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.