Ancient DNA of Grape Seeds Provides Insights Into Viticulture and Cultivation Practices in Post-Roman France

Jazmín Ramos Madrigal

Section for Evolutionary Genomics Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen Grapes (Vitis vinifera)

Domestication Wild grapes Domesticated grapes Vitits vinifera ssp. sylvestris Vitits vinifera ssp. vinifera Grapes (Vitis vinifera)

Domestication Wild grapes Domesticated grapes Vitits vinifera ssp. sylvestris Vitits vinifera ssp. vinifera

Review TRENDS in Genetics Vol.22 No.9 513

Figure 2.Themorphologicaldifferencesbetweencultivatedgrapevines(subspe- cies vinifera)andwildgrapevines(subspeciessilvestris). The differences in the leaf, flower (male and female for silvestris), the bunch at maturity and the seeds are shown.

Box 2. Genetic variation in grapevine

Three processes have had a significant impact on the development of cultivated grapevines: sexual reproduction, vegetative propaga- tion and somatic mutations. New genotypes are produced by sexual reproduction, either by crossing or self-fertilization. Because individual grapevine plants have highly heterozygous genotypes any progeny produced from seed is a novel combination of parental alleles, resulting in phenotypic variation and segregation of traits in a progeny population. The selection of a particular phenotype, Figure 1.Geneticdiversityofgrapevines.(a) Wild grapevines (indicated by the particularly a berry trait, can be a long process considering arrow), such as this male vine, can still occasionally be found growing on the the juvenile period (three-to-five years) of grapevine plants and canopy of trees in the Pyrennees in France. (b) Berry colour mutants of Pinot (left to the additional time necessary for evaluation of a trait important right); Pinot noir (black), Pinot gris (grey), Pinot blanc (white). (c) Leaf mutant of for wine production. Furthermore, many generations might be Cabernet Sauvignon; the wild-type leaf is shown on the left and the mutant on necessary to recover the desired traits. Once identified, vegetative the right. propagation (asexual) by cuttings is a method of maintaining and multiplying a highly desirable genotype so that a vineyard can be At the end of the 19th century, after several millennia of planted with a single cultivar. Cuttings are also a convenient method geographical expansion, disease-causing agents from of transporting cultivars from one region to another. Cultivars grown today are maintained by vegetative propagation. Although America reached Europe (mildews, Phylloxera) resulting clonal propagation should ensure that all plants grown from in devastation and destruction of many European vine- cuttings have the same genotype, the occurrence of a somatic yards, drastically changing the diversity of this species. As mutation in one cutting and not other cuttings might eventually lead a result, a reduction of the diversity most likely occurred to plants of the same cultivar having a slightly different genotype and sometimes a different phenotype, referred to as clonal for both cultivated and wild grapes. The extent of diversity variation. This clonal variant thus appeared rapidly over one of V. vinifera found today might be a pale reflection of what cycle of vegetative reproduction (three-to-four years). This clonal existed before the introduction of Phylloxera. European variation is more complex if the mutation is maintained in only one viticulture was saved from extinction by the introduction of cell layer of the plant, resulting in genetic chimerism. If the mutation several indigenous American, non-vinifera, Vitis species is maintained in only the L1 layer (epidermis) then it will not be passed onto progeny by sexual reproduction [51]. (Table 1) that were used as rootstocks and for breeding www.sciencedirect.com Grapes (Vitis vinifera)

Domestication Wild grapes Domesticated grapes Vitits vinifera ssp. sylvestris Vitits vinifera ssp. vinifera

Review TRENDS in Genetics Vol.22 No.9 513 Review TRENDS in Genetics Vol.22 No.9 513

Figure 2.Themorphologicaldifferencesbetweencultivatedgrapevines(subspe- cies vinifera)andwildgrapevines(subspeciessilvestris). The differences in the Figure 2.Themorphologicaldifferencesbetweencultivatedgrapevines(subspe- leaf, flower (male and female for silvestris), the bunch at maturity and the seeds cies vinifera)andwildgrapevines(subspeciessilvestris). The differences in the are shown. leaf, flower (male and female for silvestris), the bunch at maturity and the seeds are shown.

Box 2. Genetic variation in grapevine Box 2. Genetic variation in grapevine Three processes have had a significant impact on the development of cultivated grapevines: sexual reproduction, vegetative propaga- Three processes have had a significant impact on the development tion and somatic mutations. New genotypes are produced by sexual of cultivated grapevines: sexual reproduction, vegetative propaga- reproduction, either by crossing or self-fertilization. Because tion and somatic mutations. New genotypes are produced by sexual individual grapevine plants have highly heterozygous genotypes reproduction, either by crossing or self-fertilization. Because any progeny produced from seed is a novel combination of parental individual grapevine plants have highly heterozygous genotypes alleles, resulting in phenotypic variation and segregation of traits in any progeny produced from seed is a novel combination of parental a progeny population. The selection of a particular phenotype, alleles, resulting in phenotypic variation and segregation of traits in Figure 1.Geneticdiversityofgrapevines.(a) Wild grapevines (indicated by the particularly a berry trait, can be a long process considering a progeny population. The selection of a particular phenotype, arrow), such as this male vine, can still occasionally be found growing on the the juvenile period (three-to-five years) of grapevine plants and canopy of trees in the Pyrennees in France. (b) Berry colour mutants of Pinot (left to Figure 1.Geneticdiversityofgrapevines.(a) Wild grapevines (indicated by the the additional time necessary for evaluation of a trait important particularly a berry trait, can be a long process considering right); Pinot noir (black), Pinot gris (grey), Pinot blanc (white). (c) Leaf mutant of arrow), such as this male vine, can still occasionally be found growing on the for wine production. Furthermore, many generations might be the juvenile period (three-to-five years) of grapevine plants and Cabernet Sauvignon; the wild-type leaf is shown on the left and the mutant on necessary to recover the desired traits. Once identified, vegetative canopy of trees in the Pyrennees in France. (b) Berry colour mutants of Pinot (left to the additional time necessary for evaluation of a trait important the right. propagation (asexual) by cuttings is a method of maintaining and right); Pinot noir (black), Pinot gris (grey), Pinot blanc (white). (c) Leaf mutant of for wine production. Furthermore, many generations might be Cabernet Sauvignon; the wild-type leaf is shown on the left and the mutant on multiplying a highly desirable genotype so that a vineyard can be necessary to recover the desired traits. Once identified, vegetative the right. planted with a single cultivar. Cuttings are also a convenient method propagation (asexual) by cuttings is a method of maintaining and At the end of the 19th century, after several millennia of of transporting cultivars from one region to another. Cultivars multiplying a highly desirable genotype so that a vineyard can be geographical expansion, disease-causing agents from grown today are maintained by vegetative propagation. Although At the end of the 19th century, after several millennia of planted with a single cultivar. Cuttings are also a convenient method America reached Europe (mildews, Phylloxera) resulting clonal propagation should ensure that all plants grown from geographical expansion, disease-causing agents from of transporting cultivars from one region to another. Cultivars in devastation and destruction of many European vine- cuttings have the same genotype, the occurrence of a somatic grown today are maintained by vegetative propagation. Although America reached Europe (mildews, Phylloxera) resulting yards, drastically changing the diversity of this species. As mutation in one cutting and not other cuttings might eventually lead clonal propagation should ensure that all plants grown from a result, a reduction of the diversity most likely occurred to plants of the same cultivar having a slightly different genotype in devastation and destruction of many European vine- cuttings have the same genotype, the occurrence of a somatic and sometimes a different phenotype, referred to as clonal for both cultivated and wild grapes. The extent of diversity yards, drastically changing the diversity of this species. As mutation in one cutting and not other cuttings might eventually lead variation. This clonal variant thus appeared rapidly over one of V. vinifera found today might be a pale reflection of what a result, a reduction of the diversity most likely occurred to plants of the same cultivar having a slightly different genotype cycle of vegetative reproduction (three-to-four years). This clonal and sometimes a different phenotype, referred to as clonal existed before the introduction of Phylloxera. European variation is more complex if the mutation is maintained in only one for both cultivated and wild grapes. The extent of diversity variation. This clonal variant thus appeared rapidly over one viticulture was saved from extinction by the introduction of cell layer of the plant, resulting in genetic chimerism. If the mutation of V. vinifera found today might be a pale reflection of what cycle of vegetative reproduction (three-to-four years). This clonal several indigenous American, non-vinifera, Vitis species is maintained in only the L1 layer (epidermis) then it will not be passed onto progeny by sexual reproduction [51]. existed before the introduction of Phylloxera. European variation is more complex if the mutation is maintained in only one (Table 1) that were used as rootstocks and for breeding viticulture was saved from extinction by the introduction of cell layer of the plant, resulting in genetic chimerism. If the mutation www.sciencedirect.com several indigenous American, non-vinifera, Vitis species is maintained in only the L1 layer (epidermis) then it will not be passed onto progeny by sexual reproduction [51]. (Table 1) that were used as rootstocks and for breeding www.sciencedirect.com Grapes (Vitis vinifera)

Domestication Wild grapes Domesticated grapes Vitits vinifera ssp. sylvestris Vitits vinifera ssp. vinifera

Wine grapes Table grapes Modern grapes are propagated vegetatively Grape varieties = identical clones

Myles et al. PNAS 2010, 108:9 Modern grapes are propagated vegetatively Grape varieties = identical clones

Reichensteiner Muscat Blanc Gamay Schönburger

Gelber Ortlieber Ortega Chardonnay

Ehrenfelser Müller-Thurgau

Rotberger

Pinot Noir Petite Bouschet Optima Siegerrebe Alicante Trollinger Wittberger Bouschet

Riesling Teinturier du Cher

Petit Manseng Perle Aramon Osteiner Kerner Verdelho Muscat Hamburg Roter Veltliner Perlriesling Sylvaner Rotgipfler Donzillinho

Traminer Taminga Muscat of Alexandria Perle de Csaba Tinta Madeira

Flora Bequignol

Grüner Veltliner Pé agudo

Semillon Fer Servadou Sauvignon Blanc

Trousseau

Chenin Blanc SCIENCES Royalty AGRICULTURAL Sibling or equivalent Cabernet Franc Parent-offspring Trincadeiro Cabernet Sauvignon Merlot Ruby Cabernet Meslier-Saint-François Carignan Myles et al. PNAS 2010, 108:9 Colombard

Fig. 3. Network of first-degree relationships among common grape cultivars. Solid vertices represent likely parent-offspring relationships. Dotted vertices represent sibling relationships or equivalent. Arrows point from parents to offspring for inferred trios (details are provided in Materials and Methods).

Relatedness among our geographically diverse sample of vinif- To examine this in more detail, we used principal components era and sylvestris provides strong support for an origin of vinifera in analysis (PCA) to visualize relationships among individual ac- the Near East: All vinifera populations are genetically closer to cessions. Fig. 4 shows the first two principal components (PCs) eastern sylvestris than to western sylvestris (Table 1; SI Appendix, calculated from sylvestris accessions only, with vinifera cultivars Table S1). After domestication, grape growing and winemaking subsequently projected onto the axes. Whereas PC2 differ- expanded westward, reaching Western Europe by 2.800 y ago (1). entiates a subset of geographically isolated sylvestris accessions (a We find that haplotype diversity in western vinifera is slightly re- subpopulation from southern Spain and two samples from duced compared with eastern vinifera (SI Appendix, Fig. S5), Georgia), PC1 reflects a clear west-east gradient in sylvestris that suggesting that the grape experienced a modest reduction in ge- is recapitulated in the vinifera that have been projected onto PC netic diversity as it was brought to Western Europe. space. The observation that relationships among vinifera mirror Based on morphological and genetic evidence, it has been patterns of relatedness in its wild progenitor supports a scenario suggested that Western European vinifera cultivars experienced in which Western European cultivars experienced introgression introgression from local Western European sylvestris. Our finding from local wild sylvestris grapes. Alternatively, the western syl- that western vinifera are more closely related to western sylvestris vestris may have experienced gene flow from western vinifera.To than are other vinifera populations is consistent with gene flow distinguish between these two scenarios, we used a recently between wild and cultivated grapes in Western Europe (Table 1). proposed 3-population test for admixture (18). We find strong

Table 1. Population pairwise Fst estimates Sylvestris west Sylvestris east Vinifera west Vinifera central Vinifera east

Sylvestris west — Sylvestris east 0.154 — Vinifera west 0.120 0.051 — Vinifera central 0.168 0.046 0.020 Vinifera east 0.202 0.035 0.051 0.031 —

Geographic regions are defined as follows: “east” includes locations east of Istanbul, Turkey; “west” includes locations west of Slovenia, including Austria; and “central” refers to locations between them (details are pre- sented in SI Appendix,Table1).

Myles et al. PNAS Early Edition | 3of6 People have been using grapes since ~8,000 y ago

McGovern et al. PNAS 2017, 114:48 Romans Rome 100 BCE

Egyptian tomb painting depicting grape cultivation, around 1400 BCE. Image the Metropolitan Museum of Art. Ancient Egypt Questions

?

Ancient grapes/cultivars Modern cultivars (Roman grapes) Questions

?

Ancient grapes/cultivars Modern cultivars (Roman grapes)

How does the ancient grapes used by Romans and other civilizations relate to modern grapes?

How were they cultivating these grapes?

Where they transporting grapes from other regions or cultivating local wild grapes? Ancient grape seeds

Horbourg-Wihr Órleans Anne Kathrine n=1 n=3

Bay of Biscay France

Colletiére, Charavines n=1

Magalas Lattes (trading center) n=3 n=1 Nimes n=9 La Lesse-Espagnac Sauvian Marseille n=5 Roumeges, Poussan n=2 Mont Ferrier, Tourbes n=3 Ancient grape seeds

Horbourg-Wihr Órleans Anne Kathrine n=1 n=3

28 archaeological seeds

Bay of Biscay France

Colletiére, Charavines n=1

Magalas Lattes (trading center) n=3 n=1 Nimes n=9 La Lesse-Espagnac Sauvian Marseille n=5 Roumeges, Poussan n=2 Mont Ferrier, Tourbes n=3 Ancient grape seeds

Horbourg-Wihr Órleans Anne Kathrine n=1 n=3

28 archaeological seeds

Iron Age (~Bay 2,480 of Biscayyo), n=1 France

Colletiére, Charavines Roman (2,200–1,700 yo), n=19 n=1

Late medieval (1,300-900 yo), n=8 Magalas Lattes (trading center) n=3 n=1 Nimes n=9 La Lesse-Espagnac Sauvian Marseille n=5 Roumeges, Poussan n=2 Mont Ferrier, Tourbes n=3 Sequencing of ancient seeds

• aDNA extraction (archaeological seeds)

• Illumina library

• Enrichment of 10,000 selected SNPs

• High-throughput sequencing

• High quality genotypes for 21 samples

Copenhagen clean lab for aDNA processing Sequencing of ancient seeds

• aDNA extraction (archaeological seeds)

• Illumina library

• Enrichment of 10,000 selected SNPs

• High-throughput sequencing

• High quality genotypes for 21 samples

Ancient grape seed Copenhagen clean lab for aDNA processing GrapeReSeq reference panel

~789 modern domesticated varieties

112 wild grapes

~10,000 polymorphic sites

Le Paslier et al. in press GrapeReSeq reference panel

~789 modern domesticated varieties

112 wild grapes 0.1 0.0 0.1 Dim 2 (1.2%) 2 Dim

~10,000 polymorphic sites 0.2

0.4 0.3 0.2 0.1 0.0 0.1 Dim 1 (4.7%) Le Paslier et al. in press Wild grapes Wine grapes: West and Central Europe Medieval grapes Iberian Peninsula and Roman grapes Northwest Africa Table grapes Iron Age grapes Bacilieri et al. BMC Plant Biology 2013, 13:25 Balkans and East Europe GrapeReSeq reference panel

WILD ~789 modern domesticated varieties

112 wild grapes 0.1 0.0 0.1 Dim 2 (1.2%) 2 Dim

~10,000 polymorphic sites 0.2

0.4 0.3 0.2 0.1 0.0 0.1 Dim 1 (4.7%) Le Paslier et al. in press Wild grapes Wine grapes: West and Central Europe Medieval grapes Iberian Peninsula and Roman grapes Northwest Africa Table grapes Iron Age grapes Bacilieri et al. BMC Plant Biology 2013, 13:25 Balkans and East Europe GrapeReSeq reference panel

TABLE WILD ~789 modern domesticated varieties

112 wild grapes 0.1 0.0 0.1 Dim 2 (1.2%) 2 Dim

~10,000 polymorphic sites 0.2

0.4 0.3 0.2 0.1 0.0 0.1 Dim 1 (4.7%) Le Paslier et al. in press Wild grapes Wine grapes: West and Central Europe Medieval grapes Iberian Peninsula and Roman grapes Northwest Africa Table grapes Iron Age grapes Bacilieri et al. BMC Plant Biology 2013, 13:25 Balkans and East Europe GrapeReSeq reference panel

TABLE WILD ~789 modern domesticated varieties

112 wild grapes 0.1 0.0 0.1 Dim 2 (1.2%) 2 Dim WINE ~10,000 polymorphic sites 0.2

0.4 0.3 0.2 0.1 0.0 0.1 Dim 1 (4.7%) Le Paslier et al. in press Wild grapes Wine grapes: West and Central Europe Medieval grapes Iberian Peninsula and Roman grapes Northwest Africa Table grapes Iron Age grapes Bacilieri et al. BMC Plant Biology 2013, 13:25 Balkans and East Europe Ancient grapes are closer to domesticated grapes 0.1 0.0 0.1 Dim 2 (1.2%) 2 Dim 0.2

0.4 0.3 0.2 0.1 0.0 0.1 Dim 1 (4.7%) Wild grapes Wine grapes: West and Central Europe Medieval grapes Iberian Peninsula and Roman grapes Northwest Africa Table grapes Iron Age grapes Balkans and East Europe Ancient grapes are closer to West/Central Europe wine grapes 0.1 0.0 0.1 0.1 0.0 0.1 Dim 2 (1.2%) 2 Dim Dim 2 (1.2%) 2 Dim 0.2 0.2

0.4 0.3 0.2 0.1 0.0 0.1 0.3 0.2 0.1 0.0 0.1 Dim 1 (4.7%) Dim 1 (4.7%) Wild grapes Wine grapes: West and Central Europe Medieval grapes Iberian Peninsula and Roman grapes Northwest Africa Table grapes Iron Age grapes Balkans and East Europe Identity By Descent patterns between pairs of samples

♂ ♀

♀ ♀ ♂ ♂

k0 = share 0 allele IBD

k1 = share 1 allele IBD

k2 = share 2 allele IBD IBD: Identity due to common ancestry ngsrelate: Korneliussen and Moltke, 2015 IBD patterns between pairs of ancient samples

♂ ♀

♀ ♀ ♂ ♂

Identical twins = same variety

k0 = share 0 allele IBD

k1 = share 1 allele IBD

k2 = share 2 allele IBD IBD: Identity due to common ancestry ngsrelate: Korneliussen and Moltke, 2015 IBD patterns between pairs of ancient samples

♂ ♀ ancient × ancient

♀ ♀ ♂ ♂

Identical twins = same variety

k0 = share 0 allele IBD

k1 = share 1 allele IBD

k2 = share 2 allele IBD IBD: Identity due to common ancestry ngsrelate: Korneliussen and Moltke, 2015 IBD patterns between pairs of ancient samples

♂ ♀ ancient × ancient

♀ ♀ ♂ ♂

19 ancient varieties = Identical twins = same variety (genetically identical clusters) k0 = share 0 allele IBD

k1 = share 1 allele IBD

k2 = share 2 allele IBD IBD: Identity due to common ancestry ngsrelate: Korneliussen and Moltke, 2015 Distribution of ancient grape cultivars

Horbourg-Wihr Órleans n=2 n=1

Bay of Biscay France Colletiére, Charavines n=1

Magalas Lattes n=1 n=3 Nimes n=9 La Lesse-Espagnac Sauvian Roumeges, Poussan n=5 n=2 Mont Ferrier, Tourbes n=3 Mediterranean Sea Distribution of ancient grape cultivars

Horbourg-Wihr Same variety cultivated in Órleans n=2 n=1 multiple regions

Bay of Biscay France Colletiére, Charavines n=1

Magalas Lattes n=1 n=3 Nimes n=9 La Lesse-Espagnac Sauvian Roumeges, Poussan n=5 n=2 Mont Ferrier, Tourbes n=3 Mediterranean Sea Distribution of ancient grape cultivars

Horbourg-Wihr Same variety cultivated in Órleans n=2 n=1 multiple regions

There is evidenceBay of Biscay of cultivation France by cuttings during medieval Colletiére, Charavines period n=1

Magalas Lattes n=1 n=3 Nimes n=9 La Lesse-Espagnac Sauvian Roumeges, Poussan n=5 n=2 Mont Ferrier, Tourbes n=3 Mediterranean Sea Identity By Descent patterns between pairs of samples

♂ ♀

ancient × modern

♀ ♀ ♂ ♂

Identical twins = same variety

k0 = share 0 allele IBD

k1 = share 1 allele IBD

k2 = share 2 allele IBD IBD: Identity due to common ancestry ngsrelate: Korneliussen and Moltke, 2015 Identity By Descent patterns between pairs of samples

♂ ♀

ancient × modern

♀ ♀ ♂ ♂

900 y old seed = Savagnin Identical twins = same variety

kinship-coefficient ~ 0.49 k0 = share 0 allele IBD IBS0 ~ 0.98% k1 = share 1 allele IBD

k2 = share 2 allele IBD IBD: Identity due to common ancestry ngsrelate: Korneliussen and Moltke, 2015 Origin of ancient grape varieties

savagnin/traminer Origin of ancient grape varieties

Madel_08_P22 (~900 yo) = Savagnin (Traminer)

on-going clonal use for over 1,000 years

savagnin/traminer Reichensteiner Muscat Blanc Gamay Schönburger

Gelber Ortlieber Ortega Chardonnay

Ehrenfelser Origin of ancient grape varieties Müller-Thurgau

Rotberger

Pinot Noir Petite Bouschet Optima Siegerrebe Alicante Trollinger Wittberger Bouschet

Riesling Teinturier du Cher

Petit Manseng Perle Aramon Osteiner Kerner Verdelho Muscat Hamburg Roter Veltliner Perlriesling Sylvaner Rotgipfler Donzillinho

Traminer Taminga Madel_08_P22 (~900 yo) = Muscat of Alexandria Perle de Csaba Tinta Madeira Savagnin (Traminer) Traminer/Savagnin Flora Bequignol

Grüner Veltliner Pé agudo

Semillon Fer Servadou Sauvignon Blanc

Trousseau

Chenin Blanc SCIENCES Royalty AGRICULTURAL Sibling or equivalent Cabernet Franc Parent-offspring Trincadeiro Cabernet Sauvignon Merlot Ruby Cabernet on-going clonal use for over Meslier-Saint-François Carignan Colombard

1,000 years Fig. 3. Network of first-degree relationships among common grape cultivars. Solid vertices represent likely parent-offspring relationships. Dotted vertices represent sibling relationships or equivalent. Arrows point from parents to offspring for inferred trios (details are provided in Materials and Methods).

Relatedness among our geographically diverse sample of vinif- To examine this in more detail, we used principal components era and sylvestris provides strong support for an origin of vinifera in analysis (PCA) to visualize relationships among individual ac- the Near East: All vinifera populations are genetically closer to cessions. Fig. 4 shows the first two principal components (PCs) eastern sylvestris than to western sylvestris (Table 1; SI Appendix, calculated from sylvestris accessions only, with vinifera cultivars Table S1). After domestication, grape growing and winemaking subsequently projected onto the axes. Whereas PC2 differ- expanded westward, reaching Western Europe by 2.800 y ago (1). entiates a subset of geographically isolated sylvestris accessions (a We find that haplotype diversity in western vinifera is slightly re- subpopulation from southern Spain and two samples from duced compared with eastern vinifera (SI Appendix, Fig. S5), Georgia), PC1 reflects a clear west-east gradient in sylvestris that suggesting that the grape experienced a modest reduction in ge- is recapitulated in the vinifera that have been projected onto PC netic diversity as it was brought to Western Europe. space. The observation that relationships among vinifera mirror Based on morphological and genetic evidence, it has been patterns of relatedness in its wild progenitor supports a scenario suggested that Western European vinifera cultivars experienced in which Western European cultivars experienced introgression introgression from local Western European sylvestris. Our finding from local wild sylvestris grapes. Alternatively, the western syl- that western vinifera are more closely related to western sylvestris vestrissavagninmay have experienced/traminer gene flow from western vinifera.To than are other vinifera populations is consistent with gene flow distinguish between these two scenarios, we used a recently between wild and cultivated grapes in Western Europe (Table 1). proposed 3-population test for admixture (18). We find strong

Table 1. Population pairwise Fst estimates Sylvestris west Sylvestris east Vinifera west Vinifera central Vinifera east

Sylvestris west — Sylvestris east 0.154 — Vinifera west 0.120 0.051 — Vinifera central 0.168 0.046 0.020 Vinifera east 0.202 0.035 0.051 0.031 —

Geographic regions are defined as follows: “east” includes locations east of Istanbul, Turkey; “west” includes locations west of Slovenia, including Austria; and “central” refers to locations between them (details are pre- sented in SI Appendix,Table1).

Myles et al. PNAS Early Edition | 3of6 Identity By Descent patterns between pairs of samples

♂ ♀

♀ ♀ ♂ ♂ all × all

Parent-offspring k0 = share 0 allele IBD

k1 = share 1 allele IBD

k2 = share 2 allele IBD IBD: Identity due to common ancestry ngsrelate: Korneliussen and Moltke, 2015 Origin of ancient grape varieties

SAU3019_P9 SAU3019_P2 MAG2013_US4015_P6/8 SAU3019_P13/14

Servanin MAG2013_US4015_P10 Cougourlude_237 MDV14_US13525_P7 MDV14_US15152_P4/7/9 Ancient grapes Dureza Collet08_P27 Tressot Parentoffspring relationship Mondeuse Piquepoul noir Chatus MDV14_US12111_P7 Full siblings or similar Syrah MDV14_US12111_P4/9 MDV14_US12111_P2 Mondeuse blanche Dongine MANSÉS DE CAPDELL HBG7054_P3/17/18 MDV14_US12111_P5 SAU3019_P8 Chouchillon CUATENDRI

Wildbacher de Hongrie Roussette basse de Seyssel Arvine Humagne blanc Roumeg_P14 Rivairenc (Aspiran Noir) Roumeg_P9

Montfer_P23 Amigne Grappenoux CARRASQUIN

Cinsaut Alfrocheiro preto Montfer_P21/25 VERÚES DE HUARTE Pinotage Velteliner vert REDONDAL Sylvaner

Prieto picudo Pinot noir PEDRO XIMÉNEZ Roi des precoces Folgasao Teinturier DORADILLA Viosinho Madel_08_P22 Persan Chardonnay Flora PARDUCA BLANQUILINA

Riesling bleu MATURANA Tinta Maturana Chenin (Collection Oberlin) VERDEJO NEGRO Manseng Petit Blanc LADO Siegerrebe (Scheu 7957) Origin of ancient grape varieties

SAU3019_P9 SAU3019_P2 MAG2013_US4015_P6/8 SAU3019_P13/14

Servanin MAG2013_US4015_P10 Cougourlude_237 MDV14_US13525_P7 MDV14_US15152_P4/7/9 Ancient grapes Dureza Collet08_P27 Tressot Parentoffspring relationship Mondeuse Piquepoul noir Chatus MDV14_US12111_P7 Full siblings or similar Syrah MDV14_US12111_P4/9 MDV14_US12111_P2 Mondeuse blanche Dongine MANSÉS DE CAPDELL HBG7054_P3/17/18 MDV14_US12111_P5 SAU3019_P8 Chouchillon CUATENDRI

Wildbacher de Hongrie Roussette basse de Seyssel Arvine Humagne blanc Roumeg_P14 Rivairenc (Aspiran Noir) Roumeg_P9

Montfer_P23 Amigne Grappenoux CARRASQUIN

Cinsaut Alfrocheiro preto Montfer_P21/25 VERÚES DE HUARTE Pinotage Velteliner vert REDONDAL Sylvaner Late Iron age (480 BCE) Prieto picudo Pinot noir PEDRO XIMÉNEZ Roi des precoces Folgasao Teinturier DORADILLA Viosinho Madel_08_P22 Persan Chardonnay Flora PARDUCA BLANQUILINA

Riesling bleu MATURANA Tinta Maturana Chenin (Collection Oberlin) VERDEJO NEGRO Manseng Petit Blanc LADO Siegerrebe (Scheu 7957) Origin of ancient grape varieties

SAU3019_P9 SAU3019_P2 MAG2013_US4015_P6/8 SAU3019_P13/14

Servanin MAG2013_US4015_P10 Cougourlude_237 MDV14_US13525_P7 MDV14_US15152_P4/7/9 Ancient grapes Dureza Collet08_P27 Tressot Parentoffspring relationship Mondeuse Piquepoul noir Chatus MDV14_US12111_P7 Full siblings or similar Syrah MDV14_US12111_P4/9 MDV14_US12111_P2 Mondeuse blanche Dongine MANSÉS DE CAPDELL HBG7054_P3/17/18 MDV14_US12111_P5 SAU3019_P8 Chouchillon CUATENDRI

Wildbacher de Hongrie Roussette basse de Seyssel Arvine Humagne blanc Roumeg_P14 Rivairenc (Aspiran Noir) Roumeg_P9

Montfer_P23 Amigne Grappenoux CARRASQUIN

Cinsaut Alfrocheiro preto Montfer_P21/25 VERÚES DE HUARTE Pinotage Velteliner vert REDONDAL Sylvaner Late Iron age (480 BCE) Prieto picudo Pinot noir PEDRO XIMÉNEZ Roi des precoces Folgasao Teinturier DORADILLA Viosinho Madel_08_P22 Roman period (200 BCE – 300 CE) Persan Chardonnay Flora PARDUCA BLANQUILINA

Riesling bleu MATURANA Tinta Maturana Chenin (Collection Oberlin) VERDEJO NEGRO Manseng Petit Blanc LADO Siegerrebe (Scheu 7957) Origin of ancient grape varieties

SAU3019_P9 SAU3019_P2 MAG2013_US4015_P6/8 SAU3019_P13/14

Servanin MAG2013_US4015_P10 Cougourlude_237 MDV14_US13525_P7 MDV14_US15152_P4/7/9 Ancient grapes Dureza Collet08_P27 Tressot Parentoffspring relationship Mondeuse Piquepoul noir Chatus MDV14_US12111_P7 Full siblings or similar Syrah MDV14_US12111_P4/9 MDV14_US12111_P2 Mondeuse blanche Dongine MANSÉS DE CAPDELL HBG7054_P3/17/18 MDV14_US12111_P5 SAU3019_P8 Chouchillon CUATENDRI

Wildbacher de Hongrie Roussette basse de Seyssel Arvine Humagne blanc Roumeg_P14 Rivairenc (Aspiran Noir) Roumeg_P9

Montfer_P23 Amigne Grappenoux CARRASQUIN

Cinsaut Alfrocheiro preto Montfer_P21/25 VERÚES DE HUARTE Pinotage Velteliner vert REDONDAL Sylvaner Late Iron age (480 BCE) Prieto picudo Pinot noir PEDRO XIMÉNEZ Roi des precoces Folgasao Teinturier DORADILLA Viosinho Madel_08_P22 Roman period (200 BCE – 300 CE) Persan Chardonnay Flora PARDUCA BLANQUILINA Medieval (790 – 1020 CE) Riesling bleu MATURANA Tinta Maturana Chenin (Collection Oberlin) VERDEJO NEGRO Manseng Petit Blanc LADO Siegerrebe (Scheu 7957) Origin of ancient grape varieties

SAU3019_P9 SAU3019_P2 MAG2013_US4015_P6/8 SAU3019_P13/14

Servanin MAG2013_US4015_P10 Cougourlude_237 MDV14_US13525_P7 MDV14_US15152_P4/7/9 Ancient grapes Dureza Collet08_P27 Tressot Parentoffspring relationship Mondeuse Piquepoul noir Chatus MDV14_US12111_P7 Full siblings or similar Syrah MDV14_US12111_P4/9 MDV14_US12111_P2 Mondeuse blanche Dongine MANSÉS DE CAPDELL HBG7054_P3/17/18 MDV14_US12111_P5 SAU3019_P8 Chouchillon CUATENDRI

Wildbacher de Hongrie Roussette basse de Seyssel Arvine Humagne blanc Roumeg_P14 Rivairenc (Aspiran Noir) Roumeg_P9

Montfer_P23 Amigne Grappenoux CARRASQUIN

Cinsaut Alfrocheiro preto Montfer_P21/25 VERÚES DE HUARTE Pinotage Velteliner vert REDONDAL Sylvaner Late Iron age (480 BCE) Prieto picudo Pinot noir PEDRO XIMÉNEZ Roi des precoces Folgasao Teinturier DORADILLA Viosinho Madel_08_P22 Roman period (200 BCE – 300 CE) Persan Chardonnay Flora PARDUCA BLANQUILINA Medieval (790 – 1020 CE) Riesling bleu MATURANA Tinta Maturana Chenin (Collection Oberlin) VERDEJO NEGRO Manseng Petit Blanc LADO Late medieval (1125 CE) Siegerrebe (Scheu 7957) Conclusions

All seeds derive from wine grapes

Transportation and propagation by cuttings

Savagnin has been propagated for over 1,000 years Conclusions

All seeds derive from wine The methodology used grapes works well for ancient seeds

Transportation and propagation by cuttings

It can be used to identify links between ancient Savagnin has been propagated and modern cultivars for over 1,000 years Acknowledgments Tom Gilbert Nathan Wales Anne Kathrine W. Runge

Roberto Bacilieri

Laurent Bouby

Thierry Lacombe Patrice This Filipe G. Vieira Shyam Gopalakrisnan J. Alfredo Samaniego Víctor Moreno Mayar … Everyone in the EvoGenomics group

Danish National High-throughput Sequencing Center Acknowledgments Tom Gilbert Nathan Wales Anne Kathrine W. Runge

Roberto Bacilieri

Laurent Bouby

Thierry Lacombe Thanks for listening! Patrice This Filipe G. Vieira Shyam Gopalakrisnan J. Alfredo Samaniego Víctor Moreno Mayar … Everyone in the EvoGenomics group

Danish National High-throughput Sequencing Center Introgression with wild grapes

American/ Iberian - wine Balkans - V. sylvestris East - table grapes West - wine grapes Madel_P22 HBG7172_P3 Asian grapes wine grapes US12111_P2US12111_P7US12111_P5US13525_P7 US4015_P10US4015_P6US4015_P8Roumeg_P14Roumeg_P9 SAU3019_P2SAU3019_P8SAU3019_P9Montfer_P23 Collet08_P27 US12111_P4,9 HBG7054_P18,17 species US14152_P4,7,9 SAU3019_P13,14 Montfer_P21,25Cougourlude_237 Cougourlude_237 Órleans Nimes Hérault Horbourg- Colletiere Wihr Charavines

HBG7172_P3 ● sylvestris ● ● Balkans sylvestris ● SAU3019_P9 ● Eastern Mediteranean Manseng Petit Blanc ● ● sylvestris ● sylvestris ● & Caucasus sylvestris ● sylvestris ● ● Iberian Peninsula sylvestris ● sylvestris ● sylvestris ● sylvestris ● ● Italian Peninsula sylvestris ● sylvestris ● sylvestris ● ● sylvestris ● Maghreb sylvestris ● HBG7172_P17 ● sylvestris ● ● Middle & Far East sylvestris ● sylvestris ● Collet08_P27 ● sylvestris ● ● New World Vineyard sylvestris ● sylvestris ● sylvestris ● ● sylvestris ● Russia & Ukrain sylvestris ● sylvestris ● Western & Central sylvestris ● ● Montfer_P21 ● Europe sylvestris ● sylvestris ● ● sylvestris ● Ancient sample MAG2013_US4015_P10 ● sylvestris ● sylvestris ● sylvestris ● American/ Balkans - Madel_08_P22 ● Iberian - wine sylvestris ● West - wine grapes sylvestris ● V. sylvestris East - table grapes sylvestris ● sylvestris ● Madel_P22 HBG7172_P3 sylvestris ● Asian grapes wine grapes US12111_P2US12111_P7US12111_P5US13525_P7 US4015_P10US4015_P6US4015_P8Roumeg_P14Roumeg_P9 SAU3019_P2SAU3019_P8SAU3019_P9Montfer_P23 Collet08_P27 sylvestris ● sylvestris ● US12111_P4,9 HBG7054_P18,17 sylvestris ● Montfer_P21,25 sylvestris ● US14152_P4,7,9 SAU3019_P13,14 Cougourlude_237 sylvestris ● sylvestris ● species SAU3019_P14 ● sylvestris ● sylvestris ● sylvestris ● sylvestris ● Horbourg- Colletiere MAG2013_US4015_P8 ● Órleans Nimes Hérault sylvestris ● sylvestris ● sylvestris ● Wihr Charavines sylvestris ● Montfer_P25 ● sylvestris ● sylvestris ● sylvestris ● sylvestris ● MAG2013_US4015_P6 ● sylvestris ● SAU3019_P13 ● SAU3019_P8 ● HBG7054_P18 ● sylvestris ● Montfer_P23 ● Roumeg_P9 ● Roumeg_P14 ●

0.10 0.15 0.20 0.25 0.30 IBS – Pairwise distances Romans helped dispersed grapes through Europe

Romans played an important role in the spread of Viticulture 20-360 AD

Many grape varieties are mentioned in ancient Roman literature, e.g. Pinot noir is mentioned in writings from the 1st century AD

Ambrosi, et al. Guide des cépages. 1997