Earliest tea as evidence for one branch of the Silk Road across the Tibetan Plateau

Phytoliths and biomolecular components extracted from ancient plant remains from Chang’an (Xi’an, the city where the Silk Road begins) and Ngari (Ali) in western Tibet, China, show that the tea was grown 2100 years ago to cater for the drinking habits of the Western Han Dynasty (207BCE-9CE), and then carried toward central Asia by ca.200CE, several hundred years earlier than previously recorded. The earliest physical evidence of tea from both the Chang’an and Ngari regions suggests that a branch of the Silk Road across the Tibetan Plateau, was established by the second to third century CE.


Results
Study location. The Han Yangling Mausoleum (34° 26′ 37.99″ N, 108°56′ 26.84″ E, 415 m asl) is located to the north of Xi'an, on a loess platform along the north bank of the Weihe River. It was built for the Jing Emperor Liu Qi (188-141 BCE), the fourth emperor of the Western Han Dynasty, and his wife (Fig. S1a). A total of 86 outer burial pits surround the mausoleum (Fig. 1b), of which No.15 (DK15) was excavated in 1998-2005 by Shaanxi Provincial Institute of Archaeology 14,17 . Pit DK15 is 21 m in length and 2.6-2.7 m in width (Fig. 1c). Wooden boards divide the pit into eastern and western segments. Plant remains were noted as a large mass in the eastern part of the pit, measuring ~13 × 2 m in area and 2-8 cm in thickness and composed of various finer lamellae. These remains had partially decomposed, but includes grains and thin unconsolidated pieces colored brown to black. Some of the lamellae of crop remains have been identified by phytolith analysis and morphological features preserved macrofossils, including foxtail millet (Setaria italica), broomcorn millet (Panicum miliaceum), rice (Oryza sativa) and domesticated chenopod (Chenopodium giganteum) 14 (Fig. S1c-e). However, one sample (DK15-1) is composed of apparent plant leaves, gathered into a dark brown brick shape (Fig. 1d, Fig. S1b). Direct AMS 14 C dating of part of this sample has yielded ~255 ± 80 BCE (Table S1). These decomposed vegetative plant remains retained little diagnostic morphological features so taxonomic identification on this basis was not possible.
Gurgyam Cemetery (31°7′45.08″N, 80°38′ 28.27″E, 4290 m asl) is located in the capital of the ancient Zhang Zhung Kingdom, on the northern upriver bank of Sutlej River (Fig. 1a) in Ngari district, Tibet. The first tomb was accidentally discovered by the monks of Gurgyam monastery in 2005 (Fig. 2a,b), and a systematic excavation was later carried out by Chinese archaeologists in 2012 15,16 . The tomb was buried beneath the river's silt and gravel, with a square pit containing a square casket-shaped wooden coffin and a well-preserved skeleton. Burial artifacts include silk pieces with the woven Chinese characters "Wang Hou" (King and Princes) (Fig. 2c), various ceramic vessels, wooden tools, bronze vessels, and a golden mask ( Fig. 2d-g). An unidentified object found in one ceramic vessel appears to be agglomerated plant residue. This plant residue (XZ-1) (~4× 5× 3 cm, Fig. 1f) and other grave goods have been dated as second to third century CE (Table S1).
Identification of plant remains. The small plant leaves from both samples DK15-1 and XZ-1 show several morphological features that match those of tea (e.g. tea bud structures, Fig. 1e; Fig. S1b). However, no diagnostic morphological features survive that can be used to identify unequivocally these partially decayed leaves and buds as tea. Recently, the development of biomarker and calcium phytolith proxies has allowed the identification of components from decayed food and tea remains 2,13,18,19 . Further, a recent study has used both theanine and caffeine markers as a basis for tea identification 5,20 . Caffeine is an uncommon plant alkaloid but found across several unrelated tropical families, often prized for human consumption, including tea (Theaceae), coffee (Rubiaceae), cola and cacao (Sterculaceae), gurana (Sapindaceae), yerba mate (Aquifoliaceae), and Citrus flowers (Rutaceae) 21 . Theanine is an amino acid so far only reported from species in the Theaceae, especially in high levels in Camellia sinensis 21,22 . Additionally, our statistical observation of variations in calcium oxalates crystals and anatomical structures in modern tea and related Theaceae and non-Theaceae plants show that trichome bases in tea plants possess four distinctive straight and regular cracks. Indeed, in situ calcium phytoliths of druse form have the smallest diameter (11.65 ± 3.64 μ m) in our reference collection, providing morphological criteria for distinguishing tea from other plants 13 .
Therefore, the significant relative abundance of both theanine and caffeine found in the archeological samples (DK15-1, XZ-1) vis-à-vis similar retention times and mass fragmentation to SRM indicates that the two plant residues from the Han Yangling Mausoleum and Gurgyam Cemetery are examples of ancient tea. A further line of evidence comes from the analysis of phytoliths and calcium oxalate crystals (calcium phytoliths) of the decayed samples (DK15-1, XZ-1). Both archeological samples contain abundant calcium phytoliths, including the calcium oxalates druses and trichome base phytoliths. These calcium phytoliths also match the genus Camellia 13 (Fig. 5a-f,  Fig. S2a). These three diagnostic tests (for caffeine, theanine and calcium phytoliths) together confirm physical evidence for tea being imported to Xi'an in the first century BCE, and westwards into Tibet by the second century CE.

Discussion
Traditionally, tea constitutes two or three leaves and the terminal apical buds of the tea shrub 2,26 . The tea buds (also known as "tips") are the small, unopened leaves of the tea plant, and are often considered to be of better quality than the larger, older tea leaves 26,27 . "Imperial tea", or that called "fine plucked", is thus the tea bud alone and/or the two closest leaves 28 . Figures 1d,e show that the tea from Sample DK15-1 (in the mausoleum of the emperor of the Western Han Dynasty) consists almost entirely of tea buds, although the determination of tea cannot be identified simply from bud shapes (Fig. S1b). Phytolith analysis of Sample XZ-1 from Gurgyam Cemetery also reveals abundant calcium phytoliths identifiable as tea, as well as barley lemma phytoliths and unrecognizable plants (Fig. S2b-d). This indicates that the sample contains a mixture of tea, barley (Hordeum vulgare, Poaceae) and other plants. Therefore, it is likely that tea buds and/or leaves were consumed in a form similar to traditionally-prepared butter tea, in which tea is mixed with salt, tsampa (roasted barley flour) and/or ginger in the cold mountain areas of central Asia 2,29 . Of course, methods of brewing and consuming tea varied from culture to culture along the Silk Road 2,4,6 .
As we know, the genus Camellia is composed of over 110 species 30 . Among them, only one species, C. sinensis, is commercially used as a source of the beverage tea 31 . Exceptionally, some species of Camellia, e.g. C. irrawadiensis and C. taliensis, are only used for tea in specific areas in China, such as tropical South West China. C. irrawadiensis typically grows in upper Myanmar, and C. taliensis usually grows in the mountainous evergreen broad-leaved forests at altitudes from 1300 to 2700 m in southwestern Yunnan, China, and adjacent regions of northern Myanmar and Thailand 31 . It is thus highly unlikely that these species were the sources of tea in central China about 2000 years ago given their restricted geographical distribution. In addition, C. irrawadiensis contain very low levels of essential tea compounds, such as the caffeine content below 0.02% 31,32 . In our analysis, both theanine and caffeine are relatively rich, and diagnostic calcium phytoliths were found in the archeological samples, thus the species in our samples is most likely C. sinensis rather than another species. Although it does not grow in Tibet, tea has traditionally played a multi-functional role in Tibetan society, as a ritual object, stimulant, and source of nutrition and medicine 29 . Up until now, the history of tea in Tibet has only been traced back to the Chinese Tang dynasty (618-907CE) and Tibetan Tubo kingdom (a kingdom located in the southeast Tibetan region that existed from the 618-842CE) 8 . It has been claimed that tea was introduced to Tibet as part of the Tang Princess Wencheng's dowry (625-680CE) on her betrothal to the Songtsen Gambo 8 . Our findings indicate that tea, as an important component of Silk Road commerce, had been introduced to the Tibetan area by 1800 years ago, during the Zhang Zhung kingdom period 33 . This was at least four to five hundred years earlier than the the Southwest Silk Road through Yunnan which opened in the seventh century CE, and is known by historians as the "Tea Horse Road" 34 .
It is worth noting that silk materials containing the Chinese characters "Wang Hou" similar to those excavated at Gurgyam Cemetery (Fig. 2c) have also been found in Xinjiang Province. These date back to the third and fourth centuries CE 15,35,36 . Whether the silks in Gurgyam Cemetery are from eastern China or central Asia remains controversial 15,37,38 , although their presence alongside tea at Gurgyam Cemetery would suggest that they originated through trade from eastern China.
Our data indicate that the plant residues unearthed at both the Han Yangling Mausoleum and Gurgyam Cemetery are the earliest physical evidence of tea in the world. These data indicate that tea was part of trade of luxury products, alongside textiles, that moved along the Silk Road around 2000 years ago, and were traded up into Tibet. Recent archaeological work has highlighted how early intermittent exchanges between eastern China, the Tibetan Plateau and Central Asia began more than 4000 years ago, and resulted in the spread of food grains, a few

Methods
Extraction of caffeine from modern tea. A modern tea sample (~0.2 g) was weighed, ground to powder, and transferred into a tube with 4.0 ml ethanol. The sample was then extracted under sonication for 20 min. After filtering, the extraction was evaporated and dried in a stream of N 2 . The residue was re-dissolved with 3 ml CHCl 3 /H 2 O (1:2, v/v) and subjected to further sonication for 20 min. After the sample was layered, 25 μ l of the lower layer of fluid was transferred into an autosampling vial with 1 ml CHCl 3 for GC/MS analysis.
Extraction of caffeine from ancient samples. An ancient sample (~1 mg) was weighed, ground to powder, and transferred into a tube with 1.0 ml ethanol. The sample was then extracted under sonication for 20 min. After filtering, the extraction was evaporated and dried in a stream of N 2 . The residue was re-dissolved with 3 ml CHCl 3 /H 2 O (1:2, v/v) and subjected to further sonication for 20 min. After the sample was layered, the lower layer of fluid was transferred into an autosampling vial for GC/MS analysis. GC/MS analysis. GC/MS analysis was performed using a 7890A gas chromatograph and 5975C mass detector (Agilent Technologies, CA) in 70 eV electron impact (EI) mode. Analytes were separated using an Agilent HP-5MS capillary column of 30 m × 0.25 mm with a phase thickness of 0.25 μ m. A 2 μ l volume of the sample was injected in the splitless mode. The oven temperature program was as follows: an initial temperature of 160 °C for 0.5 min; an increase of temperature to 290 °C at a rate of 30 °C/min; and, finally, maintaining the temperature at 290 °C for 8 min. Helium was used as the carrier gas. The injector and aux-heater temperatures were set at 250 °C and 280 °C, respectively. Qualitative analysis was carried out under full-scan acquisition mode within the 30~300 u range. Compounds were identified based on the MS spectrum using NIST MS search software (version 2.0 f) and AMDIS software from the NIST 08 Mass Spectral Library database. Blank extraction was simultaneously carried out and tested using the same experimental procedure, for contamination control.
Extraction of theanine from ancient samples. An ancient sample (10-30 mg) was weighed, ground to powder, and transferred into a tube with 5-15 ml ultrapure water. The sample was boiled for 5-10 min, subjected to sonication for 30 min at 60 °C and then centrifuged at 3,000 g for 5 min. After filtering, the extraction was evaporated to 0.  UPLC/MS analysis. UPLC/MS was performed using a Waters ACQUITY UPLC-Xevo G2 Q-TOF mass spectrometer (Waters, USA). The chromatography was performed on a waters UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μ m). The mobile phases consisted of (A) 0.1% formic acid in water and (B) 0.1% formic acid in ACN. The UPLC elution conditions were as follows: 0 min, 100% A; 1 min, 100% A; 9 min, 100% B; 11 min, 100% B; 11.1 min, 100% A; and 13 min, 100% A. The flow rate was set at 0.3 ml/min. The column was maintained at 55 °C. An injection volume of 10 μ l was used for the reference standard and samples. MS analysis was performed using an electrospray ionization (ESI) source in positive mode. The desolvation gas flow rate was set to 750 l/h at a temperature of 500 °C. The cone gas was set to 25 l/h. The source temperature was 100 °C. The capillary and cone voltages were set at 3000 V and 25 V, respectively. MS spectra were acquired from m/z 50 to 1,200. An internal lock mass calibration at m/z 556.2771 with mass resolution > 22,000 was used during analysis. The instrument was controlled and data were processed using MassLynx 4.1 software (Waters, USA).

Analysis of phytoliths.
(1) Plant residue samples were placed in 20 ml of saturated nitric acid for over 12 h to oxidize organic materials completely. (2) Solutions were centrifuged at 2000 rpm for 10 min, decanted and rinsed twice with distilled water, and then rinsed with 95% ethanol until the supernatants were clear. (3) Phytolith sediments were transferred to storage vials. The residual subsamples were mounted onto microscope slides in Canada Balsam medium for photomicrography and in liquid medium for counting, measuring and line drawing. (4) Light photomicrography (phase-contrast, and microscopic interferometer) at 400× magnification was used to determine their anatomy and silicon structure patterns. (5) Phytolith parameters were measured using computer-assisted image analysis 18,19 .