Introduction
Fruit trees played a major role in the development of Mediterranean civilizations during the last millennia. Their evolutionary histories represent examples of plant evolution under three important drivers, geological, climatic and human, which have been defined as the Mediterranean triptych (Thompson 2020). Several tree species survived in refugia during the Pleistocene climatic changes, and suffered repeated range expansions and contractions, which shaped their genetic diversity and structure. Human activities constitute the most recent of the three Mediterranean triptych drivers, but they had great consequences on shaping global biodiversity (Boivin et al., 2016). Humans have been modifying Mediterranean ecosystems for thousands of years, profoundly altering the forests (Quézel and Médail, 2003). As a result, it is difficult to document the evolutionary history of fruit trees, which may have cultivated, feral or wild populations in the same region. However, recent phylogeographic studies have revealed that the imprints of ancestral populations preceding agriculture are still present in the genetic diversity structure of Mediterranean cultivated tree species (Gros-Balthazard et al., 2017; Besnard et al., 2018). Identifying the oldest components of the genetic legacy is essential to conserve genetic resources in the Mediterranean region, and it will also improve our understanding of the domestication process. As a general pattern, domestication in the Mediterranean started in the East and was followed by human-mediated westward dispersal of crops across the basin (Zeder, 2008; Zohary and Hopf, 2012). However, recent studies suggest that domestication in the Mediterranean was a protracted process involving local resources from several diversity centers during which genetic admixture, within or between species, played a crucial role (Fuller et al., 2011; Purugganan, 2019; Thompson, 2020).
The carob, a common tree in traditional Mediterranean orchards, has been traditionally valued, and still is, for its ability to produce food and fodder on marginal lands, especially during unfavourable years. Domestication of the carob tree is known to have aimed at increasing the pulp in the fruit (Zohary, 2002), but new uses have recently emerged, such as ecological restoration of degraded land, production of bioethanol or the use of a galactomannan obtained from the seeds as food stabilizer. A recent review outlined the potential of carob for developing health-beneficial food products (Brassesco et al., 2021). Because the propagation of carob cultivars is done by grafting, it is assumed that the origin of its cultivation is linked to the development of grafting methods c. 3,000 years ago (Zohary, 2002; Meyer et al., 2012). As for several crops, the Near East and the Eastern Mediterranean were initially proposed as the center of domestication for the carob tree (Zohary, 2002; Ramon-Laca and Mabberley, 2004). However, a recent multidisciplinary phylogeographic study based on wild and cultivated carob trees revealed the existence of four main genetic groups across the Mediterranean with a strong west-east structure (Viruel et al., 2020), which has also been documented for other Mediterranean plants (Désamoré et al. 2011; Nieto Feliner 2014; Chen et al. 2014; Migliore et al. 2018; Garcia-Verdugo et al. 2021). Using coalescent simulations based on microsatellite data, the estimated divergence times between these genetic groups pre-date the Neolithic origin of agriculture (Viruel et al., 2020). This contrast with two genetic studies focused on carob cultivars (Caruso et al. 2008; La Malfa et al. 2014), which reported a lack of geographical structure and strong genetic admixture. However, a recent study of the world’s largest carob cultivar germplasm collection based on microsatellite and plastid markers (Di Guardo et al., 2019) detected a genetic cluster in South Spain sharing ancestry with genotypes from Morocco and separated from cultivars of West Spain, Italy and the eastern Mediterranean. As postulated in Viruel et al. (2020), integrating the results from these studies on wild and cultivated carob trees supports a regional use and domestication of local carob in several parts of the Mediterranean. Di Guardo et al. (2019) also emphasized that mixed ancestry found in current cultivars was the result of the diffusion of selected productive, female or hermaphrodite genotypes via grafting. Cultivated and wild carob trees are often spatially close to each other and seeds are efficiently dispersed by cattle. Therefore, recurrent cultivated-wild genetic admixture could have determined diffuse domestication effects with potential impact throughout the whole carob geographical range including the wild trees. Nevertheless, the effects of domestication were not homogeneous across the Mediterranean basin. In Andalusia and Morocco, carob orchards were less intensive than in the rest of the distribution range (Di Guardo et al., 2019). In these two areas, the carob pods from cultivars have a low pulp content, similar to those of the wild type. By contrast, in the eastern and central Mediterranean areas, especially in Sicily, Crete and Cyprus, carob cultivation is more intensive and supports several traditional uses, suggesting an ancient history of selection and domestication. Recently, in agreement to this pattern, Baumel et al. (2018) showed, through a study of floristic diversity on a Mediterranean scale, that carob habitats were more heterogeneous in the west than in the east of the basin. To confirm the historical scenarios that have shaped the current diversity of both wild and cultivated carob, genomic-based approaches are required.
In this study, we aim to clarify carob evolutionary history and to assess the contribution of agriculture to the genome-wide diversity of carob. Our appraisal is based on two facts: carob cultivation and selection efforts were not homogeneous throughout the Mediterranean and carob populations are currently observed following a gradient of ecological conditions from natural habitats to cultivated lands. We hypothesize a stronger impact of domestication on genetic diversity in central and eastern Mediterranean populations. We also postulated a pattern of gene flow from east to west due to the spread by Greeks and Arabs of already domesticated carob trees (Ramon-Laca and Mabberley, 2004).
Our first objective was to identify geographical boundaries among carob population units with respect to genetic diversity structure. Building from the SSR polymorphism and SNP data obtained by Viruel et al. (2018, 2020), we aimed at delimiting geographically homogenous genetic groups of carob populations (hereafter called CEUs for Carob Evolutionary Units). Then, using these CEUs, we investigate carob genome-wide diversity and differentiation with data developed for the present study from a reduced-representation genomic approach, restriction associated DNA sequencing (RADseq), which was successful to decipher evolutionary history in several tree species (Hodel et al., 2017; Borrell et al. 2018; Warschefsky and von Wettberg, 2019; Hipp et al., 2020). Our second objective was to assess the potential impact of agriculture on the genome-wide diversity of the carob tree. We performed a comparative diversity analysis and searches for candidate loci under opposite statuses (natural versus cultivated) of carob populations. Our third objective was to reconstruct the history of CEUs including population splits and gene flow.