Winter sources of ascorbic acid for Pleistocene hominins in northern Eurasia

Hominins emerging from Africa in the Pleistocene required sources of vitamins in addition to sources of energy and nutrient substance (carbohydrates, proteins, and fats). Most of their vitamin requirements could be provided by eating the flesh of herbivores but vitamin C is in low concentrations in animal muscle tissue. Lack of vitamin C causes the fatal disease of scurvy. In southern Eurasia, hominins would have been able to harvest fruits and vegetables throughout the year but as they migrated further to the north, they would encounter regions in which no plants were growing in mid-winter. Vitamin C is enriched in organ meats but their mass was probably too low for adequate provision. Storage of summer crops of fruit was possible. Hominins could however fulfil ascorbic acid requirements by drinking aqueous extracts from the needles of pines and other conifers which contain adequate amounts of vitamin C to satisfy human needs. Evidence is shown of pine needle and related consumption in Paleolithic sites.


Introduction: vitamin C needs in humans
In most omnivores, some vitamins are needed as part of the food intake since they cannot be synthesized endogenously. Most vitamins including A, E, K, and the B vitamins are present in foods. In hominins and most other primates, vitamin C (ascorbic acid) is also needed. Lack of vitamin C leads to the development of a condition called scurvy, characterized by failure to form collagen, and porosity of the periosteal layers of bone. This disease is invariably fatal if not alleviated by administration of vitamin C. The symptoms of its occurrence can be recognized in fossil bones (Brickley 2020) and have been possibly recognized in at least one Neanderthal individual (Garralda and Vandermeersch 2008). Nicholls et al. (2020) show skeletal porosity attributable to scurvy in a 6th-4th c BCE infant skeleton. However, the main skeletal symptoms of scurvy are excess porosity of bone which could be produced by many other medical conditions. For hominins living in tropical or subtropical regions (up to latitude 45°N in W. Europe), fruits and green vegetables rich in vitamin C would have been available throughout the year. However, hominins ranged up into regions where even in interglacial periods the winter months are characterized by freezing temperatures and accumulation of snow. For example, remains of Homo erectus were found at Dmanisi in Georgia (Ferring et al. 2011) at c. 1.85 million years before the present; in western Europe, the first discovery of Neanderthals was at a site in northwestern Germany where, currently, snow falls every winter. At sites north of (or higher in elevation than) the limit of freezing temperatures, in order to survive, hominins must also have been able to find access to vitamin C in winter months.
The minimal requirement of vitamin C to prevent scurvy is about 10 mg/day. This is sufficiently low that many foods which are normally not considered as significant sources could provide an adequate supply. Scurvy develops after a few weeks of deprivation of vitamin C, with changes in facial complexion, roughening of the skin, and edema in the lower extremities (Speth 2019). After about 4 to 8 weeks, the victim suffers from a wide range of debilitating conditions (Hodges et al. 1969) eventually leading to death.

Availability of vitamins in foods in winter
Hominins were omnivorous and obtained their energy, protein, and other minor nutrients including vitamins from the flesh of animals, as well as from green plants and fruits (Asouti et al. 2018;Eaton et al. 1996;Kabukcu et al. 2023). In cold winter weather, green and fruit bearing plants would not have survived and hominins would have depended almost exclusively on animal-based foods for survival. In coastal settings, some marine plants and invertebrates, fish, and mammals would have also been available, and on unfrozen rivers, fish would still have been accessible.
Roots and tubers may have been a food source when the soil was not too solidly frozen to extract these. However, most roots and tubers are not a significant source of vitamin C (Kuhnlein 1990;Locato et al. 2013); potatoes, for example, contain only 8 mg/100 g as opposed to typical vegetables which contain 30-90 mg/100 g (Vanderslice et al. 1990).
All animal tissues contain, in addition to proteins and fat, some amounts of vitamins. Hockett and Haws (2003) showed that most vitamins are plentifully available in the muscle tissues of animals as well as birds and fish, but only negligible amounts of vitamin C (average value of 0.17 mg/100 g). Internal organs such as liver, brains, and kidneys of cows contain an average of 12.7 mg/100 g. Liver of some carnivores (e.g., polar bear) contains very large amounts of vitamin A, consumption of which can lead to illness (Hockett and Haws 2003;Jeghers and Marraro 1958). Toxicity from excess ingestion of vitamin A can also occur from consumption of the liver of herbivores (Mazur et al. 2019). Nevertheless, liver and the other internal organs (kidney, thymus, pancreas, brain) could have been significant dietary sources of vitamin C in the winter for ancient hominins (Guil-Guerrero 2023; Speth 2019). However, these organs make up a relatively small fraction of the total mass of proteinaceous tissues in herbivores. In cattle, the mass of liver is 1.3 wt% of that of the whole animal (Montanholi et al. 2017). The other organs are substantially smaller, about 0.2% of the animal mass (Fitzsimons et al. 2014). It is questionable whether the organs of most of the game obtained by these winter hunters would have sufficed to provide adequate vitamin C for human needs.

Stomach contents
Although plant foods were mainly unavailable to hominins in winter conditions, herbivorous mammals were still able to survive by consuming cellulose-rich plant foods that would have been unavailable as nutrients to humans. This was possible through the presence of a microbiome in their rumens (the stomach of ruminants) that was capable of converting the poly-hexose cellulose into glucose (Callewaert and Michiels 2010). Rumen contents (chyme) of herbivores could provide an important source of carbohydrate for hominins (Buck and Stringer 2014). Since the plant foods would have contained significant amounts of vitamin C, chyme might also have been an important source of this vitamin. However, Speth (2019) indicates that at the nearly neutral pH found in rumens of herbivores, vitamin C would quickly decay and no significant amounts would be available for hominins eating this material. The stomach content of an artic reindeer (Rangifer tarandus) contained only 1.0 ± 1.0 mg/100 g of vitamin C (Fediuk 2000). Chyme could not have been a major source of vitamin C for ancient hominins. Fediuk (2000) states that "some food plants were harvested and stored for winter use among groups of Inuit in Labrador, Siberia and Greenland," citing work by Eidlitz (1969), Hoygaard (1941), andPorsild (1953). In British Columbia, First Nations peoples stored berries in wooden boxes protected with fat derived from fish for use between the seasons of berry growth (Martindale and Jurakic 2016;Schwarcz et al. 2023). Other such practices may have been used widely across northern North America.

Storage of plant foods
This technique would be an optimal strategy for dealing with the lack of sources of vitamin C during the winter in prehistoric Eurasia, but is very hard to demonstrate for prehistoric people since in pre-ceramic times, the materials would have been stored in biodegradable containers.
Drying of fruits and vegetables could conserve the food value of these materials. However, vitamin C has a strong tendency to be oxidized to dehydroascorbic acid in aerobic conditions over long periods of storage (Santos and Silva 2008). This can happen during the initial drying phase and continue during storage.

Cambium layers of trees
The softer cambium layer underlying the bark of trees is significantly enriched in sugar and has served as a nutrient for hunter-gatherers in North America (Dilbone et al. 2013) and Scandinavia (Hosfield 2020;Östlund et al. 2004). Various authors through the past decades have indicated that this material (the cambium of pine and other trees) would also be a source of vitamin C (Airaksinen et al. 1986;Hansson 1995;Urbye 1937). However, modern analyses of samples of cambium from a Scots pine (Rautio et al. 2013) and a lodgepole pine (Dilbone et al. 2013) show that there is no detectable vitamin C in these materials. In 2011, the author, in collaboration with Jessica Hider and Prof. H. Schellhorn (Dept. of Biology, McMaster University), analyzed solutions made from macerated cambium extracted from several species of pine from the arboretum of the Hamilton (Ontario) Botanical Garden. Using HPLC and infrared spectrometry, we failed to detect any vitamin C (< 0.08 mg/100 g).

Pine needles
An additional possible source of vitamin C, also emerging from trees, can be found in historical documents. An early example is seen in events surrounding the first expedition of the French explorer Jacques Cartier to Quebec in 1536 (Durzan 2009;McDowell 2013). Many of the crew of this expedition contracted scurvy, but the disease was completely cured through the administration a liquid substance called "annedda," provided to them by the local Iroquois First Nations people. This is an aqueous extract from the needle-like leaves of a local tree, probably the eastern white cedar (arborvitae, Thuja occidentalis L.). Similar stories are reported of the survival of military forces fighting in northern Europe in the early eighteenth century whose scurvy was cured by "drinking a concoction of pine needles" (Tobler 1917). Other historical accounts of a similar nature also exist (e.g., Schick 1943).
Apparently, all needles or needle-like leaves of coniferous trees contain significant amounts of vitamin C. This was shown by Raal et al. (2018) who studied three different coniferous species. They macerated a mixture of one part pine needles and 20 parts water containing 1% citric acid and analyzed the resulting solution using HPLC techniques. The resulting solutions contained between 0.01 and 1.8 mg/ ml ascorbic acid. For comparison, citrus fruit juices contain about 0.25 to 0.60 mg ascorbic acid per ml, comparable to what was obtained by Raal et al. (2018).
These authors also studied the seasonal variation in ascorbic acid content of the pine needles and found that they reached a maximum concentration in the late winter ( Fig. 1). Raal et al. (2018) suggest that the trees might secrete more ascorbic acid in the colder months to protect against frost damage to the leaves. As a consequence, the concentration of vitamin C in conifer needles would increase in the depths of winter just at the time when they were being harvested by hominins.

Processing of pine needles by hominins
One concern in suggesting this source of vitamin C is the need to extract the vitamin into an aqueous solution in some kind of container. No ceramic or other solid containers would have been available to hominins long before the invention of ceramics. One might suppose that pits could have been dug into the sediments forming the floors of ancient hominin sites but openings such as this have not been described in archaeological sites from these periods.
Another possibility would have been the use of bags made of leather or possibly of plant stems and leaves sealed with thickened sap (pitch) from trees. These were very likely available to hominins from early in their history of migration out of Africa, used for carrying babies as well as tools and raw materials for tools (appendix E in Lee 1979;Tanner and Zihlman 1976). Such bags would be ideal tools for preparation of water extracts of pine needles. The extraction could be accelerated by heating, e.g., by placing heated rocks in the containers. Durzan (2009) notes that the leaves of conifers contain other nutritionally useful substances (sugars, amino acids); they could have been initially harvested as a food during the winter seasons when other plants were scarce or absent.
Another equally effective method of extracting vitamin C from pine needles would be through chewing them, since the water-soluble vitamin would dissolve in saliva. Thus, pouches of freshly harvested needles could be carried as a readily available source of both sugar and vitamin C.

Possible archaeological evidence for use of pine needles
The needles or corresponding non-deciduous leaves of conifers could have been a major source of vitamin C for prehistoric hominins in northern Eurasia. Such trees are widespread throughout this region and can be assumed to have been a major component of the flora during the Ice Ages (Willis and Van Andel 2004). In order to test this hypothesis, it would be necessary to show some preferential use of pines or other conifers during this period, but which decreased significantly during interglacial periods. This could be done by showing biological records of the presence of conifers in association with Paleolithic archaeological sites. However, pines and other conifers would have been important sources of fuel in these sites since fire was required both to keep people warm and for the purpose of cooking food (Carmody and Wrangham 2009). Therefore, the mere presence of pine wood either as pollen or as anthracologically recognizable burned wood fragments would not constitute a definitive proof of its use as a source of vitamin C. For the most part, needles would have been completely lost during diagenesis since they are organic matter. However, because wood is burned at sites, charcoal fragments remain which might include carbonized needles. In addition, most plants contain minor amounts of silica in the form of phytoliths, and those arising from pine needles might be recognizable.
The presence of charcoal derived from pine or other coniferous wood at a Paleolithic site is a minimum requirement to demonstrate the possibility of the use of needles as a source of vitamin C. This could be done using anthracology or, in rare cases, by recognizing the morphology of fossilized pine cones or other distinctive, larger scale structures. Alternately, study of phytoliths might reveal the past presence and use of pine needles. Both types of studies would be carried out on layers of sediment found at sites, either by flotation (Asouti and Austin 2005) or by treatment of sediment to release phytoliths (Cabanes 2020).
If pine needles had been chewed, then fine particles of them could have been incorporated into dental calculus, the layer of calcified dental plaque covering all teeth (Hardy et al. 2012;Henry et al. 2011). Calculus also contains a DNA signal which represents the diet of the individual as On one tooth showed that 13% of the diet-derived DNA was from a pine tree while on the other tooth, 20% was from pine. The authors inferred from this that the Neanderthals had been eating pine nuts, but we see now that it is also possible that they had been chewing pine needles. A tooth from Spy, a Belgian Neanderthal, contained no pine DNA at all. The search for pine DNA in dental calculus would be an excellent method to probe for the possible former use of pine needles as a source of vitamin C on hominins. If only a liquid extract ("tea") were consumed, it is less likely that DNA would be preserved on the tooth surface.

Anthracological investigations
Many studies have investigated the nature of the woody materials residual in Paleolithic sites (Asouti et al. 2018;Audiard et al. 2021;Audiard et al. 2019;Théry-Parisot et al. 2018;Willis and Van Andel 2004). In most instances, pine wood (Pinus sp.) is a major component of the residues although needles specifically are not mentioned.
A Neanderthal site in Portugal, occupying three coastal cave remnants at Figueira Brava, was studied by Zilhão et al. (2020). Working in horizons dated to the early part of the last Ice Age (110 ka to ~ 85 ka BP, stages MS5b to MS5c), they found charcoal particles which could be identified to the species level. The dominant species in all the deposits was pine, Pinus pinea, although other tree species were known to be growing in the vicinity of the cave. Among the charred remains were cone bracts, pine-nut shells, and pine needles (Fig. 2).
While the authors of the paper emphasize the possible use of pine nuts as a food source, needles were apparently collected along with the nut-bearing pine cones. Nothing is known of the vitamin C content of pine nuts.
At the site of Franchthi in southern Greece, Asouti et al. (2018) found abundant remains of a conifer, juniper (Juniperus sp.) in strata spanning the interval from 15,000 to 13,000 y BP (Epipaleolithic) to 8000-6000 y BP. Figure 3 shows part of their "anthracological diagram" which presents the relative frequency of plant species as determined from anthracological analyses of charcoal fragments that had been recovered from the cave in earlier studies (Hansen 1991).
Juniper, a conifer, is the most abundant species in the oldest layers and decreases to zero at a level estimated to date from 11,700 to 11,400 y BP (all dates calibrated 14 C). This is approximately the time when climate at the site warmed at the end of the last Ice Age. The only other abundant wood species in this time period was almond (Amygdalus). Vitamin C is undetectable in almonds (Yada et al. 2011). In the post-glacial period after 11 ka BP, Asouti et al. show an increase in wood fragments from Maloidea sp., trees of the apple family, while other fruit trees are present even later at the site. These data are compatible with the notion that Fig. 2 Fragments of needles from a pine tree (Pinus pinea) from unit IH6, area F in the Figuera Brava cave. From Zilhão et al. (2020) prior to 11,000 y BP, the residents of this site were obtaining vitamin C from juniper needles but that subsequently they were able to satisfy this requirement using fruit from trees and bushes, etc.
These two examples show that, using anthracological analysis of burnt wood particles from archaeological sites, it might be possible to determine more clearly if needles of pine and other conifers were present when hominins were living at sites during the past Ice Ages and could therefore have been used as sources of vitamin C. Note however that pine needles are not likely to be conserved in normal sample preparation for anthracological analysis. Fresh pine needles are on the order of 2 to 6 cm in length and < 1 mm in diameter (Fig. 2). When burned, they would break up into fragments ranging up to a few mm in length. In normal sample analysis from archaeological sites, sieves with openings greater than 1 mm would be used (Asouti et al. 2018) with the result that most of the charred needle fragments would be lost. Unburned needles, although longer and wider, would have been lost by organic diagenesis. Future studies to search for the presence of pine needles would be obliged to use even finer sieves. Wood ash density is less than that of water (Grau et al. 2015) so fragments of charred pine needles would be recovered by flotation.

Phytolith studies
Archaeological plant fossils are generally poorly preserved due to organic decay of the tissues. Inside the cells of most plants, there are found phytoliths, opaline silica particles of distinctive shapes and sizes which can be used to identify the former presence of some types of plants (Piperno 2006).
The site of Abric Romani is in a travertine cliff known as the Cinglera del Capelló, at the village of Capellades, about 50 km west of Barcelona, Spain. A series of travertine layers are intercalated with fine clastic sediment containing Mousterian artifacts as well as remains of wood logs, etc. The sequence has been dated by U-series (Bischoff et al. 1988) to between 60 and 40 ky BP. At the Middle Paleolithic site of Abric Romani, Cabanes et al. (2007) studied phytoliths extracted from layers J in this sequence for which travertine layers have been dated ranging in age from 50.4 to 43.9 ky BP. In the overlying layer I (i), Vallverdú et al. (2005) described the presence of wood charcoal from pines and also phytoliths that could come from pine. Cabanes et al. (2007) investigated the immediately underlying layer J and found abundant phytoliths and charcoal for pine wood. The authors write that "The high number of platelets also suggests the presence of pine needles. The small reference collection that we made with Pinus sylvestris confirms the presence of this sort of phytolith in dry needles of this species." We can infer from this that pine needles were a major component of the woody material brought into the site. This site was occupied in the middle of the last Ice Age (MIS 3), when at least in the winter and probably throughout the year, fresh fruits and green plants would not have been available. Therefore, the occupants of the site (Neanderthal hominins) would not have had access to plant derived vitamin C except perhaps through storage of fruits harvested in the summer. It is possible that they could have been using pine needles as a source of vitamin C.
While phytolith studies have been carried out at other Paleolithic sites (Madella et al. 2002;Rodríguez-Cintas and Cabanes 2017;Wroth et al. 2019), no other occurrences of pine needles have been identified or sought. While this appears to be an excellent method to define the possible former use of pine needles and other conifer needles/leaves  Fig. 3 Abundance of anthracologically recognizable burned wood fragments in the sediments of Franchthi Cave, Greece. Ages are calibrated. 14 C. Horizontal scales are numerical percentages of fragments of each species from the given horizon. Adapted from Asouti et al. (2018) as sources of vitamin C, it requires comparative reference samples in order to identify phytoliths from pine needles.

Conclusions
Vitamin C is not produced endogenously in hominins and therefore must be incorporated from the diet. If it is not incorporated into the diet, then hominins would suffer from the fatal disease of scurvy. For hominins living in regions where fruits and vegetables are not available during the winter, this requires that they find alternative sources. We have outlined a few of these possible sources. In Eurasia, Speth (2019) had previously shown that internal organs in herbivorous mammals were rich sources of the vitamin. The stomach contents (chyme) of herbivorous mammals had also been suggested as a possible source but experimental data (Fediuk 2000) do not support this idea. The possibility of storage of summer-grown fruits through the winter has been noted among the Inuit and First Nations of British Columbia, but there is no evidence regarding this technique having been used by Eurasian hominins.
In this paper, we point out a potentially important source of vitamin C, the needles and needle-like leaves of coniferous trees. These are generally enriched in vitamin C, becoming more enriched in mid-winter. They would have been an especially valuable source of this substance for hominins deprived of fresh plant sources. Extraction of ascorbic acid from the needles is easily carried out by suspending them in water, possibly using bags made of leather or other materials. They can also be chewed to extract the soluble vitamin. Coniferous trees are widespread across northern Eurasia and would have been readily accessible as a source of this essential nutrient, not only during Ice Ages but also in the winters of interglacials including the Holocene. This would also have been an essential nutrient for North American First Nations people during Holocene winters.
The importance of the availability of vitamin C to prehistoric hominins has been discussed by various authors (Buck and Stringer 2014;Guil-Guerrero 2023;Speth 2019). It is a significant issue because the lack of this nutrient invariably leads to the fatal illness of scurvy. The fact that hominins did survive in northern latitudes of Eurasia is evidence that had discovered adequate sources of vitamin C.
Acknowledgements The author acknowledges the assistance of Prof. Herbert Schellhorn and Jessica Hider in preliminary attempts to measure vitamin C in extracts of pine trees. He also thanks Profs. John Speth and Paul Goldberg for comments on aspects of this paper. The author is grateful to Amanda Henry and another referee for informative comments on this manuscript.
Author contribution HPS contributed all the research and writing on this project. Some work by Prof. Herbert Schellhorn and Jessica Hider is mentioned but did not contribute in a significant way to the conclusions of the paper.

Data availability
No new data sets are presented in this paper.

Declarations
Ethical approval Not relevant.

Competing interests
The author declares no competing interests.