bioRxiv preprint doi: https://doi.org/10.1101/2021.03.28.437401; this version posted March 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Title : An additional area of apple domestication with crop-wild gene flow, and also 2 cultivation of the local wild apple, in the Caucasus 3 4 Short title: An additional area of apple domestication in the Caucasus 5 6 Bina Hamid1, Yousefzadeh Hamed2, Venon Anthony3, Remoué Carine3, Rousselet Agnès3, 7 Falque Matthieu3, Shadab Faramarzi4, Giraud Tatiana5, Hossainpour Batol6, Abdollahi Hamid7, 8 Gabrielyan Ivan8, Nersesyan Anush9, Cornille Amandine3 9 10 1 Department of Forestry, Tarbiat Modares University, Noor, Iran 11 2 Department of Environmental science, Biodiversity Branch, Natural resources faculty, Tarbiat 12 Modares University, Noor, Iran 13 3 Université Paris Saclay, INRAE, CNRS, AgroParisTech, GQE - Le Moulon, 91190 Gif-sur- 14 Yvette, France 15 4 Department of Plant Production and Genetics, Faculty of Agriculture, Razi University, 16 Kermanshah, Iran 17 5 Ecologie Systematique Evolution, Universite Paris-Saclay, CNRS, AgroParisTech, Orsay, 18 France 19 6 Iranian Research Organization for Science and Technology, Tehran, Iran 20 7Temperate Fruits Research Center, Horticultural Sciences Research Institute, Agricultural 21 Research, Education and Extension Organization (AREEO), Karaj, Iran 22 8 Department of Conservation of Genetic Resources of Armenian Flora, A. Takhtajyan Institute 23 of Botany, Armenian National Academy of Sciences, Acharyan Str.1, 0040 Yerevan, Armenia 24 9 Department of Palaeobotany, A. Takhtajyan Institute of Botany, Armenian National Academy 25 of Sciences, Acharyan Str.1, 0040 Yerevan, Armenia 26 27 Corresponding authors: Amandine Cornille, [email protected], 28 [email protected] 29 30 Key words: apple, Caucasus, crop-wild gene flow, domestication, fruit tree, climate, 31 introgression. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.28.437401; this version posted March 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.28.437401; this version posted March 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Summary 2 Anthropogenic and natural divergence processes in crop-wild fruit tree complexes are less 3 studied than in annual crops, especially in the Caucasus, a pivotal region for plant domestication. 4 We investigated anthropogenic and natural divergence processes in apples in the Caucasus from 5 using 26 microsatellite markers amplified on 508 wild and cultivated samples. We found two 6 specific Iranian cultivated populations that were differentiated from Malus domestica, the 7 standard cultivated apple worldwide, suggesting a specific local domestication process in Iran. 8 Some Iranian apple cultivars belonged to the Caucasian wild apple gene pools, indicating that 9 farmers also use local wild apple for cultivation. Substantial wild-crop and crop-crop gene flow 10 were also inferred. We identified seven genetically differentiated populations of wild apples 11 (Malus orientalis) in the Caucasus. Niche modeling indicated that these populations likely 12 resulted from range changes linked to the last glaciation. This study pinpoints Iran as a key region 13 in the evolution and domestication of apple and further demonstrates the role of gene flow during 14 fruit tree domestication as well as the impact of climate change on the natural divergence of a 15 wild fruit tree. The results also provide a practical base for apple conservation and breeding 16 programs in the Caucasus. 17 18 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.28.437401; this version posted March 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Introduction 2 Crop-wild complexes provide good models for understanding how anthropogenic and natural 3 factors shape population divergence in the presence of gene flow. Indeed, crops are the result of a 4 recent anthropogenic divergence process, i.e. domestication, which began around 10,000 years 5 ago, and which has often been followed by subsequent crop-wild gene flow (Cornille et al. 2012, 6 2014; Diez et al. 2015; Gaut et al. 2015; Brandenburg et al. 2017; Besnard et al. 2018; Chen et 7 al. 2019; Flowers et al. 2019). On the other hand, wild species allow the study of natural 8 divergence over a longer timescale. Indeed, wild species have often undergone shifts in 9 distribution following past climate changes associated with glacial periods, and range contraction 10 has often led to population differentiation and divergence (Hewitt 1990, 1996; Petit et al. 2004; 11 Schmitt 2007; Excoffier et al. 2009; Jezkova et al. 2011). Understanding the evolutionary 12 processes shaping the natural and anthropogenic divergence of crop-wild complexes is not just an 13 academic exercise: it will also help assess the devenir of wild resources. Because of the socio- 14 economic importance of crop plants, protecting the wild relatives of crops, beyond the need for 15 preserving biodiversity (Bacles & Jump 2011), will allow us to manage the genetic resources for 16 future breeding strategies in the face of global changes (e.g. climate change, emerging diseases) 17 (Castañeda-Álvarez et al. 2016; Zhang et al. 2017; Bailey-Serres et al. 2019). 18 Fruit trees present several historical and biological features that make them fascinating 19 models for investigating anthropogenic and natural divergence with gene flow. The origin of fruit 20 trees is linked to the emergence of some of the most ancient civilizations (Zohary & Spiegel-Roy 21 1975; Vavilov 1992). Several wild-crop fruit tree complexes are now spread across the world and 22 can be good model systems to study anthropogenic and natural divergence in trees. Fruit trees are 23 also characterized by high levels of gene flow during divergence, which is expected considering 24 the typical life history traits of trees (Petit & Hampe 2006; Oddou-Muratorio & Klein 2008; 25 Cornille et al. 2013a, b). Population genetic studies of natural divergence processes associated 26 with the last glacial maximum in Europe, North America and Asia in wind-dispersed trees (e.g. 27 Abies, Pinus, Fraxinus, Quercus, Betula (Lascoux et al. 2004; Petit et al. 2004)) and animal- 28 dispersed trees (Cornille et al. 2013b) demonstrated high levels of gene flow between populations 29 as well as high dispersal capabilities. These studies also revealed the location of single (Tian et 30 al. 2009; Bai & Spitkovsky 2010; Zeng et al. 2011) or multiple (Tian et al. 2009; Qiu et al. 2011) 31 glacial refugia where most temperate tree species persisted during the last glacial maximum, and bioRxiv preprint doi: https://doi.org/10.1101/2021.03.28.437401; this version posted March 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 from which populations recolonized higher or lower latitudes during the Holocene post-glacial 2 expansion (Giesecke et al. 2017). Population genetic and genomic studies also revealed the 3 prominent role of gene flow during the anthropogenic divergence of fruit trees. Domestication of 4 several emblematic fruit tree crops, such as grape and apple, occurred with substantial crop-crop 5 and wild-crop gene flow, and without a bottleneck (Arroyo-García et al. 2006; Myles et al. 2011; 6 Cornille et al. 2012; Meyer et al. 2012a; Diez et al. 2015; Decroocq et al. 2016; Duan et al. 2017; 7 Liu et al. 2019). These studies thus revealed that domestication of fruit trees involved a specific 8 process that is different from that of annuals, and which can be explained by the long lifespan, 9 long juvenile phase and self-incompatibility system of trees (Gaut et al. 2015; Fuller 2018). 10 However, studies of natural and anthropogenic divergence processes in crop-wild fruit tree 11 complexes are still scarce in the geographic regions that were pivotal in the divergence history of 12 these complexes. 13 The Caucasus ecoregion harbors a remarkable concentration of economically important 14 plants and their wild relatives, in particular wheat, rye, barley and also fruit trees including 15 walnut, apricot and apple (Gabrielian & Zohary 2004; Yousefzadeh et al. 2012; Asanidze et al. 16 2014a). This region covers Georgia, Armenia, Azerbaijan, the North Caucasian part of the 17 Russian Federation, the northern-eastern part of Turkey and the Hyrcanian Mixed Forests region 18 in northwestern Iran (Nakhutsrishvili et al. 2015; Zazanashvili et al. 2020). Two refugia for 19 temperate plants are recognized in this region (Yousefzadeh et al. 2012; Bina et al. 2016): the 20 Colchis refugium in the catchment basin of the Black Sea, and the Hyrcanian refugium at the 21 southern edge of the Caucasus. Glacial refugia are known to harbor higher levels of species and 22 genetic diversity (Hewitt 2004), and this is the case for the Colchis and Hyrcanian Mixed Forests 23 refugia. The geography of the Caucasus, with two parallel mountain chains separated by 24 contrasted climatic zones makes this region a good model for investigating natural divergence 25 processes associated with the last glacial maximum. Furthermore, it has been suggested that Iran, 26 with its close proximity to Central Asia - the center of origin of emblematic fruit trees - and its 27 historic position on the Silk Trade Routes, is a possible secondary domestication center for apple, 28 grape and apricot (Decroocq et al. 2016; Liang et al. 2019; Liu et al. 2019).