in the east [44][45][46][47] , and J. excels subsp. polycarpos (K. Koch) Takht., found further to the east with a Transcaucasian-Central-Asian distribution. Based on random amplified polymorphic DNA (RAPD) molecular markers, these are considered as two taxa of separate species, J. excelsa and J. polycarpos, respectively 48 .
J. oxycedrus subsp. oxycedrus with reddish-brown cones about 1 cm across had recorded the number of seeds in a female cones with two whorls of ovules with 3-6 52 , as described for J. communis by Schulz et al. 53 . Female cones with four and six ovules have been found in J. oxycedrus subsp. oxycedrus and in J. communis 53,54 . The abnormal type of female cone showed less than three seeds in one particular cone which has developed a restricted number of ovules, and the lack of success of pollination and fertilization of a normal 3-ovule cone 55 .
J. communis is a very variable species with differences in morphology and habitat over an extensive circumpolar geographical range, with five 44 or seven 48 varieties. The female cones may be dry and woody (e.g. Cupressus) or succulent (e.g. Juniperus, Thuja) and have cone scales arranged in opposite pairs or in threes with one to many ovules 56 . Seeds number of J. communis per cone and the filled seeds number per cone varied significantly between geographical regions and among populations within regions 57,58 . The cone containing 1-3, rarely 4, seeds per cone of J. communis distribution range in Europe collected for 31 populations/seven distinct regions 57 . Through 4000 ripe seed cones from 50 J. communis shrubs collected randomly (60-100 cones per shrub), the seeds number per cone were ranged from 1to 5 59 . Female strobili normally contain three ovules and thus produce 1-3 seeds (although up to 6 is possible) 60 . Seed cones of common juniper usually have three seeds (varying 1-6) 56 . Filled seeds of J. communis contain a well-developed, firm, off-white (sometimes brownish) embryo and megagametophyte and, therefore, are scored as probably viable, while the empty seeds are entirely empty, contain shrivelled contents, or are embryo-less and, therefore, are scored as nonviable 61 . With more than 95%, the ripe cones contain seeds number up to 3, while 5% produced from 4 to 5 seeds per cone from J. communis shrubs collected from Mishu-Dagh Altitudes in North West of Iran 59 . Ripe cone production correlated positively to seed set and seed  www.nature.com/scientificreports/ predation but was independent of the percentages of empty and filled seeds 62 . The loss of seed per cone were due to predispersal seed predation and the abortion of seed 57 . Also, it was observed that populations from the Mediterranean mountains (south-east Spain) has the highest values in the seeds number/cone but the lowest values in the number of filled seeds per cone 57 . Furthermore, the proportion of three-seeded cones was greater in the open than under forest shade (43.8% and 2.6%, respectively) 63 . J. communis L. subsp. communis produces a large proportion of empty seeds. From the literature, the ripen cones are usually mentioned with empty places with no seeds. But it turns out that there are five chambers, but only three seeds 61,64,65 . Also, the seed quality of J. communis can be affected by the environment or forest ecology and nutrient, where the lower seed productivity can be a result of limited pollen availability or lower pollen quality and pollen growth rates [66][67][68] . In contrast, the opposite was observed in plants growing in nutrient-poor environments 67 . Seeds number of the J. communis berries are dependant on seed quality that can be observed when cutting the berries. Through visual assessment of some vesicles filled and other vesicles empty seeds (entirely empty) or shrivelled contents, or embryo-less, the number in the majority of cones is five vesicles but not all of them contain the seeds 61 .   www.nature.com/scientificreports/ We propose some possibilities for the use of the archaeological juniper cones in focus of the context; being buried with utensils, they could be used for the preparation of medical prescriptions, or they could be used for the flavoring of food. The other possibility is related to the wrestlers' statuette and the juniper intake by athletes to increase endurance.
In order to explain how and why the archaeobotanical materials have survived and preserved in an excellent conditions (Fig. 2c), for more than 2 millenniums and how they opposed both abiotic and biotic degradation. The location of the excavation site and soil condition were taken into consideration. Plant fossils are generally preserved in environments that are very low in oxygen (e.g., anaerobic sediment) as most decomposers (e.g., fungi and some bacteria) require oxygen for metabolism. Plant fossils are commonly preserved in fine-grained sediment such as sand, silt, or clay. Organic material may also be protected in fine textured clay soils than coarse sandy ones. Silt is the main constituent of soil texture that leads to poor drainage and a significant water holding capacity due to its texture taking into account the depth of buried metals and water level on the site 69 . The silt soil of Sais site, where the archaeobotanical materials were found, was advantageous for the preservation process. The rate of biological degradation of organic materials in soil was also affected by their molecular structure, while cellulose is consumed preferentially over lignin and other poly-phenols present in plant. Both organic and inorganic matters are degraded in burial environments. Long-term burial changed the appearance and the chemical nature of the buried metal objects, resulting in the formation of corrosion of metals, and in some cases the complete destruction of the artefacts 70,71 . There are different parameters, which affect the corrosion process, i.e., the metallurgy of the artefacts and the characteristics of the burial soil [72][73][74] . Soluble anions such as Cland SO4in high amount in burial environment cause severe corrosion in the long-term. In fact, the presence of high amount of soluble salt results in increasing conductivity of the soil and accelerating electrochemical reactions leading to corrosion of archaeological copper alloys 71 . The presence of soluble sulphate may due to the presence of calcium sulphate phases in the composition of soil because of gypsum used as a binder or plaster in the architecture. The presence of sulphide (metallic sulphide) and its oxidation forms sulphuric acid, acidifies the soil and decreases pH 75 . Increasing acidity reduces organic degradation. In addition to the amount of corrosive anions in the soil, pH, the concentration of soluble salts and texture of the soil affect the preservation condition. Basic copper sulphates are stable in acidic conditions. By changing the pH of the environment to an alkaline condition, they will transform to more stable compounds 76 . These products will transform into green coppertrihydroxychlorides (basic copper chlorides) in the presence of high concentration of soluble chloride ions 77,78 . This product is responsible for the green hue of the archaeobotanical specimens. The antimicrobial www.nature.com/scientificreports/ effect of copper has been known for centuries [79][80][81] , so the presence of copper fragments in the find played a role in preventing organic decomposition. From the microanalysis results and mapping of elements distribution, it was found that silica and copper precipitated in cell walls (Fig. 6), while chloride precipitated in the cell voids. Mineralization of plants by metals has previously been recorded 15 . This usually occurs when minerals carried in solution (silica, carbonate, chloride, etc.,) are deposited around plant cell surfaces or in the cell wall and intercellular spaces, encasing the plant structure 14,82 and called structural preservation. The presence of a hard coat and antioxidants in the plant are also possible causes of good preservation 83 . The initial silica deposition begins within cell walls rather than in the cell lumina. The initial silica precipitation involves the affinity of silicic acid for hydroxylgroups in hollocelluloses and lignin. This phenomenon was also observed in the studied specimens. The silicification sequence in early stages is called "organic templating" [84][85][86] . It can be concluded that there have been many factors affected the preservation condition of the archaeobotanical material, resulting in initial stages of fossilization and mineralization. The unique preservation mode is greatly enhanced by the presence of metal fragments in addition to burial environment.

Conclusion
In this study, unknown archaeobotanical materials from Sais archaeological site in Egypt, were identified. They show similar cone shapes and anatomical features of (Juniperus sp.). CT-Scanning and SEM-EDS investigations were used for detailed comparison with modern juniper cones. The archaeobotanical cones composed of five rounded to oval seeds in cone shaped 0.8-1.3 cm diameter. The unique preservation condition is discussed as regards the burial environment; the kind and texture of soil, soluble anions such as Cland SO4 -, pH and the presence of metals.