Wild grapevine ( Vitis sylvestris C.C.Gmel.) wines from the Southern Caucasus region

Grapevine domestication took place in the Caucasus area known as the Cradle of Viticulture, within or near the geographical area known as the Vavilov Triangle. The phytogenetic resources of Vitis sylvestris C.C.Gmel. have been previously collected and characterized, but the study on micro vinifications of wild grapevines from the Caucasus is new. In the present document, seven grape samples from female individuals of wild grapevine growing in the South Caucasus region were investigated to assess their oenological profile. Wine samples were obtained from the grapes collected from various populations of Armenia, Azerbaijan and Georgia in October 2013 and fermented by the native yeasts. Parameters determined in the wines were, among others, the concentration of ethanol (3.63 % - 10.15 %), pH (3.30 - 4.20), total acidity (1.2 - 10.7 g/L of tartaric acid), total polyphenol index (1.81 - 89.8) and colour intensity (2.59 - 20.76). This wide range of values is due to the different environmental conditions, the level of ripeness of harvested grapes and their genetic diversity. These data were compared with those obtained in micro vinifications of wild grapevines in Western Europe and wines of several international cultivars. The results of our research demonstrated, that the must of wild grape could be used to improve traditional wines giving them more colouration.

Fossils of this autochthonous vine for Eurasia appear within sediments dated from the end of the Pliocene (Sémah and Renault-Miskovsky, 2004). At present, these wild populations are disseminated in natural ecosystems from the Iberian Peninsula to Hindu Kush (Arnold et al., 2002). Some populations of this liana can be also found in the African Maghreb (Ocete et al., 2007). Their main habitats are river-bank forests, river mouths, flood plains, colluvial positions on the slopes of hills and mountains and coasts between the parallels 49º N (Rhine River, Germany) and 30º N (Ourika River, Morocco) (Iriarte et al., 2013). In such places, soils are often renewed by flooding (Arnold, 2002;Maghradze et al., 2010). Pallas (1799Pallas ( -1801, a German naturalist at the service of Empress Catherine II of Russia, reported the presence of countless wild grapevine populations in the Southern Caucasus. There were several individuals with large logs, some of them with the thickness of a ship's mast; their branches climbed on the surrounding trees. Bunches of grapes were harvested by the inhabitants of the region, sometimes, when the entire grape became raisin after winter frost, in the spring season. Eyriés (1841) indicated that the grapevine grows in the gullies and plains of Southern Caucasus as in their primitive homeland. Thus, suggesting this area to be part of a centre of domestication for grapevine, which is consistent with recent molecular data: "The close association of Georgian wild grapevines with Georgian cultivated accessions strongly supports their involvement in the initial domestication of grapevine" (Riaz et al., 2018).
The Caucasus became even more relevant for understanding Vitis sylvestris diversity after the choice of a neotype for this taxon by Ferrer-Gallego et al. (2019) who designated the specimen collected in Georgia (Alazani river basin, Jumaskure, 41°21.588′ N, 46°35.934′ E) by Ia Pipia, which is preserved in the Herbarium of the Institute of Botany, Ilia State University (TBI barcode TBI1052417!).
The South Caucasus region is situated between the Black and Caspian seas, across several countries, notably Armenia, Azerbaijan and Georgia, and is an important refuge area for numerous plant species including sweet chestnut, walnut and wild grapevine (Aradhya et al., 2017;Krebs, 2019;Ramishvili, 1988;Ramishvili, 2001). Several wild relatives of domesticated fruit species are present there in relic habitats in the Greater Caucasus mountain range (Huglin and Schneider, 1986;Vavilov, 1931). It constitutes the territory with the highest Eurasian grapevine diversity (wild and cultivated) (Haxthausen, 1856;Kolenati, 1846;Negrul, 1938;Vavilov, 1926) and it is part of the grapevine's "Fertile Triangle" or "Vavilov´s Triangle" (Figure 1) (Robinson et al., 2013). The South Caucasus region has been postulated as the cradle of viticulture and winemaking (McGovern, 2003;, McGovern et al., 2017Zohary, 2000).
In South Caucasus Region wild grapevine climbs on numerous tree and shrub species in open woodland (Ocete et al., 2018). Uses of Caucasian wild grapevine include medicine; agriculture (pollination of female cultivars) and food (male flowers flavouring wines in Azerbaijan, and unripe fruits (verjuice) in marinades and special sauces (Maghradze et al., 2015b).
The Eurasian wild grapevine is considered a threatened plant genetic resource due to the overexploitation of riverine forests, and the establishment of orchards and public works. The importation of fungal diseases from North America, such as downy and powdery mildews strongly reduced natural populations (Ocete et al., 2015). Furthermore, after Phylloxera infestation, there was a massive incorporation of North American Vitis species in Eurasian vineyards. They were used as root-stocks and in genetic improvement projects addressed at obtaining direct producer hybrids (French-American hybrids). Both kinds of plants showed a heavy invasive character as feral plants in wild habitats, highly competitive in the same habitats David Maghradze et al. where lived autochthonous Eurasian wild grapevine (Ocete et al., 2007;Terpó, 1974).
Wild grapevine reproduces mainly by seed, differing from the established vegetative propagation of cultivars (Iriarte et al., 2013;Revilla et al., 2010), and presents a higher level of genetic diversity, particularly in South Caucasian Region (Pipia et al., 2015). Genetic studies including haplotype distribution based on plastid DNA sequences show high levels of variation in wild grapevine (V. vinifera subsp. sylvestris) from the Greater Caucasus region (Pipia et al., 2012). In natural wild populations mutations affecting male vines can originate hermaphrodite individuals (Picq et al., 2014). Early farmers selected hermaphrodite grapevines, presumably due to their higher production of grapes and easier cultivation, to establish the first vineyards outside river-bank forests (Forni, 2006Scienza, 2004;This et al., 2006). However, some degree of dioecy coexisted in cultivation. The South Caucasus Region houses numerous female cultivars (97 out of 725 for the whole area, 53/414 in Georgia, 22/144 in Azerbaijan, and 22/171 in Armenia) (Negrul, 1970). In the years of intensive development of viticulture in Azerbaijan, it was carried out artificial pollination of functionally female grapevine varieties (Ag shany, Khatuni, Tavkveri, Nimrang and others) with pollen of male inflorescences of wild grapes to enhance the productivity of vineyards (Efendiyev, 1972).
Shulaveri-Shomu culture existed on the territory of present-day Georgia, Azerbaijan and Armenia. The culture is dated to the 6 th or early 5 th millennia BC and is thought to be one of the earliest known Neolithic cultures. Some of the first wine production artefacts were found in the archaeological sites of Shulaveri Gora and Gadachrili Gora in South Georgia with other evidence of agricultural activities dated c. 8000 BP (Chilashvili, 2004;McGovern, 2003;McGovern et al., 2017) (Figure 2). Archaeological excavations in the Areni-1 cave complex in south-eastern Armenia revealed installations and artefacts dating to around 6000 BP that are strongly indicative of wine production (Barnard et al., 2011).
It is necessary to remark that liquid products other than wine were obtained from grapes during the Prehistory and Antiquity. Grape must was used to improve ceramic pastes, at least, from the Bronze Age and grape vinegar was a very important food preserver used in beverages such as the "posca" consumed by Roman legions (Ocete et al., 2011c). The population of ancient Azerbaijan used wild grapes in food. Over time, local residents began to move wild grapevine closer to its homes and cultivate it. Remains of wild grapevine were found among the rocks of the ancient Gobustan and in the Khachmass region of Azerbaijan (Babayev, 1988).
The grapevine cross, or Saint Nino's cross, is a major symbol of the Christian Georgian Orthodox Church. Saint Nino of Cappadocia, who preached in Georgia in the 4 th century AD, is represented as a girl holding up a cross made with shoots of grapevine tied using her own hair (Maghradze et al., 2015a).
The Eurasian wild grape produces a rather astringent, small fruit with numerous seeds, hardly the kind of grape for making good wine. Its sugar content is relatively low and acids are high, as compared with the domesticated Eurasian cultivars, and the skin of its fruit is tough (McGovern, 2003). Therefore, it could be expected that wine obtained from these grapes would differ in certain analytic parameters from common wines.
According to the philosophy of the COST FA 1003 Action: "East-West Collaboration for Grapevine Diversity and Exploration and Mobilization of Adaptive Traits for Breeding" (2010 -2014) an expedition to collect and conserve plant genetic resources of grapevines from the South Caucasian Region was carried out in 2013.
Georgian cultivated and wild grapevine has been described (Chkhartishvili and Ocete et al., 2012) and genetically characterized (De Lorenzis et al., 2015;Ekhvaia et al., 2014;Imazio et al., 2013;Imazio et al., 2013), but not the winemaking with wild grapevine of this country, likely in Azerbaijan  and Armenia .
We believe it is important to make it clear that wild grapevines in the Caucasus are an important genetic resource for all the reasons above stated. The wild grapevines of the Caucasus have been studied and characterized genetically and morphologically but there is a lack of data of the characteristics of the wine they provide.
The wild grapes have been vinified since ancient times and are still used for this purpose both in the study area and in other places where wild grapevine grows (for example in Sardinia).
For all this, the aim of this work is: to characterize the wine that is obtained from wild grapes harvested in the several populations of Azerbaijan, Georgia and Armenia; to establish a preliminary characterization on the oenological potential of wild grapes within this geographical area; to know better the likely compositions of the wines produced before grapevine domestication.

|Sampling
Harvest of grapes took place at the second middle of October 2013 in Armenia, Azerbaijan and Georgia in the wild grapevine populations free of the presence of feral cultivars and American root-stocks ( Figure 3).
The coordinates of the different populations sampled along river-bank forests and flood plains are shown in Table 1 (Ocete et al., 2018).
All-female plants had red suborbicular berries, with diameter inferior to 1 cm. The skin of the grape is blue-black or dark red-violet (Table 1, Figure 3). The surface is covered with a thick wax layer.

Wine production and analysis
Bunches containing a considerable proportion of ripe grapes were selected among those available for harvest. High heterogeneity in the fruit set and ripening process observed in the same cluster (millerandage) is characteristic of wild grapevine populations (Trad et al., 2017). The removal and separation of ripe grape berries from the stems (destemming) were done manually. Of each sample, 50 berries preselected as ripe were weighed to calculate what percentage is transformed into must. Only ripe berries were pressed using manual machinery. Given the small number of grapes available, only one sample from each locality (Table 1)  * We follow the rules of IPGRI-UPOV-OIV (1997). ** Given that wild grapevine plants are climbing on different tree supports, the upper branches receive abundant light (Ocete et al., 2018). ***Climate data from NOAA (2020)  Slope orientation, altitude and sun exposition** North facing, 421 m a. s.l., shaded West facing, 277 m a. s. l., sun-exposed Steep slope west facing, 652 m a. s. l., sun-exposed Average precipitation (mm)*** 495 370 650 Azerbaijan Georgia tation was carried out in the laboratory in glass jars, the first four weeks, and then transferred to bottles for transport, with the own yeasts that carried the berries (spontaneous fermentation), for a maximum of 15 days, with a fixed temperature of 20 ºC and daily stirring of the must with the skins of the berries. There was no addition of potassium metabisulfite. The samples were analyzed following the methods proposed by the OIV (2015)  -Reducing sugars: Autoanalyzer FCSA Q05 with Quaatro 39 (SEAL, Norderstedt, Germany -AXFLOW, Arsta, Sweden).

Comparison
To determine relationships within the wines obtained we calculated the pairwise differences between samples in form of a dissimilarity matrix.
The crude matrix consisted of 8 variables (ethanol content (% vol), total acidity (g/l), pH, tartaric acid (g/l), L-malic acid (g/l), colour intensity, total polyphenol index and reducing sugars (g/l)) and 18 units (defined using meansd, mean, and mean+sd values for each of the 6 samples). The matrix of chemical parameters was used to compute a dissimilarity matrix using DARwin V.6.0.17 (2018-04-25) (Perrier et al., 2003;Perrier and Jacquemoud-Collet, 2006 To realistically represent individual relations, a hierarchical tree was constructed to describe the relationships between units (samples) based on the common agglomerative heuristic that proceeds by successive ascending agglomerations. For updating dissimilarity during the tree construction, the Ward criterion was adopted, which searches at each step for a local optimum to minimize the within-group or equivalently to maximize the between-group inertia. For the graphic representation, we have opted for the software Figtree version 1.4.3. (Rambaut, 2014). Analytical data of comparison samples were obtained from De Gianni (

RESULTS AND DISCUSSION
The must yield per kilogram of grapes harvested was situated between 16-17 %, due to the low proportion of pulp in the fruits. A wine volume of less than 250 ml was obtained in each of the micro fermentations, so the method of distillation with electronic densitometry was not applicable to calculate the ethanol concentration (v/v). Overall, the ethanol content measurement results were extremely low for a beverage that could be called wine (Table 2). This may be due to a low sugar content in the grapes.
Given that between the wine production in Georgia and the analysis in Spain, a period of several weeks elapsed (c. 40 days), it is likely that spontaneous malolactic fermentation occurred, which would explain why tartaric and malic acids represent only up to 50 % of total acidity.
The fact that the grapes have been fermented with local natural yeasts can influence the analytical characteristics of the experimental wines. Therefore, the differences between the wines are due not only to different origins and environmental conditions but to different yeasts as well.
Data on micro vinifications (Table 2) can be summarized as follows:

Azerbaijan
Wine 1 (Guruchai River 1). After fermentation, the percentage of ethanol recorded in this sample was 5.78%. This wine had good total acidity and showed a normal concentration of tartaric acid (Almela et al., 1996). The colour intensity was very low, similar to a rosé wine.
Wine 2 (Guruchai River 2). This wine showed a higher percentage of ethanol, 10.15 %. It is the maximum found in this region of the South Caucasus. Total acidity is adequate. The total polyphenol index is high, the colour intensity is good, 10.60 (it could be appropriate for a good quality red wine obtained from cultivars).
Wine 3 (Guruchai River 3). This wine showed a lower concentration of ethanol, 4.62 %. It has a low concentration of tartaric acid. The total polyphenol index could not be carried out due to the small volume of the sample.
Wine 4 (Guruchai River 4). This sample has a high total acidity, a low to normal amount of tartaric acid and only 5.04 % ethanol concentration. The intensity of the colour and the polyphenol index are normal according to its maturity level.

Georgia
Wine 5 (Ktsia River 1). The concentration of ethanol is 5.21 % vol. The intensity of colour and the polyphenol index present very good values. In this case, the phenolic maturity has been more in advance than the technological one as suggested by the sugars/acidity values ratio.
Wine 6 (Mtkvari River 1). The ethanol concentration is 7.2 % vol. The colour intensity and polyphenol index present decidedly acceptable values.

Armenia
Wine 7 (Debet River 1). The berries of this sample were so small, and with hardly any pulp, that barely any must volume was achieved and several determinations could not be completed. It showed the lowest percentage of alcohol of all microvinifications. Due to the few parameters determined (Table 2), it is not included in the comparison.
The ethanol percentage of normal samples varies between 4.62 % and 10.15 % (the abnormal sample 7 presented 3.63 %). The colour intensity varies between 2.59 and 20.76. It is necessary to remark that a wine is considered red, after the malolactic fermentation, when its intensity of the colour is 3.5 at least, for instance by the Regulatory Council of the Denomination of Origin Rioja (Spain) (Riojawine, 2019).
In general, ethanol levels and, sometimes, colour intensity values in Caucasus wines from wild grapevines are lower than those registered in micro vinification with wild grape samples from Sardinia (Italy) (Lovicu et al., 2009) and Andalusia, La Rioja, Castille and León and Navarre (Spain) (Ocete et al., 2011a;Ocete et al., 2011b). In the case of Spain, the maximum ethanol content was 14 %, registered in a sample harvested in Cáceres province (Extremadura) (Ayala et al., 2011) and the top colour intensity was 26.4 determined on a micro vinification from the Ega River (Álava province, Basque Country) (Meléndez et al., 2015).
Concerning the classification, colour intensity and total polyphenol index determine three main groups (Figure 4) (cf. Table 2).
Group I is characterized by the highest values of total polyphenol index, 50 -90 (mean 66) and colour intensity, 10 -21 (mean 17.2). Total polyphenols and colour intensity are lower and similar for Groups II and II (17-31 for polyphenol index and 3-5 for colour intensity). Group II presents a lower tartaric acid content (0.2 -0.9 g/L) in comparison with Group I (1.2 -3.5 g/L) and Group III (1.4 -2.7 g/L). Group II, also, presents an extremely low total acidity (0.8 -1.6 g/L) and a higher pH (5.6 -5.7). Finally, ethanol content was found not useful to recognize groups. Group I (Figure 4) include South Caucasian wild grapevine samples: two from Georgia and one from Azerbaijan. Whose compositions show similarities with some of the wild grapevine samples from Spain (Ayala et al., 2011;Ocete et al., 2011a;Ocete et al., 2011b), Korean wines (Kang et al., 2008) and Vitis rotundifolia wines (Morris and Brady, 2004;Talcott, 2004).
Guruchai River samples 1, 3, 4, which form clusters II and III, produced wines that have shown similarities with those of Vitis vinifera cultivars and most wild Eurasian grapevine samples from Spain.
It is worth to highlight that, from a molecular marker perspective, South Caucasian populations belong to chlorotypes C and D, whereas Spanish ones belong to chlorotype A (Arroyo- García et al., 2006;De Andrés, et al., 2012).
All samples present reducing sugars not transformed in ethanol, at different concentrations. The high total polyphenol index and high acidity could be assumed responsible for the disruption of the normal action of yeasts. However, these are not significantly different from the levels in wines from cultivars. Moreover, Wine 3 has 1.7 g/L of reducing sugars and low polyphenols and acids content (Table 2). Therefore, we cannot associate this level of sugars with problems in fermentation due to the total polyphenol index and high acidity.
At the time of carrying out the analyses, the laboratory did not have the instruments for the study of aroma, so these data are not available, despite their interest. It would also be interesting to produce more wine to perform a sensory analysis. However, several points make it very difficult: the extremely low number of grapes produced by wild strains in their natural habitats of South Caucasus during episodes of drought, the inter-annual irregularity in the harvest and the difficult access to some of the populations.
The use of wild grapevine has been frequented for producing wine throughout history. Currently, the Eurasian wild grapevine is in the list of Endangered Plant Species of Georgia since 1982 (Chemonics, 2000). In Azerbaijan, people have always produced red and white wines. An interesting wine is the so-called "gora sharab", traditional of the region Guba-Khachmaz, Shaki-Zaqatala, Garabagh. For making this wine people use cultivated and wild grapes gathered in forest and riversides (Salimov and Musayev 2012).
In the Azerbaijan Research Institute of Viticulture and Wine-making buds and pollen of wild grapes are used as a flavour for preparing flavoured dessert wine like «nectar». This wine is characterized by a particular taste and unique flavour (Amanov, 2001;Amanov, 2005).
From unripe berries of wild grapevine, local habitants prepare healing juice, called «gora suyu» or «gara suyu». This juice is successfully used in the treatment of diabetes. Grapes contain numerous polyphenols, including the stilbene resveratrol, the flavanol quercetin, catechins, and anthocyanins that have shown potential for reducing hyperglycaemia, improving β-cell function, and protecting against β-cell loss.
Therefore, with a low mean glycaemic index and glycaemic load, grapes or grape products may provide health benefits to type 2 diabetics (Rasines-Perea and Teissedre, 2017;Zunino, 2009).
An infusion of fresh leaves of the wild vine is widely used for the treatment of rheumatism (as a bath), as well as for improving eyesight (Damirov and Shukurov, 1985).
In Sardinia, a traditional wine is known as «vinu de marxani» or "vino de volpe" is made with the wild grape. Until the middle of the 20th century, shepherds of the mountainous area of Sulcis made their own wine with these wild grapevines, which they called vino de caoprai (Lovicu et al., 2009;Lovicu, 2013). Therefore, it has been traditional to make wine completely with wild grapes.
The potential contribution of wild grapes (wine 1, wine 2, wine 5) to lower the pH of the must by increasing the acid content, facilitating good wine conservation, is extremely limited by the considerable drop in alcohol that this addition can produce. Red wild grape wines (wine 2, wine 5 and wine 6), despite their high polyphenolic content that could help improve the preservation of base wines and add a higher concentration of anthocyanins, are of little use as improvers of wines made with cultivated varieties, for the same reason.

CONCLUSIONS
The wild grapevine populations cited in the present paper could be useful to make deeper oenological studies, such as the analysis of anthocyanin fingerprints. Wild grapevines with fruits rich in colour could be used to intensify the colour in red wines, as long as their low ethanol content can be resolved.
These wines (wines 2, 5 and 6) for their content in polyphenols could be used for improving the conservation of organic wines.
It is desirable that in these countries the traditional wine of the wild grapevine continues to be made and eventually added to the conventional local wines, which would confer certain original characteristics to the wines from the domesticated cultivars of the Eurasian grapevine.