Genomic evidence of genuine wild versus admixed olive populations evolving in the same natural environments in western Mediterranean Basin.

Crop-to-wild gene flow is a mechanism process widely documented, both in plants and animals. This can have positive or negative impacts on the evolution of admixed populations in natural environments, yet the phenomenon is still misunderstood in long-lived woody species, contrary to short-lived crops. Wild olive Olea europaea L. occurs in the same eco-geographical range as domesticated olive, i.e. the Mediterranean Basin (MB). Moreover, it is an allogamous and anemophilous species whose seeds are disseminated by birds, i.e. factors that drive gene flow between crops and their wild relatives. Here we investigated the genetic structure of western MB wild olive populations in natural environments assuming a homogenous gene pool with limited impact of cultivated alleles, as previously suggested. We used a target sequencing method based on annotated genes from the Farga reference genome to analyze 27 western MB olive tree populations sampled in natural environments in France, Spain and Morocco. We also target sequenced cultivated olive tree accessions from the Worldwide Olive Germplasm Bank of Marrakech and Porquerolles and from an eastern MB wild olive tree population. We combined PCA, sNMF, pairwise FST and TreeMix and clearly identified genuine wild olive trees throughout their natural distribution range along a north-south gradient including, for the first time, in southern France. However, contrary to our assumption, we highlighted more admixed than genuine wild olive trees. Our results raise questions regarding the admixed population evolution pattern in this environment, which might be facilitated by crop-to-wild gene flow.

Characterization of Worldwide Olive Germplasm Banks of Marrakech (Morocco) and Córdoba (Spain): Towards management and use of olive germplasm in breeding programs.

Olive (Olea europaea L.) is a major fruit crop in the Mediterranean Basin. Ex-situ olive management is essential to ensure optimal use of genetic resources in breeding programs. The Worldwide Olive Germplasm Bank of Córdoba (WOGBC), Spain, and Marrakech (WOGBM), Morocco, are currently the largest existing olive germplasm collections. Characterization, identification, comparison and authentication of all accessions in both collections could thus provide useful information for managing olive germplasm for its preservation, exchange within the scientific community and use in breeding programs. Here we applied 20 microsatellite markers (SSR) and 11 endocarp morphological traits to discriminate and authenticate 1091 olive accessions belonging to WOGBM and WOGBC (554 and 537, respectively). Of all the analyzed accessions, 672 distinct SSR profiles considered as unique genotypes were identified, but only 130 were present in both collections. Combining SSR markers and endocarp traits led to the identification of 535 cultivars (126 in common) and 120 authenticated cultivars. No significant differences were observed between collections regarding the allelic richness and diversity index. We concluded that the genetic diversity level was stable despite marked contrasts in varietal composition between collections, which could be explained by their different collection establishment conditions. This highlights the extent of cultivar variability within WOGBs. Moreover, we detected 192 mislabeling errors, 72 of which were found in WOGBM. A total of 228 genotypes as molecular variants of 74 cultivars, 79 synonyms and 39 homonyms as new cases were identified. Both collections were combined to define the nested core collections of 55, 121 and 150 sample sizes proposed for further studies. This study was a preliminary step towards managing and mining the genetic diversity in both collections while developing collaborations between olive research teams to conduct association mapping studies by exchanging and phenotyping accessions in contrasted environmental sites.

Evolutionary transcriptomics reveals the origins of olives and the genomic changes associated with their domestication.

The olive (Olea europaea L. subsp. europaea) is one of the oldest and most socio-economically important cultivated perennial crop in the Mediterranean region. Yet, its origins are still under debate and the genetic bases of the phenotypic changes associated with its domestication are unknown. We generated RNA-sequencing data for 68 wild and cultivated olive trees to study the genetic diversity and structure both at the transcription and sequence levels. To localize putative genes or expression pathways targeted by artificial selection during domestication, we employed a two-step approach in which we identified differentially expressed genes and screened the transcriptome for signatures of selection. Our analyses support a major domestication event in the eastern part of the Mediterranean basin followed by dispersion towards the West and subsequent admixture with western wild olives. While we found large changes in gene expression when comparing cultivated and wild olives, we found no major signature of selection on coding variants and weak signals primarily affected transcription factors. Our results indicated that the domestication of olives resulted in only moderate genomic consequences and that the domestication syndrome is mainly related to changes in gene expression, consistent with its evolutionary history and life history traits.

Cultivated Olive Diversification at Local and Regional Scales: Evidence From the Genetic Characterization of French Genetic Resources.

Molecular characterization of crop genetic resources is a powerful approach to elucidate the origin of varieties and facilitate local cultivar management. Here we aimed to decipher the origin and diversification of French local olive germplasm. The 113 olive accessions of the ex situ collection of Porquerolles were characterized with 20 nuclear microsatellites plus their plastid haplotype. We then compared this collection to Mediterranean olive varieties from the Worldwide Olive Germplasm Bank of Marrakech, Morocco. High genetic diversity was observed within local French varieties, indicating a high admixture level, with an almost equal contribution from the three main Mediterranean gene pools. Nearly identical and closely related genotypes were observed among French and Italian/Spanish varieties. A high number of parent-offspring relationships were also detected among French varieties and between French and two Italian varieties ('Frantoio' and 'Moraiolo') and the Spanish variety ('Gordal Sevillana'). Our investigations indicated that French olive germplasm resulted from the diffusion of material from multiple origins followed by diversification based on parentage relationships between varieties. We strongly suggest that farmers have been actively selecting olives based on local French varieties. French olive agroecosystems more affected by unexpected frosts than southernmost regions could also be seen as incubators and as a bridge between Italy and Spain that has enhanced varietal olive diversification.

Controlling for genetic identity of varieties, pollen contamination and stigma receptivity is essential to characterize the self-incompatibility system of Olea europaea L.

Bervillé et al. express concern about the existence of the diallelic self-incompatibility (DSI) system in Olea europaea, mainly because our model does not account for results from previous studies from their group that claimed to have documented asymmetry of the incompatibility response in reciprocal crosses. In this answer to their comment, we present original results based on reciprocal stigma tests that contradict conclusions from these studies. We show that, in our hands, not a single case of asymmetry was confirmed, endorsing that symmetry of incompatibility reactions seems to be the rule in Olive. We discuss three important aspects that were not taken into account in the studies cited in their comments and that can explain the discrepancy: (i) the vast uncertainty around the actual genetic identity of vernacular varieties, (ii) the risk of massive contamination associated with the pollination protocols that they used and (iii) the importance of checking for stigma receptivity in controlled crosses. These studies were thus poorly genetically controlled, and we stand by our original conclusion that Olive tree exhibits DSI.

Elucidation of the genetic architecture of self-incompatibility in olive: Evolutionary consequences and perspectives for orchard management.

The olive (Olea europaea L.) is a typical important perennial crop species for which the genetic determination and even functionality of self-incompatibility (SI) are still largely unresolved. It is still not known whether SI is under gametophytic or sporophytic genetic control, yet fruit production in orchards depends critically on successful ovule fertilization. We studied the genetic determination of SI in olive in light of recent discoveries in other genera of the Oleaceae family. Using intra- and interspecific stigma tests on 89 genotypes representative of species-wide olive diversity and the compatibility/incompatibility reactions of progeny plants from controlled crosses, we confirmed that O. europaea shares the same homomorphic diallelic self-incompatibility (DSI) system as the one recently identified in Phillyrea angustifolia and Fraxinus ornus. SI is sporophytic in olive. The incompatibility response differs between the two SI groups in terms of how far pollen tubes grow before growth is arrested within stigma tissues. As a consequence of this DSI system, the chance of cross-incompatibility between pairs of varieties in an orchard is high (50%) and fruit production may be limited by the availability of compatible pollen. The discovery of the DSI system in O. europaea will undoubtedly offer opportunities to optimize fruit production.

Evolutionary forces affecting synonymous variations in plant genomes.

Base composition is highly variable among and within plant genomes, especially at third codon positions, ranging from GC-poor and homogeneous species to GC-rich and highly heterogeneous ones (particularly Monocots). Consequently, synonymous codon usage is biased in most species, even when base composition is relatively homogeneous. The causes of these variations are still under debate, with three main forces being possibly involved: mutational bias, selection and GC-biased gene conversion (gBGC). So far, both selection and gBGC have been detected in some species but how their relative strength varies among and within species remains unclear. Population genetics approaches allow to jointly estimating the intensity of selection, gBGC and mutational bias. We extended a recently developed method and applied it to a large population genomic dataset based on transcriptome sequencing of 11 angiosperm species spread across the phylogeny. We found that at synonymous positions, base composition is far from mutation-drift equilibrium in most genomes and that gBGC is a widespread and stronger process than selection. gBGC could strongly contribute to base composition variation among plant species, implying that it should be taken into account in plant genome analyses, especially for GC-rich ones.

How anthropogenic changes may affect soil-borne parasite diversity? Plant-parasitic nematode communities associated with olive trees in Morocco as a case study.

BACKGROUND: Plant-parasitic nematodes (PPN) are major crop pests. On olive (Olea europaea), they significantly contribute to economic losses in the top-ten olive producing countries in the world especially in nurseries and under cropping intensification. The diversity and the structure of PPN communities respond to environmental and anthropogenic forces. The olive tree is a good host plant model to understand the impact of such forces on PPN diversity since it grows according to different modalities (wild, feral and cultivated olives). A wide soil survey was conducted in several olive-growing regions in Morocco. The taxonomical and the functional diversity as well as the structures of PPN communities were described and then compared between non-cultivated (wild and feral forms) and cultivated (traditional and high-density olive cultivation) olives. RESULTS: A high diversity of PPN with the detection of 117 species and 47 genera was revealed. Some taxa were recorded for the first time on olive trees worldwide and new species were also identified. Anthropogenic factors (wild vs cultivated conditions) strongly impacted the PPN diversity and the functional composition of communities because the species richness, the local diversity and the evenness of communities significantly decreased and the abundance of nematodes significantly increased in high-density conditions. Furthermore, these conditions exhibited many more obligate and colonizer PPN and less persister PPN compared to non-cultivated conditions. Taxonomical structures of communities were also impacted: genera such as Xiphinema spp. and Heterodera spp. were dominant in wild olive, whereas harmful taxa such as Meloidogyne spp. were especially enhanced in high-density orchards. CONCLUSIONS: Olive anthropogenic practices reduce the PPN diversity in communities and lead to changes of the community structures with the development of some damaging nematodes. The study underlined the PPN diversity as a relevant indicator to assess community pathogenicity. That could be taken into account in order to design control strategies based on community rearrangements and interactions between species instead of reducing the most pathogenic species.

A large set of 26 new reference transcriptomes dedicated to comparative population genomics in crops and wild relatives.

We produced a unique large data set of reference transcriptomes to obtain new knowledge about the evolution of plant genomes and crop domestication. For this purpose, we validated a RNA-Seq data assembly protocol to perform comparative population genomics. For the validation, we assessed and compared the quality of de novo Illumina short-read assemblies using data from two crops for which an annotated reference genome was available, namely grapevine and sorghum. We used the same protocol for the release of 26 new transcriptomes of crop plants and wild relatives, including still understudied crops such as yam, pearl millet and fonio. The species list has a wide taxonomic representation with the inclusion of 15 monocots and 11 eudicots. All contigs were annotated using BLAST, prot4EST and Blast2GO. A strong originality of the data set is that each crop is associated with close relative species, which will permit whole-genome comparative evolutionary studies between crops and their wild-related species. This large resource will thus serve research communities working on both crops and model organisms. All the data are available at http://arcad-bioinformatics.southgreen.fr/.

Plasticity in Vegetative Growth over Contrasted Growing Sites of an F1 Olive Tree Progeny during Its Juvenile Phase.

Climatic changes impact fruit tree growth and severely limit their production. Investigating the tree ability to cope with environmental variations is thus necessary to adapt breeding and management strategies in order to ensure sustainable production. In this study, we assessed the genetic parameters and genotype by environment interaction (GxE) during the early tree growth. One hundred and twenty olive seedlings derived from the cross 'Olivière' x 'Arbequina' were examined across two sites with contrasted environments, accounting for ontogenetic trends over three years. Models including the year of growth, branching order, environment, genotype effects, and their interactions were built with variance function and covariance structure of residuals when necessary. After selection of a model, broad sense heritabilities were estimated. Despite strong environmental effect on most traits, no GxE was found. Moreover, the internal structure of traits co-variation was similar in both sites. Ontogenetic growth variation, related to (i) the overall tree form and (ii) the growth and branching habit at growth unit scale, was not altered by the environment. Finally, a moderate to strong genetic control was identified for traits at the whole tree scale and at internode scale. Among all studied traits, the maximal internode length exhibited the highest heritability (H2 = 0.74). Considering the determinant role of this trait in tree architecture and its stability across environments, this study consolidates its relevance for breeding.

QTL mapping of flowering and fruiting traits in olive.

One of the challenge fruit growers are facing is to balance between tree production and vegetative growth from year to year. To investigate the existence of genetic determinism for reproductive behaviour in olive tree, we studied an olive segregating population derived from a cross between 'Olivière' and 'Arbequina' cultivars. Our strategy was based on (i) an annual assessment of individual trees yield, and (ii) a decomposition of adult growth units at the crown periphery into quantitative variables related to both flowering and fruiting process in relation to their growth and branching. Genetic models, including the year, genotype effects and their interactions, were built with variance function and correlation structure of residuals when necessary. Among the progeny, trees were either 'ON' or 'OFF' for a given year and patterns of regular vs. irregular bearing were revealed. Genotype effect was significant on yield but not for flowering traits at growth unit (GU) scale, whereas the interaction between genotype and year was significant for both traits. A strong genetic effect was found for all fruiting traits without interaction with the year. Based on the new constructed genetic map, QTLs with small effects were detected, revealing multigenic control of the studied traits. Many were associated to alleles from 'Arbequina'. Genetic correlations were found between Yield and Fruit set at GU scale suggesting a common genetic control, even though QTL co-localisations were in spe`cific years only. Most QTL were associated to flowering traits in specific years, even though reproductive traits at GU scale did not capture the bearing status of the trees in a given year. Results were also interpreted with respect to ontogenetic changes of growth and branching, and an alternative sampling strategy was proposed for capturing tree fruiting behaviour. Regular bearing progenies were identified and could constitute innovative material for selection programs.

Construction of core collections suitable for association mapping to optimize use of Mediterranean olive (Olea europaea L.) genetic resources.

Phenotypic characterisation of germplasm collections is a decisive step towards association mapping analyses, but it is particularly expensive and tedious for woody perennial plant species. Characterisation could be more efficient if focused on a reasonably sized subset of accessions, or so-called core collection (CC), reflecting the geographic origin and variability of the germplasm. The questions that arise concern the sample size to use and genetic parameters that should be optimized in a core collection to make it suitable for association mapping. Here we investigated these questions in olive (Olea europaea L.), a perennial fruit species. By testing different sampling methods and sizes in a worldwide olive germplasm bank (OWGB Marrakech, Morocco) containing 502 unique genotypes characterized by nuclear and plastid loci, a two-step sampling method was proposed. The Shannon-Weaver diversity index was found to be the best criterion to be maximized in the first step using the Core Hunter program. A primary core collection of 50 entries (CC50) was defined that captured more than 80% of the diversity. This latter was subsequently used as a kernel with the Mstrat program to capture the remaining diversity. 200 core collections of 94 entries (CC94) were thus built for flexibility in the choice of varieties to be studied. Most entries of both core collections (CC50 and CC94) were revealed to be unrelated due to the low kinship coefficient, whereas a genetic structure spanning the eastern and western/central Mediterranean regions was noted. Linkage disequilibrium was observed in CC94 which was mainly explained by a genetic structure effect as noted for OWGB Marrakech. Since they reflect the geographic origin and diversity of olive germplasm and are of reasonable size, both core collections will be of major interest to develop long-term association studies and thus enhance genomic selection in olive species.

Primary domestication and early uses of the emblematic olive tree: palaeobotanical, historical and molecular evidence from the Middle East.

Our knowledge of the origins of olive tree domestication in the Middle East and on the processes governing its extension and persistence in different vegetation types from prehistory through antiquity to modern times derives from diverse sources, spanning the biological sciences to the humanities. Nonetheless, it lacks a robust overview that may lead to floating interpretations. This is especially true in the Middle East, considered as the cradle of agriculture, and where the evolutionary history of this emblematic tree is intertwined with that of civilizations. Olive fruit, oil and wood have been, since Prehistoric times, characteristic products of the lands bordering the Mediterranean Sea. In the domestic economy of these countries, the olive tree gradually became a traditional tree crop since the first oil extraction, through the emergence of regional commerce that accompanied the rise and fall of early Near-Middle Eastern urbanism, until the development of modern trade, with an oil production estimated at circa 3000000 tons per year. The rising importance of the olive tree in human life has turned the tree into an endless source of fascination in the Aegean and Eastern Mediterranean, a symbol and a sacred tree, widely cited in the Bibles, the Koran, and in ancient literature. Here we argue that advances in radiocarbon chronology, palaeobotany, genetics, and archaeology-history have profoundly refined the history of olive trees in the Middle East. This review shows that the heartland of primary olive domestication must be enlarged to the Levant and not only focus on the Jordan Valley. The domestication of the olive tree is a long and ongoing process, linked to the early production of oil and the development of the olive trade. We also suggest that the olive tree became a particular icon, a sacred tree, during the Biblical period in the Levant.

Loss of genetic diversity as a signature of apricot domestication and diffusion into the Mediterranean Basin.

BACKGROUND: Domestication generally implies a loss of diversity in crop species relative to their wild ancestors because of genetic drift through bottleneck effects. Compared to native Mediterranean fruit species like olive and grape, the loss of genetic diversity is expected to be more substantial for fruit species introduced into Mediterranean areas such as apricot (Prunus armeniaca L.), which was probably primarily domesticated in China. By comparing genetic diversity among regional apricot gene pools in several Mediterranean areas, we investigated the loss of genetic diversity associated with apricot selection and diffusion into the Mediterranean Basin. RESULTS: According to the geographic origin of apricots and using Bayesian clustering of genotypes, Mediterranean apricot (207 genotypes) was structured into three main gene pools: 'Irano-Caucasian', 'North Mediterranean Basin' and 'South Mediterranean Basin'. Among the 25 microsatellite markers used, only one displayed deviations from the frequencies expected under neutrality. Similar genetic diversity parameters were obtained within each of the three main clusters using both all SSR loci and only 24 SSR loci based on the assumption of neutrality. A significant loss of genetic diversity, as assessed by the allelic richness and private allelic richness, was revealed from the 'Irano-Caucasian' gene pool, considered as a secondary centre of diversification, to the northern and southwestern Mediterranean Basin. A substantial proportion of shared alleles was specifically detected when comparing gene pools from the 'North Mediterranean Basin' and 'South Mediterranean Basin' to the secondary centre of diversification. CONCLUSIONS: A marked domestication bottleneck was detected with microsatellite markers in the Mediterranean apricot material, depicting a global image of two diffusion routes from the 'Irano-Caucasian' gene pool: North Mediterranean and Southwest Mediterranean. This study generated genetic insight that will be useful for management of Mediterranean apricot germplasm as well as genetic selection programs related to adaptive traits.

Genetic structure and core collection of the World Olive Germplasm Bank of Marrakech: towards the optimised management and use of Mediterranean olive genetic resources.

The conservation of cultivated plants in ex-situ collections is essential for the optimal management and use of their genetic resources. For the olive tree, two world germplasm banks (OWGB) are presently established, in Córdoba (Spain) and Marrakech (Morocco). This latter was recently founded and includes 561 accessions from 14 Mediterranean countries. Using 12 nuclear microsatellites (SSRs) and three chloroplast DNA markers, this collection was characterised to examine the structure of the genetic diversity and propose a set of olive accessions encompassing the whole Mediterranean allelic diversity range. We identified 505 SSR profiles based on a total of 210 alleles. Based on these markers, the genetic diversity was similar to that of cultivars and wild olives which were previously characterised in another study indicating that OWGB Marrakech is representative of Mediterranean olive germplasm. Using a model-based Bayesian clustering method and principal components analysis, this OWGB was structured into three main gene pools corresponding to eastern, central and western parts of the Mediterranean Basin. We proposed 10 cores of 67 accessions capturing all detected alleles and 10 cores of 58 accessions capturing the 186 alleles observed more than once. In each of the 10 cores, a set of 40 accessions was identical, whereas the remaining accessions were different, indicating the need to include complementary criteria such as phenotypic adaptive and agronomic traits. Our study generated a molecular database for the entire OWGB Marrakech that may be used to optimise a strategy for the management of olive genetic resources and their use for subsequent genetic and genomic olive breeding.

Genomic profiling of plastid DNA variation in the Mediterranean olive tree.

BACKGROUND: Characterisation of plastid genome (or cpDNA) polymorphisms is commonly used for phylogeographic, population genetic and forensic analyses in plants, but detecting cpDNA variation is sometimes challenging, limiting the applications of such an approach. In the present study, we screened cpDNA polymorphism in the olive tree (Olea europaea L.) by sequencing the complete plastid genome of trees with a distinct cpDNA lineage. Our objective was to develop new markers for a rapid genomic profiling (by Multiplex PCRs) of cpDNA haplotypes in the Mediterranean olive tree. RESULTS: Eight complete cpDNA genomes of Olea were sequenced de novo. The nucleotide divergence between olive cpDNA lineages was low and not exceeding 0.07%. Based on these sequences, markers were developed for studying two single nucleotide substitutions and length polymorphism of 62 regions (with variable microsatellite motifs or other indels). They were then used to genotype the cpDNA variation in cultivated and wild Mediterranean olive trees (315 individuals). Forty polymorphic loci were detected on this sample, allowing the distinction of 22 haplotypes belonging to the three Mediterranean cpDNA lineages known as E1, E2 and E3. The discriminating power of cpDNA variation was particularly low for the cultivated olive tree with one predominating haplotype, but more diversity was detected in wild populations. CONCLUSIONS: We propose a method for a rapid characterisation of the Mediterranean olive germplasm. The low variation in the cultivated olive tree indicated that the utility of cpDNA variation for forensic analyses is limited to rare haplotypes. In contrast, the high cpDNA variation in wild populations demonstrated that our markers may be useful for phylogeographic and populations genetic studies in O. europaea.

Grafting versus seed propagated apricot populations: two main gene pools in Tunisia evidenced by SSR markers and model-based Bayesian clustering.

Apricot was introduced into the Mediterranean Basin from China and Asian mountains through the Middle-East and the Central Europe. Traditionally present in Tunisia, we were interested in accessing the origin of apricot species in the country, and in particular in the number and the location of its introductions. A set of 82 representative apricot accessions including 49 grafted cultivars and 33 seed propagated 'Bargougs' were genotyped using 24 microsatellite loci revealing a total of 135 alleles. The model-based Bayesian clustering analysis using both Structure and InStruct programs as well as the multivariate method revealed five distinct genetic clusters. The genetic differentiation among clusters showed that cluster 1, with only four cultivars, was the most differentiated from the four remaining genetic clusters, which constituted the largest part of the studied germplasm. According to their geographic origin, the five identified groups (north, centre, south, Gafsa oasis and other oases groups) enclosed a similar variation within group, with a low level of differentiation. Overall results highlighted the distinction of two apricot gene pools in Tunisia related to the different mode of propagation of the cultivars: grafted and seed propagated apricot, which enclosed a narrow genetic basis. Our findings support the assumption that grafting and seed propagated apricots shared the same origin.

Traditional agroecosystems as conservatories and incubators of cultivated plant varietal diversity: the case of fig (Ficus carica L.) in Morocco.

BACKGROUND: Traditional agroecosystems are known to host both large crop species diversity and high within crop genetic diversity. In a context of global change, this diversity may be needed to feed the world. Are these agroecosystems museums (i.e. large core collections) or cradles of diversity? We investigated this question for a clonally propagated plant, fig (Ficus carica), within its native range, in Morocco, but as far away as possible from supposed centers of domestication. RESULTS: Fig varieties were locally numerous. They were found to be mainly highly local and corresponded to clones propagated vegetatively. Nevertheless these clones were often sufficiently old to have accumulated somatic mutations for selected traits (fig skin color) and at neutral loci (microsatellite markers). Further the pattern of spatial genetic structure was similar to the pattern expected in natural population for a mutation/drift/migration model at equilibrium, with homogeneous levels of local genetic diversity throughout Moroccan traditional agroecosystems. CONCLUSIONS: We conclude that traditional agroecosystems constitue active incubators of varietal diversity even for clonally propagated crop species, and even when varieties correspond to clones that are often old. As only female fig is cultivated, wild fig and cultivated fig probably constitute a single evolutionary unit within these traditional agroecosystems. Core collections, however useful, are museums and hence cannot serve the same functions as traditional agroecosystems.

Cyto-nuclear discordance in the phylogeny of Ficus section Galoglychia and host shifts in plant-pollinator associations.

BACKGROUND: Hybridization events are relatively common in vascular plants. However, the frequency of these events is unevenly distributed across the plant phylogeny. Plant families in which individual species are pollinated by specific pollinator species are predicted to be less prone to hybridization than other families. However, exceptions may occur within these families, when pollinators shift host-plant species. Indeed, host shifts are expected to increase the rate of hybridization events. Pollinators of Ficus section Galoglychia are suspected to have changed host repeatedly, based on several cases of incongruence between plant phylogeny and taxonomy, and insect phylogeny and taxonomy. We tracked cyto-nuclear discordance across section Galoglychia as evidence for hybridization. To achieve a proper global view, we first clarified the monophyly of section Galoglychia as it had been questioned by recent phylogenetic studies. Moreover, we investigated if fig size could be a factor facilitating host shifts. RESULTS: Phylogenetic chloroplast and nuclear results demonstrated the monophyly of section Galoglychia. Within section Galoglychia, we detected several cases of statistically significant cyto-nuclear discordance. Discordances concern both terminal nodes of the phylogenetic trees and one deep node defining relationships between subsections. Because nuclear phylogeny is congruent with morphological taxonomy, discordances were caused by the chloroplast phylogeny. Introgressive hybridization was the most likely explanation for these discordances. We also detected that subsections pollinated by several wasp genera had smaller figs and were pollinated by smaller wasps than subsections pollinated by a single wasp genus. CONCLUSION: As hypothesized, we discovered evidences of past hybridization in Ficus section Galoglychia. Further, introgression was only detected in subsections presenting incongruence between plant and pollinator phylogenies and taxonomy. This supports the hypothesis that host shift is the cause for plant-pollinator incongruence. Moreover, small fig size could facilitate host shifts. Eventually, this study demonstrates that non-coding chloroplast markers are valuable to resolve deep nodes in Ficus phylogeny.