Discussion
Intense research on plant phylogenetics and phylogeography over the last
two decades have allowed the discovery of several major biogeographical
trends in the Mediterranean basin (Garcia-Verdugo et al., 2021) and
renewed our understandings of plant domestication (Purugganan, 2019).
Following an initial focus on biogeographic refugia, recent studies have
revealed the genetic imprints of past expansions and migration
processes, some involving the entire Mediterranean basin (see reviews in
Médail and Diadema 2009; Nieto Feliner 2011; Nieto Feliner, 2014;
Migliore et al., 2018; Vargas et al., 2018; Thompson, 2020;
Garcia-Verdugo et al., 2021). Our study provides a better understanding
of the phylogeography of Mediterranean plants by revealing a new
historical scenario: the main gene pools of carob (i.e. CEUS) originated
from a biogeographic refugium probably located in southwest Morocco. Our
results also highlight that carob domestication has mainly relied on the
use of locally selected and disseminated varieties, albeit punctuated by
long-distance westward dispersal events by humans, which match major
cultural waves by Romans and Arabs.
Evolutionary history of the carob tree
Our phylogeographic investigation allows rejecting a long-standing
hypothesis that proposes an introduced origin of the carob tree in most
of the Mediterranean. An Eastern Mediterranean or Southern Arabian
origin followed by a human-mediated expansion were proposed by several
authors partly based on linguistic evidence from vernacular names,Ceratonia siliqua and C. oreothauma occurrences in
western Asia and carob agricultural practices (reviewed in Ramon‐Laca &
Mabberley, 2004). However, genetic data from SSR and plastid markers
based on a thorough population sampling across the Mediterranean (Viruel
et al., 2020) found a better explanation to account for all the data
according to which current C. siliqua populations originated from
two disjunct refugia after the Last Interglacial ca. 116 Ka ago. SSR
data revealed introgression in the Central Mediterranean and Northern
Morocco, but the strong west vs. central-east pattern based on plastid
data revealed a low human influence on the main current patterns of
genetic diversity and structure of the carob tree across the
Mediterranean. The comprehensive review of carob fossil data done by
Viruel et al., (2020) did not provide support for an eastern origin ofC. siliqua either. Instead, the fossil record shows a mostly
continuous presence of Ceratonia around the palaeo‐Mediterranean
Sea since the Oligocene with a progressive decline starting c. 20 Ma.
Compared to SSR data, RADseq allows bridging phylogenetic and population
genetic inferences (Parchman et al., 2018). Here, the inclusion ofCeratonia oreothauma , the sister species of C. siliqua ,
despite their divergence around 6.4 Ma (Viruel et al., 2020),
corroborates our previous conclusion on the importance of western
Mediterranean in the history of the carob. Our SVDquartets phylogenetic
reconstruction provides further resolution pointing to southwest Morocco
as closest to the ancestral population of C. siliqua (Figure 2).
This origin was suggested based on a slightly higher genetic diversity
revealed for both nuclear (SSR) and plastid data in Viruel et al.
(2020). However, in our previous study, coalescent‐based models tested
by an approximate Bayesian computation approach supported a two refugia
hypothesis to explain the west-east split in the genetic diversity
structure of the carob tree. Here, our phylogenetic and population
genomic inferences support a different scenario. According to
SVDquartets phylogenetic reconstruction based on nuclear genome-wide
diversity, the carob tree followed two routes of migration from south
Morocco; one northward that reached western north Africa and south Spain
(NM, SES and SWS CEUs) and another towards the east that gave rise to
the central-eastern CEUs. Both mitochondrial and plastid data extracted
from RADseq data also support the existence of the ancestral pool in
southwest Morocco. Specifically, eastern mtDNA and pDNA haplotypes are
present in the northern part of SM (Imouzzer Ida Ou Tanane area) thus
suggesting that this is the most credible source for the eastern
populations. By contrast to our previous study, our new scenario
explains the west-east split in the carob genetic diversity by two
migration routes from an ancestral population situated in south Morocco.
As shown in our previous study, species distribution modelling (SDM)
indicates that both the Last Interglacial and the Last Glacial Maximum
were periods of contraction for this species during the Pleistocene
(Viruel et al., 2020). Moreover, SDM predicted that some areas in the
North African and South European Atlantic coasts could have been
continuously suitable since the last 130 ka. Southwest Morocco has been
identified as a biogeographic refugium and even as a diversification
cradle for several taxonomic groups (e.g. Médail and Quézel, 1999;
Médail et al. 2001; Ortiz et al., 2009; Martínez-Freiría et al., 2017;
Bobo-Pinilla et al., 2018; Villa-Machio et al., 2018; Klesser et al.,
2021). Although Mediterranean phylogeographic studies focused mostly on
glacial refugia, three recent studies have highlighted South and West
Morocco as a refugium for plant populations during the LIG (Villa-Machio
et al., 2018: Bobo-Pinilla et al., 2018: Viruel et al., 2020) where an
overall pronounced climate continentality could have been buffered by
the ocean vicinity.
Footprints of domestication on the current genetic structure of the
carob tree across the Mediterranean
Although disentangling the history of cultivated plants is complex, our
phylogeographic investigation in the carob tree sheds light on the
history of agriculture. Our previous study based on SSR data (Viruel et
al., 2020) suggested that local domestication events from wild
populations were the most likely scenario. The RADseq data here
presented, depicting a strong east-west genome-wide differentiation
could not explain domestication solely based on translocations and/or
human-based dispersals from east to west. Agriculture practices in the
carob tree are based on propagation by grafting (Zohary, 2002) although
seeds could have also been transported. In either case, domestication
based only on westward propagations of cultivars from the east would
have maintained the maternal (eastern) haplotypes in the Western
Mediterranean. Instead, our results conclude that the dispersal of
selected varieties (vegetatively propagated), between remote
geographical areas, was not the main force of domestication in carob
tree.
The use of genomic data at the infraspecific level has permitted
identifying footprints of domestication in crop models where PCR-based
molecular markers had previously failed. In the case of date palm,
genomic data revealed that human-mediated dispersal imprints were
superimposed on a previous phylogeographical structure (Gros-Balthazar
et al. 2017; Flowers et al. 2019). In the carob tree, despite a moderate
differentiation (Gst = 11%), genome-wide diversity is structured into
three main genetic sources: SM, SWS+ SES and NEM + SEM (Figure 2, and
Fig. S5 in supplementary material). Although this pattern does not
suggest translocation of eastern domesticated varieties into South
Morocco or the Iberian Peninsula, it does fit with the patterns found in
geographically intermediate groups (NM, CM and NEM). These are less
differentiated, which is explained by high rates of admixture (Fig. 2).
To untangle the role of human-based dispersals in these strong genetic
admixtures, we used allelic-frequency based models aiming at estimating
the intensity and origin of dispersal events throughout the evolutionary
tree of the carob tree (Fig. 3): results of Treemix recover westwards
migrations that were mostly originated from SEM, or from central-eastern
CEUs (SEM, NEM and CM). These translocations match with the beginning of
carob agriculture in the East, its dispersal by Greeks, Romans and after
by Arabs in historical times (Ramon Laca and Mabberley, 2001; Viruel et
al., 2020). They may have contributed to genetic admixed pool used
locally for cultivation as observed in North Morocco (NM).
The second footprint of domestication was observed in CM, which is the
area among those considered in our study in which cultivated varieties
(either local or imported selections) are most diffused (Di Guardo et
al., 2019). This CEU is characterized by a slightly lower genetic
diversity and a small excess of heterozygosity whereas all other CEUs
showed a deficit. We detected a genetic group of individuals without
admixture in CM, corresponding to the monumental carobs of the Ragusa
district (Sicily, South Italy). Without being clones, these individuals,
harvested without interruption for centuries, are genetically very close
to each other and form a lineage within CM. The genetic patterns of
these ancient CM individuals have not been observed in other CEUs,
supporting again the idea that diffusion of selected genotypes at the
local scale local, rather than long-distance dispersal, played a major
role in the domestication of carob. Despite this pattern, we did not
detect any candidate loci under selection due to domestication
pressures, which could be explained by the limitations of our method and
sampling or by a low effect of domestication on the carob genome.
Compared to other perennial crop species for which candidate and
adaptive loci have been found by whole genome sequencing as well as
RADseq (Cornejo et al., 2018; Alves-Pereira et al., 2020; Groppi et al.,
2021), a relatively lower impact of selection is likely in carob.
Domestication leading to fine-tuning of gene expression patterns rather
than genome-wide evolution, as observed in olive (Gros-Balthazard et
al., 2019), maybe almost undetectable by a reduced-representation
genomics approach such as RADseq.
Conservation of genetic diversity within Carob Evolutionary Units (CEUs)
Knowing the structure of genome-wide diversity is essential for
preserving the genetic resources of cultivated species and for future
breeding (Purugganan 2019). We used an integrative approach combining
geographic and genetic differentiation to characterize evolutionary
units for Ceratonia siliqua across the Mediterranean. In a survey
including 1020 samples, seven non-overlapping CEUs were identified as
the best solution to minimize intra-group variance and obtain homogenous
groups non overlapped geographically. Four genetic clusters, identified
within carob tree populations based on a thorough sampling across the
Mediterranean using nuclear SSR and SNP data, are contained within the
seven CEUs (Figure 2): South Morocco (SM), Iberian Peninsula (SES, SWS),
Central Mediterranean (CM, NM) and Eastern Mediterranean (NEM, SEM).
These four genetic clusters exhibit moderate introgression in the West
and East CEUs, but high patterns of admixture in the Central
Mediterranean (CM, NM), more intense in NM. RADseq data further resolved
these genetic structuring across the Mediterranean by identifying seven
genetic clusters (Fig. 2 A,D), which, in some cases fully matched with a
CEU (e.g. SM, SES) or two CEUs (SWS, SES) whereas, in other cases, a
mixture of more than one genetic cluster was observed (e.g. CM). These
data permit a better interpretation of the genetic diversity patterns
between CEUs and are thus important for future designs of ex situ
conservation. Our results suggest that moderate genetic diversity is
uniformly distributed across CEUs (Table 1). Only a slightly higher
genetic diversity was estimated in Western CEUs (SWS, SES, SM) based on
SSR loci. Although Central CEUs (CM, NM) are highly admixed, these
factors did not entail an increase in genetic diversity compared to
non-admixed clusters. Conservation of genetic resources for the carob
tree should recover genetic diversity found across the Mediterranean by
preserving materials from western and eastern CEUs prioritizing the most
differentiated CEUs SM, SES + SWS, and SEM. CM, which contains three
genetic groups and for which carob cultivars have been well
characterized, specifically in Italy, should benefit from more
investigations on carob evolution under domestication.
Aknowledgments
This study is part of the DYNAMIC project supported by the French
national agency of research (ANR-14-CE02-0016) and benefited from
equipment and services from the molecular biology facility (SCBM) at
IMBE (Marseille, France). All bioinformatics and simulations were done
on the High-Performance Computing Cluster from the Pytheas informatic
facility (OSU Institut Pytheas Aix Marseille Univ, INSU-CNRS UMS 3470)
J.V. benefited from a Postdoc Fellowship funded by DYNAMIC and a Marie
Skłodowska-Curie Individual Fellowship (704464 - YAMNOMICS -
MSCA-IF-EF-ST). The authors thank for their help to complete our
sampling: Annette Patzelt (Oman Botanic Garden), Minas Papadopulos
(Department of Forests of the Republic of Cyprus), Zahra Djabeur (Oran
University), Nabil Benghanem (Tizi-Ouzou University), Gianluigi
Bacchetta (Cagliari University), Sonja Yakovlev (Paris-Sud University),
Errol Vela (CIRAD), Maria Panitsa (Patras University), and the services
of Junta de Andalucia.
Author contributions
H. S., A.B., F.M., S.L.M., M.B.K., L.O., G.N.F. and J.V. conceived,
planed the study and collected samples. F.L.M. performed the DNA
extraction and quality assessment. J.V, A.B. and V.F. performed curation
and analysis of microsatellite data. A.B. performed RADseq data curation
and SNPs filtering. A.B. and J.V provided the analysis, tables and
figures. A.B., J.V., G.N.F. and F.M. interpreted the results. A.B. and
J.V. drafted the manuscript. J.V., G.N.F., S.L.M. and M.D.G. edited the
manuscript. J.V., G.N.F. and A.B. wrote the final manuscript. H.S. was
in charge with funding acquisition and project administration. All
authors read and approved the final version.
Data Availability Stament
Full information on populations sampling and microsatellite data are
available in Viruel et al. (2020) and deposited in DRYAD
(https://doi.org/10.5061/dryad.k7m020r).
Raw RADseq reads are deposited at NCBI under Bioproject accession
(######). Assemblies from ipyRAD, data files and R scripts of
analyses are available at Zenodo (##############).
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Supporting information
Tab. S1: Statistics from four assemblies conducted on RADseq
data (36 samples) elaborated with ipyrad with varying the clustering
threshold from 0.9 to 0.96 % of similarity.
Tab. S2: pairwise Gst differentiations among CEUs based on
RADseq data. Values above the overall Gst (11%) in bold.
Fig. S1 : Design of Carob Evolutionary Units (CEUs) using
ClustGeo, which considers euclidean genetic and geographical distances.
17 SSRs and 15 SNP markers from microsatellite loci were used. The Ward
dendrogram of 56 carob populations with a partition in K=7 clusters (A)
was obtained with a normalized proportion α of explained inertia of 0.2
for the geographic distance and 0.8 for the genetic distance (B). C)
Neighbor Joining tree based on pairwise genetic differentiation (Gst SSR
markers) among the seven clusters. See main text for acronyms of CEUs.
Fig. S2 : Genome wide diversity structure of 350 carob trees
based on 10,012 RADseq loci with an overall missing data rate of 64%.
(A) PCA scatter plot of 350 carob RADseq genotypes. (B) Neighbor joining
tree of pairwise Gst differentiations among seven Carob Evolutionary
Units (CEUs).
Fig. S3: FST per loci
distribution (1 SNP by locus) with 56 loci identified as outliers by
OUTFLANK due to their unexpectedly high Fst differentiation (FDR
<0.05). The blue line is the inferred neutral distribution.
Fig. S4: Map of mtDNA and pDNA haplogroups from 14 and 21
RADseq loci respectively obtained for 190 carob trees. West and East
haplogroups match strictly for both organelle data sets except for South
Morocco (SM).
Fig. S5 : Population genetic structure of the carob according to
RADseq. A) Genetic admixture plots for 190 carob trees from k=2 to K=7
ancestral populations obtained with the snmf method (LEA package)
performed on 3,557 unlinked SNPs. The West and East lineages refer to
organellar haplogroups (Fig. S4). B) Cross-entropy criterion suggesting
two optimal solutions with K= 5 or 7.
Fig. S6: FST per loci distribution (1 SNP by locus). OUTFLANK
method did not detected any outlier (FDR <0.05). The blue line
is the inferred neutral distribution.
Figure legends
FIGURE 1: Bioinformatic pipeline to extract and filter SNPs
from RADseq data for the carob tree.
FIGURE 2 : Population genetic structure of the carob tree. A)
SVDquartets tree of seven genetically and geographically homogeneous
groups (CEUs) based on RADseq markers. Genetic admixture plots are based
on four ancestral populations for SSR markers (1020 genotypes, 17 loci)
and on 7 ancestral populations for RADseq markers (190 genotypes, 3557
neutral and unlinked SNPs). B) & C) PCA scatterplots of RADseq
genotypes -the first three components = 15.2% of variance). D) Map of
genetic admixture based on RADseq markers and 7 ancestral populations.
FIGURE 3: Evolutionary history of the carob tree reconstructed
with Treemix. Maximum likelihood trees obtained without (A) and with
gene flow (B) events explaining 96% and 99% of the variance,
respectively. The color of the arrows indicates the migration weight
which is the fraction of ancestry derived from the migration edge.
TABLE 1: Estimates of genetic diversity based on
microsatellites (17 SSR loci) and genome-wide markers (3557 SNPs) for
seven Ceratonia siliqua units (CEUs). Within each CEU, samples
were split into groups according to their origin (cultivated,
seminatural or natural habitats).