The apple is the most common and culturally important fruit crop of temperate areas. The elucidation of its origin and domestication history is therefore of great interest. The wild Central Asian species Malus sieversii has previously been identified as the main contributor to the genome of the cultivated apple (Malus domestica), on the basis of morphological, molecular, and historical evidence. The possible contribution of other wild species present along the Silk Route running from Asia to Western Europe remains a matter of debate, particularly with respect to the contribution of the European wild apple. We used microsatellite markers and an unprecedented large sampling of five Malus species throughout Eurasia (839 accessions from China to Spain) to show that multiple species have contributed to the genetic makeup of domesticated apples. The wild European crabapple M. sylvestris, in particular, was a major secondary contributor. Bidirectional gene flow between the domesticated apple and the European crabapple resulted in the current M. domestica being genetically more closely related to this species than to its Central Asian progenitor, M. sieversii. We found no evidence of a domestication bottleneck or clonal population structure in apples, despite the use of vegetative propagation by grafting. We show that the evolution of domesticated apples occurred over a long time period and involved more than one wild species. Our results support the view that self-incompatibility, a long lifespan, and cultural practices such as selection from open-pollinated seeds have facilitated introgression from wild relatives and the maintenance of genetic variation during domestication. This combination of processes may account for the diversification of several long-lived perennial crops, yielding domestication patterns different from those observed for annual species.

The apple, one of the most ubiquitous and culturally important temperate fruit crops, provides us with a unique opportunity to study the process of domestication in trees. The number and identity of the progenitors of the domesticated apple and the erosion of genetic diversity associated with the domestication process remain debated. The Central Asian wild apple has been identified as the main progenitor, but other closely related species along the Silk Route running from Asia to Western Europe may have contributed to the genome of the domesticated crop. Using rapidly evolving genetic markers to make inferences about the recent evolutionary history of the domesticated apple, we found that the European crabapple has made an unexpectedly large contribution to the genome of the domesticated apple. Bidirectional gene flow between the domesticated apple and the European crabapple resulted in the domesticated apple being currently more similar genetically to this secondary genepool than to the ancestral progenitor, the Central Asian wild apple. We found that domesticated apples have evolved over long time scales, with contributions from at least two wild species in different geographic areas, with no significant erosion of genetic diversity. This process of domestication and diversification may be common to other fruit trees and contrasts with the models documented for annual crops.

Introduction

Domestication is a process of increasing codependence between plants and animals on the one hand, and human societies on the other [1], [2]. The key questions relating to the evolutionary processes underlying domestication concern the identity and geographic origin of the wild progenitors of domesticated species [3], the nature of the genetic changes underlying domestication [4], [5], the tempo and mode of domestication (e.g., rapid transition versus protracted domestication) [6] and the consequences of domestication for the genetic diversity of the domesticated species [7], [8], [9], [10]. An understanding of the domestication process provides insight into the general mechanisms of adaptation and the history of human civilization, but can also guide modern breeding programs aiming to improve crops or livestock species further [11], [12].

Plant domestication has mostly been studied in seed-propagated annual crops, in which strong domestication bottlenecks have often been inferred, especially in selfing annuals, such as foxtail millet, wheat and barley [11], [13], [14], [15], [16], [17]. Genetic data have suggested that domestication or the spread of domesticated traits has been fairly rapid in some annual species (e.g, maize or sunflower), with limited numbers of populations or species contributing to current diversity [10], [18], [19], [20], [21], [22]. In contrast, a combination of genetics and archaeology suggested a protracted model of domestication for other annual crops, and in particular for the origin of wheat or barley in the Fertile Crescent [11], [23]. However, the genetic consequences of domestication have been little investigated in long-lived perennials, such as fruit trees [24], [25], [26]. Trees have several biological features that make them fascinating and original models for investigating domestication: they are outcrossers with a long lifespan and a long juvenile phase, and tree populations are often large and connected by high levels of gene flow [27], [28].

Differences in life-history traits probably result in marked differences in the mode and speed of evolution between trees and seed-propagated selfing annuals [27], [28], [29]. For example, outcrossing may tend to make domestication more difficult, in part because the probability of fixing selected alleles is lower than in selfing crops [6], [13]. The combination of self-incompatibility and a long juvenile phase also results in highly variable progenies, making breeding a slow and expensive process, and rendering crop improvement difficult. The development of vegetative propagation based on cuttings or grafting has been a key element in the domestication of long-lived perennials, allowing the maintenance and spread of superior individuals despite self-incompatibility [30]. However, the use of such techniques has further decreased the number of sexual cycles in tree crops since the initial domestication event, adding to the effect of long juvenile phases in limiting the genetic divergence between cultivated trees and their wild progenitors [30], [31], [32], [33]. Thus, domestication can generally be considered more recent, at least in terms of the number of generations, in fruit tree crops than in seed-propagated selfing annuals.

Given the slow process of selection and the limited number of generations in which humans could exert selection, the protracted nature of the domestication process in trees has probably resulted in limited bottlenecks [25], [31] and in a weaker domestication syndrome [34] than in seed-propagated annuals. Nevertheless, many cultivated fruit trees clearly display morphological, phenotypic and physiological features typical of a domestication syndrome, such as large fruits and high sugar or oil content [32], [35]. Many aspects of fruit tree domestication have been little studied [25]. Consequently, most of the hypotheses concerning the consequences of particular features of trees for their domestication/diversification remain to be tested. Recent studies on grapevines, almond and olive trees have provided illuminating insights, such as the importance of outcrossing and interspecific hybridization [36], [37], [38], but additional studies of other species are required to draw more general conclusions.

Here, we investigated the origins of the domesticated apple Malus domestica Borkh., one of the most emblematic and widespread fruit crops in temperate regions [35]. A form of apple corresponding to extant domestic apples appeared in the Near East around 4,000 years ago [39], at a time corresponding to the first recorded uses of grafting. The domesticated apple was then introduced into Europe and North Africa by the Greeks and Romans and subsequently spread worldwide [35]. While the ancestral progenitor has been clearly identified as being M. sieversii, the identity and relative contributions of other wild species present along the Silk route that have contributed to the genetic makeup of apple cultivars remain largely unknown. This is surprising given the potential importance of this knowledge for plant breeding and for our understanding of the process of domestication in fruit trees.

The wild Central Asian species M. sieversii (Ldb.) M. Roem has been identified as the main contributor to the M. domestica genepool based on similarities in fruit and tree morphology, and genetic data [40], [41], [42], [43]. The Tian Shan forests were identified as the geographic area in which the apple was first domesticated, on the basis of the considerable intraspecific morphological variability of wild apple populations in this region [44], [45]. Nucleotide variation for 23 DNA fragments even suggested that M. sieversii and M. domestica belonged to a single genepool (which would be called M. pumila Mill.), with phylogenetic networks showing an intermingling of individuals from the two taxa [43]. Some authors have also suggested possible contributions of additional wild species present along the Silk Route: M. baccata (L.) Borkh, which is native to Siberia, M. orientalis Uglitz., a Caucasian species present along western sections of the ancient trade routes, and M. sylvestris Mill. (European crabapple), a species native to Europe [46], [47], [48], [49]. These hypotheses were based on the history of human migration and trade, the lack of phylogenetic resolution between M. domestica and these four wild species [41], [42], genetic evidence of hybridization at a local scale between domesticated apple and M. sylvestris [40], and the recent finding of sequence haplotype sharing between M. sylvestris and M. domestica [50]. However, such secondary contributions remain a matter of debate, mostly due to the difficulty of distinguishing introgression from incomplete lineage sorting [43], [50], [51]. The three wild species occurring along the Silk Route all bear small, astringent, tart fruits. None of these species has the fruit quality of M. sieversii, but they may have contributed other valuable horticultural traits, such as later flowering, resistance to pests and diseases, capacity for longer storage or climate adaptation. The organoleptic properties of the fruits of these wild species may also have been selected during domestication, for the preparation of apple-based beverages, such as ciders [46], [52]. Cider apples are indeed smaller, bitter and more astringent than dessert apples and bear some similarity to M. sylvestris apples. There is also evidence to suggest that Neolithic and Bronze Age Europeans were already making use of M. sylvestris [39].

In this study, we used a comprehensive set of apple accessions sampled across Eurasia (839 accessions from China to Spain; Figure 1 and Figure S1; Table S1) and 26 microsatellite markers distributed evenly across the genome to investigate the following questions: 1) Is there evidence for population subdivision within and between the five taxa M. domestica, M. baccata, M. orientalis, M. sieversii and M. sylvestris? 2) How large is the contribution of wild species other than the main progenitor, M. sieversii, to the genome of M. domestica? 3) Does M. domestica have a genetic structure associated with its different possible uses (i.e., differences between cider and dessert apples)? 4) What consequences have domestication, subsequent crop improvement and vegetative propagation by grafting had for genetic variation in cultivated apples? Most of our samples of M. domestica corresponded to cultivars from Western Europe (Figure 1 and Figure S1), as almost all the cultivars available in modern collections (including American, Australasian cultivars) are of European ancestry and this region is therefore the most relevant area for the detection of possible secondary introgression from the European crabapple.