Ecology

Comparison of spectral water index methodsFigure 3 shows the results of different spectral water index methods. Through overlay analysis with the original image and detailed visual analysis, it was found that AWEIsh had the best extraction performance and could accurately identify the boundary of the water body. NDWI, MNDWI, and EWI had different degrees of leakage extraction; NDWI and EWI had an evident leakage extraction in the northwest corner of the image, and the water leakage extraction of MNDWI was mainly concentrated in the middle of the image. There was a lot of water body misidentified in WI, especially in the southeast corner of the image.Figure 3Results of water extraction from different spectral water index methods. (a) The original image. The threshold values and extracted water bodies from (b) NDWI, (c) MNDWI, (d) EWI, and (f) AWEIsh methods determined by the OTSU algorithm. (e) The extraction result of WI.Full size imageBased on the visual interpretation of the water boundary, 100 test samples were selected and the confusion matrix32 was calculated to obtain the extraction accuracy of the water body from three aspects: commission error, omission error, and overall accuracy (Table 1). As seen from the table, the overall accuracy of AWEIsh was the highest, attaining a value of 99.14%, with extremely low commission and omission errors. WI had the lowest overall accuracy and the highest commission error, and could not distinguish water bodies and low reflectivity features effectively. The overall accuracies of NDWI, MNDWI, and EWI were similar. Comparing the results of the visual interpretation and quantitative analysis, the rapid extraction model of surface water based on the Google Earth Engine utilizing AWEIsh index was used for assessing the spatiotemporal changes of water bodies.Table 1 Accuracy comparison of different spectral water index methods.Full size tableTime series analysis of typical reservoir areaTo understand the inter-annual variation trend and intra-annual variation of the reservoir area in the dry zone of Sri Lanka, time series analysis was conducted with the Maduru Oya Reservoir as the case study area. The Maduru Oya Reservoir is the second largest reservoir in Sri Lanka, located in the east-central region, which is the main water source for irrigation and drinking, and has a high incidence of chronic kidney disease of unknown aetiology (CKDu). Figure 4 shows the inter-annual and intra-annual variations of Maduru Oya Reservoir area.Figure 4Observed area change in the Maduru Oya Reservoir. (a) Inter-annual variation of the Maduru Oya Reservoir area; (b) Intra-annual variation of the Maduru Oya Reservoir area in 2017.Full size imageFigure 4 shows that the inter-annual fluctuation of Maduru Oya Reservoir area is slight, while the intra-annual fluctuation is significant. From 1988 to 2018, the reservoir area showed an overall increasing trend with slight float; the smallest area was recorded in 1992 (27.43 km2) and the largest area in 2013 (42.97 km2) (Fig. 4a). The rainy season in the dry zone of Sri Lanka occurs from October to February, and the dry season occurs from March to September. In 2017, the maximum area of the Maduru Oya Reservoir was noted in February, and the minimum area was noted in September. The area in February was 2.24 times bigger than that of September, with a difference of 31.58 km2. The maximum area of reservoirs or lakes generally occurs at the end of the wet season (February), and the minimum area occurs at the end of the dry season (September)2, which is consistent with the occurrence of maximum and minimum area in the Maduru Oya Reservoir in 2017(Fig. 4b). The area of the reservoir increased significantly in May during the dry season. According to meteorological data33, there were persistent strong winds and torrential rains in Sri Lanka in May 2017, resulting in an abnormal increase in the reservoir area. Generally, the period in which the area increased was from October to February (rainy season), while March to September (dry season) was the period in which the area decreased regardless of the influence of abnormal weather factors. The intra-annual fluctuation of the reservoir was severe, and there was a risk of drought and flooding at the same time. This observation implied that the seasonal regulation of water resources must be focussed in the future.Analysis of spatiotemporal change of inland lakes and reservoirsTo systematically analyze the spatiotemporal variation characteristics of inland water in Sri Lanka in recent years, and considering the cloud cover of Landsat-5/8 images, 1995, 2005 and 2015 were selected as the study year with an interval of 10 years. The distribution information of surface water in three stages was obtained by running the rapid extraction model of surface water in the Google Earth Engine. According to statistics, the surface water areas of Sri Lanka in 1995, 2005, and 2015 were 1654.18 km2, 1964.86 km2, and 2136.81 km2, respectively. In the past 20 years, the water area of Sri Lanka has increased significantly. To further analyse the spatiotemporal changes of inland lakes and reservoirs, a 5-m buffer data of rivers in 2015 were produced in ArcGIS10.3 software; further, the area corresponding to the river channels were removed from the three images and only the lagoon areas were preserved. Lagoons are ubiquitous in the coastal areas of Sri Lanka, with flood discharge, aquaculture, coastal protection, and other functions34. The results consisting of the extracted lakes, reservoirs, and lagoons are shown in Fig. 5.Figure 5Water extraction results for Sri Lanka in 1995, 2005, and 2015. The administrative boundary data of Sri Lanka comes from the Humanitarian Data Exchange (HDX) open platform (https://data.humdata.org). The maps were generated by geospatial analysis of ArcGIS software (version ArcGIS 10.3; http://www.esri.com/software/arcgis/arcgis-for-desktop).Full size imageThe overall water area of lakes and reservoirs in Sri Lanka showed an increasing trend from 1995 to 2015, and the lagoon area increased over these 20 years (Fig. 5). Because the lagoon does not belong to inland freshwater sensu stricto, the corresponding statistical analysis was not included in the following step. According to statistics, the total area covered of lakes and reservoirs in Sri Lanka were 1020.41 km2, 1270.53 km2, and 1417.68 km2 in 1995, 2005, and 2015 respectively. In the past 20 years, the area of lakes and reservoirs in Sri Lanka has increased by a considerable margin, attaining a value of 397.27 km2. To further analyse the spatiotemporal variation of inland lakes and reservoirs, they were divided into four grades according to their area: I ( More

Field studiesPhotosynthetic carbon fixation was investigated at eight different stations in the Pearl River estuary of the South China Sea (Fig. 1a and Supplementary Table 1), where the phytoplankton assemblages were dominated by diatoms45 during the time of our investigation (June 2015). Samples were collected from 10 to 20 m depths and transferred immediately into 50 mL quartz tubes and sealed to prevent gas exchange. The samples were inoculated with 100 μL of 5 μCi (0.185 MBq) NaH14CO3 solution for 2.15 h. All the incubations were carried out under incident solar radiation, attenuated with neutral density filters to simulate light intensities at the sampling depths, and the temperature was controlled with flow-through surface seawater.After incubation, the cells were filtered onto glass-fiber filters (25 mm, Whatman GF/F, USA) and stored at −20 ° C until measurement, during which the filters were exposed to HCl fumes overnight and dried (20 °C, 6 h) to remove unincorporated NaH14CO3 as CO2. The incorporated radioactivity was measured by liquid scintillation counting (LS 6500, Beckman Coulter, USA), and photosynthetic carbon fixation rates were estimated as previously reported46. Since the measurements were carried out under varying and low light levels similar to in situ levels at depths of 10 and 20 m, we normalized the photosynthetic rates to light intensity (μmol C (μg Chl a)−1 h−1 (μmol photons m−2 s−1)−1) to obtain the light use efficiency of photosynthesis (PLUE). This was done to allow for a meaningful comparison among different stations according to the linear relationship of photosynthetic carbon fixation under low solar irradiance levels46, which lies within the range of sunlight levels used in the present fieldwork ( More

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Environmental isotopic baseline of the Yingpan cemeteryMultiple body tissues of Yingpan Man, including his serial dentine, bone collagen, hair keratin and muscle, were isotopically analyzed. The δ13C measurements show a mix of C3 and C4 dietary inputs with values that range from − 17.3‰ to − 13.7‰, while the δ15N results are remarkably elevated and range from 14.9‰ to 19.9‰. According to the isotopic literature, plant δ15N values above 20‰ are relatively rare in terrestrial environments22,26,27,28. However, the δ15N values of the plants, animals and Yingpan Man are remarkably elevated (up to 23.3‰) compared to isotopic results from other parts of China and Europe (Fig. 3)12. This suggests that the entire ecosystem of this portion of Xinjiang is 15N-enriched. In addition, seeds of Nitraria pamirica having elevated δ15N results (up to 27.9‰) were found in the western region of the Pamir Plateau near the Afghanistan border23. Grains of wheat and barley from Shichengzi (ca. 202 BC–220 AD; ~ 400 km from Yingpan) yielded δ15N values that range from 14.6‰ to 19.8‰ (mean ± SD = 17.2 ± 1.5‰; n = 10) and 13.4‰ to 19.8‰ (mean ± SD = 17.3 ± 1.9‰; n = 12), respectively29. This evidence suggests that the entire Tarim Basin and greater Xinjiang has some of the most elevated terrestrial δ15N values in Eurasia. This is also supported by past isotopic work in China, which demonstrated a correlation between human δ15N values and annual mean precipitation, with individuals from Xinjiang having the highest δ15N values of all regions studied12. The cause of this 15N-enrichment must be at least partially environmental based on past isotopic studies27,30. The extreme aridity of the Yingpan cemetery site, which is located in the Taklamakan Desert, the driest region of China and characterized by little rainfall of 0 to 100 mm/yr and high evapotranspiration rates  > 2500 mm/year (Fig. 1)31, results in a large plant 15N-enrichment by intensive evaporation of 15N-depleted ammonia (NH4+) from the soil. These elevated δ15N results are then translated up the food chain to the domestic animals and humans. This is compelling evidence that Yingpan man was born and raised in a 15N-enriched environment that was extremely arid, and that he consumed wheat/barley, broomcorn millet and grape that were grown locally.Breastfeeding, weaning and childhood dietary patterns of Yingpan ManIsotopic analysis of dentine serial sections in human teeth permit an investigation of individual dietary patterns over the time period when the teeth were developing24. For Yingpan Man, his first molar (M1), represents the period of his life from birth to approximately 10 years old32. The first five 1 mm dentine sections from the M1 crown, corresponding to the first ~ 2.2 years of his life, show a steady decrease in δ15N of 2.5‰ (Fig. 4). This is evidence that Yingpan Man was fully weaned off breastmilk at or before the age of ~ 2.2 years old (Fig. 4)33,34, and is similar to findings of the Zhou Dynasty (1122–771 BC) sites of Boyangcheng35, Xiyasi and Changxinyuan36.Archaeological evidence for infant feeding practices is rarely preserved in Xinjiang. However, two extraordinary feeding vessels (one made of goat breast skin, the other made from an ox horn) were previously discovered with the burial of a 10-month old infant mummy in the southern Tarim Basin4,7. This infant dates to ~ 1000 BC and was found at the Zhagunluke site which is ~ 460 km from the Yingpan cemetery (Fig. 1). In addition, historical documents also provide supporting evidence that children were fed with animal milk in ancient Xinjiang. For example, the Kharosthī scripts (written in a version of the north Indian Prakrit language) are a collection of contracts, letters and other documents (e.g. wood tablets, leather, silk, paper, etc.) that detail life, events, trading, taxes and agricultural practices during the third to fifth century AD of the Shanshan Kingdom (~ 250 km from Yingpan, see Fig. 6)37. It is recorded in these documents that a “milk fee” of cattle or camel was paid to the adoptee when children were adopted in ancient Shanshan, and this exchange was not only protected by the law but also needed to be ensured by witnesses37,38. Thus, there may have been a common tradition of feeding infants with milk from domestic animals (e.g. goat, cattle, camel, etc.) during the weaning process in ancient Xinjiang.Figure 6Yingpan Man’s grave goods mirroring the cultural contact across the Eurasian continent (~ 300 AD). (a) Decorative patterns of vase and flowers on the sideboards of Yingpan Man’s coffin. (b) Decorative patterns of nude putties, goats, bulls and pomegranate trees on Yingpan Man’s woolen caftan. (c) Yingpan Man’s white hemp mask with golden diadem (possibly related to the death masks from Tashtyk culture). (d) Woolen trousers with decorative patterns of double quatrefoil florals surrounded by lozenges made up of solid circles and flowers. (e) Miniature funerary summer coat. (f) Miniature funerary winter robe. (g) Embroidered armband. (h) Yellow painted wood coffin with decorative patterns. (i) A broken brocade that was decorated with Chinese characters of “Shou” and “You”. (j) Silk fish. (k) Crowing cockerel pillow. (l) Perfume sachet. (m) A tufted carpet with decorative patterns of a male lion. (Map was generated using GMT 5.2.1. The original pictures of artifacts were previously published14 and provided by Wenying Li. Pictures were modified using Adobe Photoshop CC 2015 V.1.2. The final layout was created in Adobe Illustrator CC 2019 V.23.1.1).Full size imageIn contrast, the δ13C results of the first four dentine sections show little variability, which indicates that Yingpan Man consumed other foods/liquids from an early age and was not exclusively breastfed for a significant amount of time after birth (Fig. 4; Fig. S4)34. Between 4 and 9 mm, there is an increase in the δ13C values of 1.7‰, which corresponds to a late weaning and childhood diet from approximately 1.8 to 4.2 years old, where more millet was consumed (Fig. 4; Fig. S4). This millet possibly took the form of a gruel as there is little evidence that Yingpan Man consumed increased amounts of animal protein during this period. Similar isotopic patterns in dentine representing individuals that consumed increasing amounts of millet during weaning and early childhood were found at the Late Neolithic (4500 BP) Gaoshan site in Sichuan Province39. This suggests that millet may have had a long history of being used as a weaning or childhood food in China. From 9 to 14 mm, the dentine serial sections show a decline in δ13C values of 1.4‰ which indicates a period of increasing C3 foods in the diet from approximately 4.2 to 6.6 years old (Fig. 4; Fig. S4). Then from 14 to 21 mm the δ13C values increase again by 1.3‰, evidence of a change back to millet consumption from approximately 6.6 to 10 years old. Thus, over the first 10 years of life, Yingpan Man had frequent dietary shifts between C3-based and C4-based foods (Fig. 4; Fig. S4).Seasonal diet of Yingpan ManIn agreement with his dentine serial sections, the δ13C results of Yingpan Man’s hair display fluctuations between C3-based (e.g. wheat, barley) and C4-based (millet) foods (Fig. 5a). These hair δ13C results appear to follow a periodic trend, and suggest that his diet changed during different seasons of the year. In contrast, apart from the last 6–7 months of life, Yingpan Man’s hair δ15N values show little variation (Fig. 5b). However, a strong correlation was found between the variability of both the hair δ13C and δ15N values (Sperman’s r = − 0.534; sig = 0; n = 46; Fig. S5a), but not between the δ13C and δ34S values (Sperman’s r = 0.100; sig = 0.539; n = 40; Fig. 5; Fig. S5b). The lack of significant changes in the δ15N values is evidence that Yingpan Man’s protein consumption was relatively constant for approximately the last 4 years before death. This constant protein consumption, but variable intake of C3 and C4 plants, could suggest that in addition to directly consuming different amounts of wheat/barley and millet, Yingpan Man may have actually consumed domestic animals (goats, sheep, cattle) that were foddered on these crops at different times of the year. This possibility could explain the periodic variability in the δ13C values as well as the lack of variability in the δ15N values. In addition, the δ34S hair results also show little variation in the serial sections (~ 1.5‰). Yingpan Man’s δ34S results have a terrestrial range between 10.4‰ and 11.9‰40,41,42 and are similar to past δ34S values from the Proto-Shang site of Nancheng in Hebei Province43. While few δ34S studies have been reported for China for comparison, this lack of isotopic variability in sulfur suggests that Yingpan Man was likely not a Silk Road traveler, but stayed close to the local area during the last years of his life42. Future research involving strontium analysis on Yingpan Man’s hair serial sections and teeth will hopefully support or refute these findings44.Regional evidence in support of seasonal dietary changes comes from apatite δ13C and δ18O results of serial sections of tooth enamel of domesticates from the pastoral sites of Dali, Begash and Tasbas in Kazakhstan (~ 750 km from Yingpan)45. Specifically, data from early and middle Bronze Age sheep, goats and cattle displayed periodic patterns in their δ13Capa and δ18Oapa results that reflect the consumption of more millet during winter and more C3 plants during summer months, based on environmental inputs of body water45. A combination of radiocarbon dates and the application of the dietary mixing model (MixSIAR) identified early sheep and goats from this region (e.g. Dali, 2705–2545 cal BC) to be winter foddered with up to 44–50% of millet intake45. During later periods, this reliance on millet fodder during the winter months increased with some goats from Begash having 50–60% millet in their diets. Further, the relative contribution of millet to the diet of sheep and goats from the early phase (2345–2080 cal BC) of Begash reached up to 68–74% of the whole diet during winter months, especially from November to December45. This key study indicates that there is a long history and precedent for seasonal C3 and C4 feeding of domestic animals in Central Asia, and supports the isotopic findings of a seasonal diet in the hair of Yingpan Man.In addition, isotopic analysis of sequential hair samples from mummies recovered from the Oglakhty cemetery in the Minusinsk Basin of southern Siberia, Russia (~ 900 km north of Yingpan) also show seasonal dietary variation with millet and fish consumed during the late summer and autumn46. These mummies of the Tashtyk culture date to the same period as Yingpan Man (third to fourth centuries AD), and interestingly were also buried with white painted gypsum funerary masks that are similar to the one that Yingpan Man was wearing46,47. This unique burial tradition could suggest links such as trade or an association between Yingpan Man and the Tashtyk culture and additional research is necessary to explore this possibility in more detail (Fig. 6).Historical documents such as the Kharosthī scripts provide additional valuable information about seasonal diets and the foods consumed by the inhabitants of ancient Xinjiang37. These texts describe how the people of the Tarim Basin cultivated mainly wheat, millet and barley as their main cereal crops and that grapes were carefully managed for the production of wine37. Autumn (around the 10th month of the year), was mentioned as the time for harvesting crops and trading crops as well as wine and animals, and paying debts37. Thus, autumn would have been the time of the year with the most abundant amount of food resources, especially C3 foods like fruit, vegetables and wine. This is still true today, as the harvesting of agricultural products in the Tarim Basin mainly occurs in the month of October48. In contrast, during the winter months food resources would have been scarce with only non-perishable crops like millets and wheat available to guarantee the food supply “in the harsh winters of Inner Asia”45. Thus, more C4 foods were likely consumed during the winter and spring months while more C3 foods were consumed during the summer and autumn months.If this information is applied to Yingpan Man’s hair δ13C results, it would suggest that the δ13C values decreased during the summer and autumn months (June to October) and likely reach a nadir during the middle of autumn or October. Therefore, Yingpan Man’s hair sections which are 8 to 10 cm, 18 to 25 cm and 36 to 43 cm from his scalp reflect the period of middle autumn (between August to October) while the hair sections which are 4 to 6 cm, 13 to 15 cm and 29 to 33 cm from his scalp represent late winter (December to February). This would suggest that Yingpan Man died ~ 4 months after the last drop in his δ13C hair values or in spring, possibly March or April (Fig. 5). In addition, the clothes in which Yingpan Man was buried, as well as his associated wardrobe, also provide information about the timing of his death14,15. The miniature robe placed on his chest was designed for winter, as it was long, double layered and the interior was lined with sheep’s wool (Fig. 2f). Whereas the miniature coat placed near his wrist was designed for summer as it was shorter and made only of a single layer of silk (Fig. 2p). Yingpan Man’s caftan, in which he was buried, was double layered with the outer layer made of wool and the inner lining made of silk, and this was likely designed either for the spring or autumn (Fig. 2k)14. Thus, Yingpan Man’s burial clothes combined with the isotopic and historical evidence indicate that Yingpan Man died sometime during the spring months14,15.To better illustrate Yingpan Man’s seasonal dietary variation, two hair sections respectively representing the highest (Sample A: − 14.0 ± 0.1‰, 15.2 ± 0.1‰) and lowest δ13C values (Sample B: − 17.3 ± 0.2‰, 15.8 ± 0.1‰) were analyzed by a Bayesian mixing model with the application of FRUITS (Food Reconstruction Using Isotopic Transferred signals)49,50. As displayed in Fig. S6, isotopic data for millet, wheat/barley, grape and sheep/goat from the Yingpan cemetery were incorporated into the mixing model as feasible dietary sources for Yingpan Man in both scenarios (Sample A and Sample B). The isotopic fractionation between human hair and diet is corrected with an offset of 4.0 ± 0.5‰ for δ13C values51,52,53 and 4.5 ± 0.5‰ for δ15N values51,54. The relative contribution of different macronutrients is defined in the mixing model according to published records with 74 ± 4% of the carbon originating from protein and 26 ± 4% originating from carbohydrates and lipids and all of the nitrogen originating from protein55,56. The high hair δ13C value of − 14.0‰ (Sample A) represents a heavy reliance on millet consumption (39–67%; median = 53%) and a low amount of dietary input from wheat/barley (0–51%; median = 12%), grape (1–46%; median = 25%) and sheep/goat (0–30%; median = 4%), which likely reflects Yingpan Man’s diet during the winter and spring months (Fig. S6 and Table S13). In contrast, the low hair δ13C value of − 17.3‰ (Sample B) likely represents a summer/autumn diet with a decline in C4 foods, like millet (8–44%; median = 28%), as well as increased reliance on C3 foods like wheat/barley (1–71%; median = 21%) and sheep/goat (0–81%; median = 9%) (Fig. S6 and Table S13). However, the importance of sheep/goat and wheat/barley is likely under-estimated here given their small sample size and highly elevated δ15N values. Nonetheless, the FRUITS mixing model indicates that the varying consumption of plant foods, especially millet, is clearly responsible for the δ13C shifts of Yingpan Man’s diet.The last months of Yingpan ManThe hair δ13C results provide an estimate for the time of year Yingpan Man likely died. In addition, the hair δ15N and δ34S values can provide evidence for how Yingpan Man may have died. The last ~ 6 cm (closest to the scalp) of Yingpan Man’s hair show a general rise in δ15N by ~ 1‰ (Fig. 5b). This pattern is uncharacteristic compared to the other hair δ15N results that display little variation. This unique 15N-enrichment could represent a period of catabolic wasting due to the recycling of tissue proteins as a result of a prolonged illness25,57,58. Additional support for some sort of disease or period of illness comes from the fact that there is little change in the δ13C values but a slight decrease in the δ34S values during the last ~ 6 cm of Yingpan Man’s hair. Tissue catabolism is known to cause an increase in δ15N but little change in δ13C values in human hair25. Further, δ34S results are known to decrease in the red blood cells and serum (by ~ 1.5‰) of patients suffering from liver cancer59. As the last ~ 6 cm of hair displayed a slight decrease in δ34S by ~ 1‰, with the largest decline during the last month of life, this evidence in conjunction with the δ13C and δ15N values suggests that Yingpan Man did not die suddenly but likely suffered some type of debilitating sickness over the last months of his life before he succumbed. However, as most wasting diseases or illnesses leave no traces on human skeletons, it is difficult to define the specific disease that caused Yingpan Man’s death, and a detailed paleo-pathological study of Yingpan Man is needed in the future.The grave goods buried with Yingpan Man also suggest he may have suffered a compromised health status before death. In particular, a piece of tattered yellow brocade decorated with brown and blue images of vines, animals, birds, flowers, as well as the Chinese characters of “Shou” and “You” was found placed at a prominent position on the right side of Yingpan Man’s head (Fig. 7)14. According to Chinese historical literature sources, “You” means “blessing” or “blessed”. While, “Shou” means “long live” or “healthy” (Fig. 7)60. Similar grave goods with Chinese characters e.g. “Yan Nian Yi Shou Da Yi Zi Sun” (meaning “live longer and benefit the descendants”), “Chang Le Ming Guang Cheng Fu Shou You” (meaning “always be happy, be bright, be lucky and be blessed”), “Yong Chang” (meaning “always be prosperous”), were also frequently unearthed from contemporary and later cemeteries of the Tarim Basin, especially the nearby sites of the Loulan culture (third century BC to 448 AD, renamed as Shanshan in 77 BC, ~ 250 km from Yingpan), as grave goods that carry good wishes for the dead15. However, Yingpan Man’s brocade shows significant traces of wear or “rubbing” (Fig. 7). This is interesting as this brocade was not complete or new but well-worn to the point of being tattered and frayed, yet it was still placed at a very important position in Yingpan Man burial—just beside his head14. This suggests that this brocade may have been an important “lucky” health charm for Yingpan Man that was frequently used either by himself or by those who cared for him before burial. Thus, both isotopic and archaeological evidence suggest Yingpan Man suffered some type of illness during the last ~ 6 months of his life, likely in winter, and that he succumbed to this illness in the following spring.Figure 7Photo and sketch showing details of the embroidered brocade that was found with Yingpan Man burial. (The original picture and sketch were previously published14 and provided by Wenying Li. Pictures were modified using Adobe Photoshop CC 2015 V.1.2. The final layout was created in Adobe Illustrator CC 2019 V.23.1.1).Full size imageWho was Yingpan Man?The social identity and status of Yingpan Man is enigmatic given the various cultural components of his grave goods and since the collection of his funerary objects are unique compared to all of the other burials found in the same cemetery14. This has created much controversy and debate about the social identity of Yingpan Man7,14,15,16. Thus, “Who was Yingpan Man?” and “Why was he buried here, in a normal unmarked grave with such lavish and exotic grave goods?” is an active topic of debate.The physical anthropology of Yingpan Man was investigated but not formally published. According to which, the metric and nonmetric index of traits of his skeletal remains suggest a mix of both European and Mongolian features (Dong Wei, personal communication). However, facial reconstruction conducted by Dong Wei (personal communication) indicates that Yingpan Man’s facial structure is more characteristic of the features from Western Eurasia. This is consistent with the image of the face painted on his death mask and the fact that he wears a golden diadem across his forehead, which is more traditionally associated with Greece4,7. However, Yingpan Man’s white hemp mask is similar in style to the white painted gypsum masks of the Tashtyk culture from the Minusinsk Basin of Russia46. In addition, Yingpan Man and the mummies of the Oglakhty cemetery of Tashtyk date to nearly the same period (third to fourth centuries AD), and the polychrome silk cloth from the Tarim Basin has also been discovered in Oglakhty46. This evidence, while circumstantial, could suggest some form of association or that links with the Tashtyk may have taken place during the lifetime of Yingpan Man, possibly through trade or familial relationships (Fig. 6).Other components of Yingpan Man’s burial provide important clues about his social identity and cultural affiliations in life61. The styles and types of grave goods of Yingpan Man display an unique mix of both Eastern and Western cultures and traditions that were likely common to inhabitants of Silk Road trading towns in Xinjiang during the third to fourth centuries AD (Fig. 6)14,15,16. However, there appears to be a strong eastern component in some of his funerary arrangements. For example, the burial practices associated with Yingpan Man: covering his face, filling his nose, burying his body fully clothed, covering it with a silken burial shroud, as well as using miniature funeral objects as grave goods are in accordance with the suggested burial rites of “Yan” (meaning “covering”), “Zhen” (meaning “filling”), “She Min Mu” (meaning “covering the eyes”), “Qin” (meaning “quilt” or “burial shroud”) mentioned in the Confucian literature of Yili (meaning “Rites”, formed during the Zhou Dynasties (1046 BC to 256 BC))15,62. In addition, the styles and designs of some of his grave goods are indicative of Chinese spiritual beliefs. For instance, the diamond and circle-shaped designs of decorative patterns on the cover and sideboards of Yingpan Man’s coffin are argued by some scholars to be the traditional “Lianbi” pattern (meaning “linked jades”) which symbolizes the jade burial suits (“Jinlü Yuyi”) that were popular in Han Dynasty burials of high status nobles in central and southern China (e.g. Nanyuewang tomb, Mancheng tomb)15. In particular, as jades are believed to be the ideal material for embalming in ancient China63, the “Lianbi” pattern on Yingpan Man’s coffin carries the symbolic meaning of preserving his body forever so that his soul and spirt will reach heaven (Fig. 6; Fig. S1)15.The brocade “health charm” found to the right side of Yingpan Man also shows a clear affiliation with China, as it was decorated with the Chinese characters of “Shou” and “You”. This is important as some historical documents suggest that Kharosthī was the common language in the Tarim Basin at this time37,38, but Yingpan Man was likely more accustomed to Chinese spiritual beliefs according to this brocade (Figs. 6, 7). Moreover, the crowing cockerel pillow is also interesting as it was recorded in the Han Dynasty literature of Lunheng (meaning “On Balance”, compiled by Chong Wang during the Eastern Han Dynasty in 88 AD), that the dead “turns into ghosts” and “hurt the living ones”64, and that a crowing cockerel was believed to be able to expel evil spirts and ghosts65. Thus, a crowing cockerel pillow was frequently used in funerary practices in China since the Han Dynasty66, and it is still a common grave good today in some areas of modern China (e.g. Shandong and Guizhou Provinces)65.The eight decorative pearls attached to the crowing cockerel pillow are also important status markers regarding the identity of Yingpan Man (Fig. 8). In Xinjiang, pearls would have been long distance imported products from the coastal regions which are  > 3000 km from the Yingpan cemetery. Ancient China, Egypt, Persia, Greece and India were known to have produced and prized pearls67. However, according to Chinese historic literature sources: Shangshu (meaning “the Book of Documents”; written by pre-Qin philosophers during the Zhou Dynasty ~ 1000 BC)68, Hanshu (meaning “the Book of Han”; written by Gu Ban during the Eastern Han Dynasty in 105 AD)69 and Hou Hanshu (meaning “the Book of Later Han”; written by Ye Fan during the Southern Dynasties from 432 to 445 AD)70, pearls were known as royal tributes in China since the Xia Dynasty (~ 2000 BC), and were mainly produced in the coastal cities of southern China, such as Panyu (modern Guangzhou City in Guangdong Province), Hepu (modern Hainan Province), Zhuya (modern Guangxi and Guangdong Province), etc.67 These coastal regions of China were connected to the Tarim Basin area via the Silk Road trading routes and were possibly the source for the pearls found on Yingpan Man’s pillow (Fig. 6). In particular, a silk pillow fully covered with unpolished natural pearls (placed beneath the tomb owner’s head, weighing ~ 470 g) and a lacquer box of pearls (weighing 4,117 g) were unearthed from the tomb of the King of the Nanyue Kingdom (a vassal state of Han) in modern Guangzhou71. This demonstrates a similar preference for pearls as decorations on pillows or as grave goods for high status individuals.Figure 8Crowing cockerel pillow. (a–d) Photo and sketch of the crowing cockerel pillow from Yingpan Man burial. (e–h) Decorative patterns of the four mythical beasts on Han Dynasty eaves tiles. (The original pictures and sketches were previously published14 and provided by Wenying Li. Pictures were modified using Adobe Photoshop CC 2015 V.1.2. The final layout was created in Adobe Illustrator CC 2019 V.23.1.1).Full size imageMoreover, the decorative patterns found on Yingpan Man’s crowing cockerel pillow are remarkable. These include the images of: a monkey-shaped face, a griffin-shaped beast and a net-shaped pattern as well as the images of the mythical beasts of the “blue dragon”, “red sparrow” and “white tiger” (Fig. 8d)17,47,61,72. Together with another mythical beast known as the “black turtle-snake”, these four mythical beasts were believed to be the guardians of the four cardinal directions and also represents four different colors and elements according to traditional Chinese cosmology (Fig. 8e–h). The dragon guarding the East and representing the color blue and the element of wood, the sparrow guarding the South and representing the color red as well as the element of fire, the tiger guarding the West and representing the color white and the element of metal, and the turtle-snake guarding the North and representing the color black and the element of water62,73. Thus, the images of these four mythical beasts were frequently integrated into the designs of cities, buildings, cemeteries and objects of ancient China for their symbolic function of protection63. In this context, the net-shaped pattern on Yingpan Man’s crowing cockerel pillow is likely a symbol of the four cardinal directions, while the monkey shaped face possibly symbolizes a human, that is to be protected. Notably, here, in the design of Yingpan Man’s crowing cockerel pillow, the image of the northern guardian of the turtle-snake is replaced by the Greek mythical beast known as Griffin (Fig. 8d)16,47,74. This change in imagery and symbolism could suggest that contact between the East and the West was via a northern route and that this was associated with some type of Greek influence or the Griffin. According to historical literature sources, a large number of Greeks migrated into Central Asia with Alexander the Great’s eastward expedition3,5,6. Hellenistic kingdoms such as Bactria (a.k.a. Daxia in the Chinese literature, located in the area of Pamir Plateau in modern Afghanistan, ~ 1900 km from Yingpan) subsequently became centers of Greek influence in this area and exported elements of Hellenistic culture to surrounding kingdoms that were located along the main routes of the Silk Road, such as Dayuan (located in the Fergana Valley, ~ 1300 km from Yingpan)2,6,75. Some ancient cities in the Tarim Basin area, such as Yingpan, Maideke and Yuansha, are also argued to have been influenced by Hellenistic elements as their city walls were circular in shape and this was clearly a unique architecture compared to the square-shaped traditional Chinese city walls63,76. It is highly probably that these Hellenistic elements were transported to the ancient city of Yingpan by the Silk Road trading routes which followed a north–south direction in this region, and therefore would support that Greek elements would be associated with the northern direction in this region of Xinjiang. In conclusion, Yingpan Man’s crowing cockerel pillow displays an unprecedented combination of Chinese spiritual beliefs incorporated with western motifs, and symbolizes the intertwining of Eastern and Western cultures in this part of Central Asia (Figs. 6, 8).Even more significant uses of western components in Yingpan Man’s burial is also visualized in other grave goods, e.g. his: carpet, coffin, caftan. The decorative pattern of the male lion on Yingpan Man’s tufted woolen carpet is clearly an imported element as the lion was originally from Africa, Southern Europe, West Asia and India. Lions were not introduced into China until 87 AD, being sent to Emperor Zhang of the Eastern Han Dynasty as a gift from Pacorus II, the King of the Anxi Empire (a.k.a. Parthian Empire (247 BC to 224 AD, replaced by the Persian in 226 AD, located in modern Iran))70. In particular, this exquisite lion-decorated carpet suggests a very high social status for Yingpan Man as lions were deified in ancient China since the Han Dynasty and were used as symbols of power, authority and royalty as it was in other areas of the Eurasia (Fig. 2a)15,61. Moreover, it is also argued by some scholars that the motif of the lion on this carpet is from the Buddhist art of India, as lions are frequently mentioned in Buddhist stories and a Buddhist temple was also found at the site of Yingpan (Fig. 6)17,77.In addition, though the main decorative patterns on Yingpan Man’s coffin are the “Lianbi” pattern, images of vines, leaves, pomegranate flowers and vases were also depicted inside of the diamond patterns (Fig. S1)15. Among which, the image of pomegranate flowers is clearly an imported element from the West as pomegranates were originally domesticated in the middle East ~ 5000 years ago and were not introduced into China until the Han to Jin Dynasties, or with the flow of goods along the Silk Road78. In particular, the pomegranate was used as a symbol of health, fertility and rebirth as mentioned in many ancient cultures, especially in Greek and Turkish myths78. Thus, this is particularly interesting as the image of pomegranate appears not only on Yingpan Man’s coffin as flowers on the headboard and footboard (Fig. 6; Fig. S1), but also on his caftan as decorative patterns of fruited-trees (Figs. 6, 9). Specifically, the decorative patterns on Yingpan Man’s woolen caftan consists of six sets of nude puttis and animals with fruited pomegranate trees standing in between (Fig. 9)14. In particular, each of these six sets of images is composed of a symmetrical pair of confronting muscular puttis or animals (goats or bulls) that are either leaning away from or toward each other14. The nude puttis are holding either a spear, sword or a shield with capes swirling from their shoulders, while the animals of goats and bulls are in the pose of jumping and the bulls have laurel wreaths around their waists (Fig. 9)14. A similar design of stance and composition of figures was also discovered in a mosaic floor from the Villa of Good Fortune at Olynthos, Greece (paired female figures with weapons, fourth century BC)79 as well as another mosaic floor from Pella, the Macedonian capital (two nude youths with capes flying on their shoulder and weapons held in their hand, about to attack an animal in between of them, 325 to 300 BC)79. Thus, it is suggested that this caftan was the work of a weaver familiar with both Western and Eastern motifs as the character and poses of the nude puttis are clearly Western in style and appearance, the fruited pomegranate tree is believed to be a Persian motif, while the paired facing animals of goats and bulls are similar to the animal art of Central Asia8. However, this caftan was completed using the technique of double-weaving, the lining and belt were made of silk14, while the design of the side slits are indicative of a localized adaptation for horse-riding15. Moreover, analysis using high performance liquid chromatography (HPLC) on the red and yellow threads of this caftan suggest that they were dyed in a local workshop with indigenous materials of Rubia Tinctorum and Populus Pruinosa Schrenk, respectively80,81. In conclusion, the design and style of Yingpan Man’s caftan is unprecedented, and it is a masterpiece that combines both Greek/Roman, Persian, Central Asian, Chinese and local elements (Fig. 6).Figure 9Photo and sketch of the decorative patterns on Yingpan Man’s caftan. (The original pictures and sketches were previously published14 and provided by Wenying Li. Pictures were modified using Adobe Photoshop CC 2015 V.1.2. The final layout was created in Adobe Illustrator CC 2019 V.23.1.1).Full size imageThe opulence and fine quality of the objects buried with Yingpan Man indicate that he must have had a high social status before death14,47. Given the importance of the town of Yingpan as a trading center on the Silk Road, the excavators who discovered Yingpan Man suggested that he was a wealthy merchant from the West14. Others have proposed that Yingpan Man might have been a Sogdian merchant since the Sogdians (an Iranian-speaking people whose homeland lay near Samarkand in what is now Uzbekistan) were the richest traders along the route7. However, given the relatively young age of Yingpan Man before his death (~ 30 years old), it is unlikely that he amassed all his fortune and high social status only through trade, and possibly by inheritance or military feats. The government of the Jin Dynasty established the administrative organization of “Xiyu Zhangshi Fu” (meaning “Chief Governor of the Western Regions”) in ancient Xinjiang, and the capital city of “Xiyu Zhangshi Fu” was located nearby Lop Nur and is very close to the ancient city of Yingpan (~ 185 km away)15,16. In addition, comparison of Yingpan Man’s burial to other contemporary burials from Gansu also suggest that Yingpan Man was possibly a military official from the government of Central China16. More supporting evidence comes from the embroidered armband that was buried with Yingpan Man as colorful armbands were suggested to be used by soldiers for the protection from evil forces in ancient Xinjiang (Fig. 6)15. An additional explanation is that Yingpan Man was a noble or even a king of the nearby state named Shan (a.k.a. Moshan)76, and the ancient city of Yingpan was suggested to be the capital city of this state15. An alternative explanation is that Yingpan Man belonged to a local noble family who were displaced from Bactria to the southern Tarim Basin after civil strife in the Kushan Empire at the end of the second century AD, given the popularity of Kushan arts in this area during the Han to Jin Dynasties82,83. The isotopic evidence presented here, in particular the hair δ34S results, add additional information to Yingpan Man’s identity. The lack of δ34S isotopic variability (10.4‰ to 11.9‰) over the last ~ 3–4 years of life indicates that Yingpan Man was not a Silk Road traveler or merchant, at least during this period of his life. Thus, Yingpan Man appears to have been a local, possibly a governmental official or royal to this region of the Tarim Basin, perhaps from the nearby state of Shan. This might suggest why he was buried in the Yingpan cemetery as it was purported to be capital of this ancient state. More

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Natural diets of tropical birds vary within speciesWe collected 62 regurgitated samples (using the tartar emetic method 22) from multiple tropical bird species representing four bird orders (Columbiformes–Pigeons, Coraciiformes–Kingfishers, Psittaciformes–Parrots, and Passeriformes–Passerines). First, we characterized diet components visually and then through metabarcoding of 52 of these samples using universal primers targeting invertebrates (Cytochrome c oxidase subunit I: COI gene) and plants (Internal transcribed spacer 2: ITS2 gene) (Table S1 and Fig. 2). Through visual identification, we identified plant material in 26 samples. The most common visually identified invertebrate orders were Araneae (spiders—27 samples), and Coleoptera (beetles—27 samples) (Table S2). Metabarcoding sequences were analysed using the OBITools software25. Overall, we found 47 plant operational taxonomic units (OTUs—97% sequence similarity threshold) and 180 invertebrate OTUs (Table S3). Plant items were dominated by the orders Rosales (27.7% OTUs), Fabales (8.5% OTUs), and Sapindales (8.5% OTUs). Except for four OTUs, all plants were identified to the genus level. Of the invertebrate OTUs, 54 belonged to feather mites (known feather symbionts), endoparasites, and rotifers (likely due to accidental consumption along with drinking water), and these OTUs were removed from further analyses, leaving 126 potential dietary invertebrate OTUs. Invertebrate samples were dominated by the classes Insecta (67.5% OTUs) and Arachnida (28.6% OTUs). At the order-level, dietary items were mainly represented by Araneae (spiders—28.6% OTUs), Hemiptera (true bugs—15.9% OTUs), Diptera (flies—14.3% OTUs), and Lepidoptera (moths and butterflies—10.3% OTUs). However, 77% of the invertebrate OTUs could not be identified to genus level, highlighting the limited research on genotyping invertebrate communities in Papua New Guinea.Figure 2Natural diets of wild birds vary between individuals of the same species and the results of the two identification methods of dietary components (visual identification and metabarcoding). Relative abundances based on the presence/absence of data of different dietary components are indicated in colours. Only invertebrates are separated into taxonomic orders as visual identification is unable to identify plant orders. Individuals depicted with asterisks had both crop microbiome and diet samples (dataset 1), while black font represents individuals with both cloacal microbiomes and diet samples (dataset 2). Individuals are clustered according to the species (each species is given a six-letter code name) and their literature-based dietary guilds. The order of the species is indicated with illustrations (Columbiformes–Pigeons, Coraciiformes–Kingfishers, Passeriformes–Passerines and Psittaciformes–Parrots), while ‡ represents diet samples with a complete consensus between the two identification methods.Full size imageDiet item identification differed markedly between visual and metabarcoding methods (Fig. 2, Tables S2 and S3). The diet components of individuals also varied notably within species (Figs. 2 and S1). Only diets of 12 out of 52 individuals were fully congruent between the two methods (Fig. 2). Of these 12 samples, eight had only plant material. Identification of invertebrate orders also differed between the two methods (Fig. 2, Table 1). Both methods identified the arthropod orders Hemiptera, Diptera, Orthoptera (crickets and locusts), and Araneae in the same samples (Fig. 2 and Table 1), while metabarcoding detected lower proportions of Coleoptera than the visual identification (Table 1).Table 1 Comparison between diet items identified in the regurgitated samples from the two approaches (visual identification and metabarcoding).Full size tableComparison of microbiomes and consumed diet itemsFor subsequent comparisons of diets and microbiomes, we utilised individual datasets from both visual identification (diet components identified at the order level) and metabarcoding (both OTU and order level), and a combination (order level) of both approaches (for details see “Methods” section on identifying prey items). Due to differences between the diet identification methods, a combination of the results was used to circumscribe the full diversity of consumed diets and to account for inherent biases associated with the two methods (i.e., the inability to identify plant material and smaller body parts of invertebrates visually, and extraction and sequencing biases associated with metabarcoding). We separated the microbiome dataset into three datasets due to sequencing limitations: dataset 1 included 12 birds with successfully sequenced crop microbiomes and diets identified using both methods, dataset 2 included 27 birds with successfully sequenced cloacal microbiomes and diets, and dataset 3 included 17 birds for which we obtained successfully sequenced crop and cloacal microbiomes (Table S1). Prior to subsequent analyses, each microbiome dataset was rarefied to even sequencing depths using the sample with the lowest number of sequences26 (Fig. S2).Crop microbiome similarity did not align with the consumed diet similarity (dataset 1)Out of the collected crop samples (N = 62), samples from only 19 individuals were successfully sequenced for their microbiomes. Of these individuals, we acquired diet samples for 12 individuals. Bacterial 16S rRNA MiSeq sequences were analysed using the DADA2 pipeline27 within QIIME228. There were 351,867 bacterial sequences (mean ± SD: 29,322 ± 33,009) in the crop microbiomes prior to rarefaction (Table S4). After rarefaction, bacterial sequences were identified to 615 amplicon sequence variants (ASVs—100% sequence similarity). Crop microbiomes were dominated by Proteobacteria (53.6%), Actinobacteria (18.9%), and Firmicutes (17.9%). Alpha diversities of individual microbiomes were calculated using the diversity function in the microbiome package29 and they did not differ significantly between host orders [Chao1 richness: Kruskal Wallis (KW) χ2 = 4.559, df = 3, p = 0.2271; Shannon’s diversity index: χ2 = 2.853, df = 3, p = 0.4149], or literature-based dietary guilds (Chao1 richness: KW χ2 = 4.317, df = 2, p = 0.1155; Shannon’s diversity index: KW χ2 = 2.852, df = 2, p = 0.2403) (Fig. S3).The compositional differences of crop microbiomes were investigated with the adonis2 function in the vegan package30 using permutational multivariate analyses of variance tests (PERMANOVA). These analyses revealed that the bird host order did not influence the crop microbiome composition (PERMANOVA10,000 permutations: Bray–Curtis: F = 1.251, R2 = 0.0993, p = 0.1911; Jaccard: F = 1.154, R2 = 0.0962, p = 0.2191) (Fig. S1). The effect of feeding guild was masked by host order as they are strongly correlated in this dataset. Furthermore, the lack of an effect of host taxa on crop microbiomes may be a result of the small sample sizes.We further investigated whether alpha diversity of the crop microbiomes was influenced by the diet item diversity of individuals. The Chao1 richness estimates of the microbiomes and the richness of the consumed diet items (number of different diet items based on the combined results) of individuals were not significantly correlated (Table S5), suggesting that the diet richness does not impact crop microbiome richness. However, Shannon’s diversity index of crop microbiomes and diet diversity were marginally significantly negatively associated (Table S5). This suggests that despite the lack of an association between diet and microbiome richness, crop microbiome evenness could be influenced by diet diversity.We then explored the association between the crop microbiome composition and the consumed diets, investigating correlations between Bray–Curtis and Jaccard dissimilarities of microbiomes, and Jaccard dissimilarity of diets using Mantel tests in the vegan package30. The compositional similarity of the diets based on any of the methods (visual, metabarcoding—both OTU and order-level separately, and combined) did not correlate significantly with crop microbiome compositions (Table 2 and Fig. S4). We observed similar non-significant associations between diets and microbiomes when investigating host orders separately (Table S6). This suggests that overall crop microbiomes of individuals are not completely modelled by the composition of the consumed diets.Table 2 Results of Mantel tests between the crop (dataset 1) and the cloacal (dataset 2) microbiome similarities (measured with both Bray–Curtis and Jaccard distances) and the consumed diet similarities (measured with Jaccard distances).Full size tableHost-taxon specific cloacal microbes are associated with different diet items (dataset 2)We obtained 27 individuals from 15 bird species with successfully sequenced cloacal microbiomes and diet samples (based on both metabarcoding and visual identification). Prior to rarefying, we acquired 818,272 bacterial sequences from the cloacal swab samples (mean ± SD: 30,306 ± 20,903) (Table S7). After rarefaction, bacterial sequences were assigned to 1,324 ASVs that belonged to Actinobacteria (35.9%), Proteobacteria (32.6%), Firmicutes (21.2%) and Tenericutes (5.0%). Cloacal microbiome alpha diversity did not differ significantly between different bird orders (Chao1 richness: KW χ2 = 2.624, df = 3, p = 0.4532; Shannon’s diversity: χ2 = 6.595, df = 3, p = 0.0861) or literature-based dietary guilds (Chao1 richness: KW χ2 = 1.128, df = 3, p = 0.7703; Shannon’s diversity: KW χ2 = 1.673, df = 3, p = 0.6429) (Fig. S5).However, cloacal microbiome beta diversity was significantly influenced by host bird order (PERMANOVA10,000 permutations: Bray–Curtis: F = 2.159, R2 = 0.2055, p  More

Patterns of species abundanceIn their analysis of the fossil assemblage of VM4, Luzón et al.1 indicate that herbivorous taxa comprise the bulk of the fauna. Their data, compiled in Table 1, show that herbivore remains represent 94.2% (1492/1578) of NISP and 78.8% (41/52) of MNI values for large mammals. These figures are close to those of VM3, 93.5% (6570/7027) and 84.4% (287/340), respectively (Table 1). A χ2 test shows that these differences are not statistically significant (p  > 0.3 in both cases). Among herbivores, Luzón et al.1 indicate that E. altidens is the species most abundantly preserved, both in frequency of remains and number of individuals, followed by cervids, bison, caprines, and megaherbivores (i.e., elephant, rhino, and hippo). This is also the situation in VM3 according to data compiled in Table 1: for example, the NISP value of E. altidens represents 31.8% (124/390) of the remains of large mammal identified in VM4 and 49.6% (2937/5924) in VM3. Although this difference is statistically significant (χ2 = 46.408, p  0.75). The difference based on NISP values seems high, but it falls within the range expected from variations in abundance data from different years for the ungulate prey more common in Serengeti, where the frequencies of Thomson’s gazelle, wildebeest, and zebra fluctuated in the late sixties between 18.9–56.3%, 21.3–42.8%, and 11.1–15.7%, respectively15,16. Finally, P. brevirostris is the species most represented among carnivores in both assemblages according to NISP values (Table 1), 26.8% (15/56) in VM4 and 30.0% (122/407) in VM3 (χ2 = 0.241, p  > 0.6), followed by canids, ursids and felids.The distribution of NISP and MNI values among taxa in VM4 and VM3 was further analysed using an approach based on contingency tables. The table for NISP values shows a significant χ2 value (Table 2, left part). This results from some differences in taxa abundance between the assemblages compared, which are reflected in the adjusted residuals: remains of megaherbivores and carnivores (excluding hyaenas) are represented in VM4 by higher frequencies than those expected from a random, homogeneous distribution, while they are underrepresented in VM3. This applies to the estimates obtained for VM4 using the data of Luzón et al.1 and our own data (Tables S1, S2). The NISP values estimated for P. brevirostris by Luzón et al.1 suggest a higher frequency of this carnivore in VM4 than in VM3, as indicated by the adjusted residual. However, the abundance of hyaena remains in our dataset for VM4 does not depart significantly from the expectations, as happens in VM3. Given that the database of Luzón et al.1 includes less than half of the remains of large mammals included in our database (Table 2), this suggests that the high frequency of P. brevirostris reported in VM4 results from poor sampling. The remains of other carnivores are more abundantly represented in VM4 than in VM3. However, it must be noted that a study of 24 dens of the three living hyaenas showed that the abundance of carnivore remains is highly variable, even among dens of the same species17. The distribution of MNI values among taxa in VM4 and VM3 (Table 2, right part) does not differ from the expectations of a random distribution according to the low χ2 value of the contingency table. Only the adjusted residual for megaherbivores, which are slightly over-represented in VM4 according to the data of Luzón et al.1, is statistically significant, while their abundance in VM3 is slightly lower than expected. Moreover, the probabilities of obtaining in the randomization tests the cumulative χ2 values observed for the NISP and MNI values of each species (p  0.97, respectively; Fig. S4) are equivalent to those obtained with their groupings in Table 2.Table 2 Contingency tables for the abundance of large mammals in the assemblages of the two excavation quarries of Venta Micena compared in this study, VM4 (a: data published by Luzón et al.1 for the fossils unearthed during the years 2005 and 2019–2020; b: unpublished data analysed by M.P. Espigares for the fossils of 2005 and 2013–2015) and VM3 (updated from Ref.9).Full size tableIn summary, the comparison of the faunal assemblages from both excavation quarries (Tables 1, 2) only shows some minor differences in taxa abundance for horse, megaherbivores, and carnivores other than the hyaena, as well as the presence in VM3 of some remains of two small ungulates (a roe deer-sized cervid and a chamois-sized bovid) and two small carnivores (Table 1), which are not reported by Luzón et al.1. Given their comparatively low number of specimens studied at VM4, it is reasonable to expect that the latter taxa, which are poorly represented in VM3, will also appear in VM4 during future excavations.Age mortality profilesLuzón et al.1 indicate that two megaherbivores, elephant Mammuthus meridionalis and rhino Stephanorhinus aff. hundsheimensis, show frequencies of non-adults that are close to, or even higher than, those of adults, as happens in VM3 (Table 1). However, the low MNI counts for these species in VM4 do not allow to state this: for example, elephants are represented by a juvenile and an adult, which gives a frequency of 50% of non-adults; with a sample size of only two individuals, the 95% confidence interval calculated with a binomial approach for this percentage is 1.3–98.7% (Table 1). In S. hundsheimensis, the frequency of non-adults, 80% (4/5), has also a very wide confidence interval (28.4–99.5%). In three species of medium-to-large sized ungulates, E. altidens, the ancestor of water buffalo Hemibos aff. gracilis and P. verticornis, Luzón et al.1 report similar frequencies of adults and non-adults, while they indicate that Bison sp. shows a lower frequency of juveniles (Table 1). This is true for horse and deer (58.3% and 42.9% of non-adults, respectively), but Hemibos is only recorded by one adult individual, which means that the percentage of non-adults for this species is not reliable. Luzón et al.1 calculate the percentage of 33% non-adult bison over a sample of only three individuals, of which one is a juvenile: the confidence interval for non-adults (0.8–90.6%) comprises the frequencies for horse and megacerine deer (Table 1), which rules out their suggestion of a lower frequency of juveniles for this bovid. In contrast to VM4, the abundances of non-adult horse, bison and megacerine deer are similar in VM3 (Table 1), where they are represented by higher MNI counts (which makes their percentages reliable). A similar reasoning can be applied to the claim of Luzón et al.1 that adults outnumber calves and juveniles among smaller herbivores such as the Ovibovini Soergelia minor, the Caprini Hemitraus albus and the cervid Metacervocerus rhenanus: in these species, MNI counts are very low to calculate reliably the percentage of juveniles (see their confidence intervals in Table 1). In fact, Luzón et al.1 acknowledge this limitation when they write that “the total number of individuals in each species is too low to draw reliable conclusions on the resulting patterns” and “a prime-dominant, L- or U-shaped mortality profile cannot be clearly discerned”. The situation in VM3 is quite different (Table 1): MNI counts for the two ungulates better represented in the assemblage, E. altidens and P. verticornis, allowed to reconstruct U-shaped attritional mortality profiles (Fig. 2b), which evidenced that the hypercarnivores focused on young and old individuals in the case of large prey6,7.Patterns of skeletal abundanceThe limitations and inaccuracies cited above result from the small sample analysed by Luzón et al.1 in VM4 (1578 remains of large mammals of which only 420 could be determined taxonomically and anatomically, compared to 8150 and 6331 remains in VM3, respectively: Table 1). These limitations apply also to their inferences on the skeletal profiles of ungulates. For example, they indicate that species of herbivore size class 2 (50–125 kg: M. rhenanus, H. albus, and S. minor) show biased skeletal profiles, with a predominance of teeth and elements of the forelimb over those of the hindlimb. In VM3, these ungulates also show higher frequencies of teeth than of bones, which has been interpreted as evidence of the transport by P. brevirostris of small-to-medium sized ungulates as whole carcasses to their denning site, where the giant hyaenas fractured the bones for accessing their medullary cavities and this resulted in their underrepresentation compared to teeth7,8,9,10. In the case of the major limb bones of these species in VM4, the elements of the forelimb (12.9%, 13 bones out of 101 determined remains) are twice as abundant as those of the hindlimb (6.9%, 7 bones), but these percentages do not differ statistically (χ2 = 2.028, p = 0.1544), which indicates the effects of poor sampling. In the species of herbivore size class 3 (125–500 kg), Luzón et al.1 indicate that they are well represented by all anatomical elements (e.g., craniodental elements account for ~ 30% of the remains, while both axial and appendicular elements show frequencies  > 20%). This pattern is like the one reported in VM3 for medium-to-large sized ungulates7,8,9,10. However, Luzón et al.1 indicate a bias in the disproportionate amount of posterior limb remains compared to anterior limb specimens, which in their opinion contrasts with the more balanced representation of these elements observed in VM3. Specifically, the number of forelimb bones (13.8%, 54 out of 392 bones) is about half the abundance of hindlimb bones (25.3%, 99 bones). This difference is statistically significant (χ2 = 16.460, p  More