Molecular Ecology. 2022;31:4095–4111. |4095wileyonlinelibrary.com/journal/mecReceived: 21 September 2021 | Revised: 13 May 2022 | Accepted: 8 June 2022DOI: 10.1111/mec.16563 ORIGINAL ARTICLEGenome-wide footprints in the carob tree (Ceratonia siliqua) unveil a new domestication pattern of a fruit tree in the MediterraneanAlex Baumel1| Gonzalo Nieto Feliner2| Frédéric Médail1| Stefano La Malfa3|Mario Di Guardo3| Magda Bou Dagher Kharrat4| Fatma Lakhal-Mirleau1|Valentine Frelon1| Lahcen Ouahmane5| Katia Diadema6| Hervé Sanguin7, 8|Juan Viruel91Aix Marseille University, Avignon University, CNRS, IRD, IMBE, Institut Méditerranéen de Biodiversité et d'Ecologie marine et continentale, Faculté des Sciences et Techniques St-Jérôme, Marseille, France2Real Jardín Botánico (RJB), CSIC, Madrid, Spain3Department of Agriculture, Food and Environment (Di3A), University of Catania, Catania, Italy4Laboratoire Biodiversité et Génomique Fonctionnelle, Faculté des Sciences, Université Saint-Joseph, Campus Sciences et Technologies, Beirut, Lebanon5Faculté des Sciences Semlalia, Laboratoire de Biotechnologies Microbiennes Agrosciences et Environnement, Université Cadi Ayyad Marrakech, Marrakech, Morocco6Conservatoire Botanique National Méditerranéen de Porquerolles (CBNMed), Hyères, France7CIRAD, UMR PHIM, Montpellier, France8PHIM, Univ Montpellier, CIRAD, INRAE, Institut Agro, IRD, Montpellier, France9Royal Botanic Gardens, Kew, Surrey, UKThis is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.© 2022 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.CorrespondenceAlex Baumel, Aix Marseille University, Avignon University, CNRS, IRD, IMBE, Institut Méditerranéen de Biodiversité et d'Ecologie marine et continentale, Faculté des Sciences et Techniques St-Jérôme, Marseille, France.Email: alex.baumel@imbe.frFunding informationAgence Nationale de la Recherche, Grant/Award Number: ANR-14- CE02- 0016; H2020 Marie Skłodowska-Curie Actions, Grant/Award Number: 704464 - YAMNOMICS - MSCA-IF- EF- STHandling Editor: Ana CaicedoAbstractIntense research efforts over the last two decades have renewed our understand-ing of plant phylogeography and domestication in the Mediterranean basin. Here we aim to investigate the evolutionary history and the origin of domestication of the carob tree (Ceratonia siliqua), which has been cultivated for millennia for food and fodder. We used >1000 microsatellite genotypes to delimit seven carob evolutionary units (CEUs). We investigated genome-wide diversity and evolutionary patterns of the CEUs with 3557 single nucleotide polymorphisms generated by restriction-site asso-ciated DNA sequencing (RADseq). To address the complex wild vs. cultivated status of sampled trees, we classified 56 sampled populations across the Mediterranean basin as wild, seminatural or cultivated. Nuclear and cytoplasmic loci were identified from RADseq data and separated for analyses. Phylogenetic analyses of these genomic- wide data allowed us to resolve west-to- east expansions from a single long-term refu-gium probably located in the foothills of the High Atlas Mountains near the Atlantic coast. Our findings support multiple origins of domestication with a low impact on the genetic diversity at range-wide level. The carob was mostly domesticated from
4096 |BAUMEL EtAL.1 | INTRODUCTIONFruit trees played a major role in the development of Mediterranean civilizations during the last millennia (Zohary & Hopf, 2012). Several species survived in refugia during the Pleistocene climatic changes and suffered repeated range expansions and contractions, which shaped their genetic diversity and structure. Their evolutionary histories represent examples of plant evolution under three im-portant drivers (geological, climatic and human), which have been defined as the Mediterranean triptych (Thompson,2020). Human activities represent the most recent driver of the Mediterranean triptych on shaping global biodiversity (Boivin et al.,2016). Indeed, humans have profoundly modified Mediterranean ecosystems for thousands of years, resulting in a continuum between forest and agrosystems (Quézel & Médail, 2003). As a result, it is difficult to document the evolutionary history of fruit trees, which may have cultivated, feral and wild populations in the same region (Besnard et al., 2018 ). In exceptional cases, intense dispersal by humans across the Mediterranean has led to the lack of robust genetic structure, for example in the chestnut (Fineschi et al., 2000) or the stone pine (Vendramin et al., 2008 ). By contrast, recent phy-logeographic studies have revealed that imprints of ancestral populations preceding agriculture are still present in the genetic diversity and structure of some Mediterranean cultivated tree species, such as the olive tree and the wild date palm (Besnard et al., 2018 ; Gros- Balthazard et al., 2017). Identifying the ancestral genetic legacy is essential to properly conserve genetic resources in the Mediterranean region, and to improve our understanding of the domestication process.As a general pattern, the domestication of plants in the Mediterranean started in the East, in the Fertile Crescent, and was followed by human-mediated westward dispersals of crops across the Mediterranean basin (Zeder, 2008 ; Zohary & Hopf, 2012). However, recent studies suggest that the cultivation of useful plants was not a rare phenomenon throughout the Mediterranean and may have involved local resources from several diversity centres in a pro-tracted process during which genetic admixture, within or between species, played a crucial role (Fuller et al., 2011; Purugganan,2019; Thompson,2020).The carob (Ceratonia siliqua, Fabaceae) is a common tree in tradi-tional Mediterranean orchards which has traditionally been valued, and still is, for its ability to produce food and fodder in marginal lands, notably during unfavourable years. Thus, domestication of the carob tree aimed at increasing pulp in the fruit (Zohary,2002). New uses have recently emerged, such as producing bioethanol or obtain-ing galactomannan from seeds as a food stabilizer. A recent review outlined the potential of carob for developing health-beneficial food products (Brassesco et al., 2021). The carob tree has also been the subject of trials for the ecological restoration and afforestation of degraded lands (Domínguez et al., 2010). Cultivars are propagated clonally by grafting; branches (scions) from selected productive trees are grafted on carob trees (rootstocks) often obtained from seedlings. Then it is assumed that the spread of its cultivation and domestication was linked to the development of grafting methods ca. 3000 years ago (Meyer et al., 2012; Zohary, 2002). As for several crops, the Near East and the Eastern Mediterranean regions were initially proposed as the centre of domestication for the carob tree (de Candolle, 1883 ; Ramón-Laca & Mabberley, 2004; Zohary, 2002). Due to the lack of international breeding programmes, each country possesses its own carob varieties and few international exchanges of selected varieties have been reported so far. The countries with the highest numbers of cultivated varieties are Spain, Portugal and Italy (Tous et al., 2013), although a lack of geographical pattern ex-plaining the genetic structure was initially found for Spanish and Italian cultivars (Caruso et al., 2008 ; La Malfa et al., 2014). A recent study of the world's largest germplasm collection detected genetic structure between cultivars from south Spain and Morocco, and separated from cultivars of northeast Spain, using microsatellite and plastid markers (Di Guardo et al., 2019). This finding was congruent with studies including wild carob populations (Viruel et al., 2018 , 2020), which recognized four main genetic groups across the Mediterranean and a strong west–east structure, as documented for several Mediterranean plants (Chen et al.,2014; Désamoré et al., 2011; García-Verdugo et al.,2021; Migliore et al., 2018 ; Nieto Feliner,2014). The scenario resulting from integrating the results from these studies supports the regional use, cultivation and domes-tication of wild carob trees in several parts of the Mediterranean. The mixed ancestry found in current cultivars was probably the locally selected wild genotypes and scattered long-distance westward dispersals of domesticated varieties by humans, concomitant with major historical migrations by Romans, Greeks and Arabs. Ex situ efforts to preserve carob genetic resources should prioritize accessions from both western and eastern populations, with emphasis on the most differentiated CEUs situated in southwest Morocco, south Spain and eastern Mediterranean. Our study highlights the relevance of wild and seminatural habitats in the conservation of genetic resources for cultivated trees.KEYWORDSbiogeography, genetic diversity, microsatellite, origin, phylogeography, RADseq
