Aim Phylogeography of fruit trees is challenging due to recurrent exchanges between domesticated and wild populations. Here we tested the eastern refugium hypothesis (ERH) for the carob tree, Ceratonia siliqua, which supports its natural and domestication origins in the eastern Mediterranean and a feral origin in the west. Location Mediterranean basin. Taxon Ceratonia siliqua L., Leguminosae. Methods A phylogenetic reconstruction based on two nuclear and one plastid sequences was performed to estimate the divergence time between the carob tree and its sister species, Ceratonia oreothauma. Variation from four plastid regions and 17 nuclear microsatellite loci were used to decipher genetic structure in the carob tree and to test coalescent‐based models by an Approximate Bayesian computation (ABC) approach. We assessed our hypotheses by examining palaeobotanical records and hindcasting the past distribution of the carob tree at Mid‐Holocene, Last Glacial Maximum (LGM) and Last Interglacial (LIG) using species distribution modelling. Results The split between C. oreothauma and C. siliqua was estimated at 6.4 Ma, and a first divergence within C. siliqua at 1.3 Ma. After a continuous presence since the Oligocene, Ceratonia was rarely found in the fossil record during the Pleistocene but present in the western and the eastern Mediterranean. Plastid and nuclear markers, characterized by low allelic richness, revealed a strong west‐east genetic structuring. ABC analyses rejected the ERH. Main conclusions Our study supports a severe population decline during LIG. The strong west–east divergence and the occurrence of four lineages within C. siliqua provided support for a new hypothesis of multiple domestications of the carob tree from native populations throughout the Mediterranean basin.

Premise of the StudySimple sequence repeat (SSR) or microsatellite markers have been used in a broad range of studies mostly scoring alleles on the basis of amplicon size as a proxy for the number of repeat units of an SSR motif. However, additional sources of variation within the SSR or in the flanking regions have largely remained undetected.MethodsIn this study, we implemented a next‐generation sequencing–based genotyping approach in a newly characterized set of 18 nuclear SSR markers for the carob tree, Ceratonia siliqua. Our aim was to evaluate the effect of three different methods of scoring molecular variation present within microsatellite markers on the genetic diversity and structure results.ResultsThe analysis of the sequences of 77 multilocus genotypes from four populations revealed SSR variation and additional sources of polymorphism in 87% of the loci analyzed (42 single‐nucleotide polymorphisms and five insertion/deletion polymorphisms), as well as divergent paralog copies in two loci. Ignoring sequence variation under standard amplicon size genotyping resulted in incorrect identification of 69% of the alleles, with important effects on the genetic diversity and structure estimates.DiscussionNext‐generation sequencing allows the detection and scoring of SSRs, single‐nucleotide polymorphisms, and insertion/deletion polymorphisms to increase the resolution of population genetic studies.

In this study, we investigated whether indigenous arbuscular mycorrhizal (AM) fungi could improve the tolerance of Cupressus atlantica against water deficit. We tested a gradient of watering regime spanning from 90% to 25% of soil retention capacity of water on mycorhized and non-mycorhized seedlings in pot cultures with sterilized and non-sterilized soils. Our result showed a positive impact of AM fungi on shoot height, stem diameter and biomass as well as on the growth rate. We also observed that inoculation with AM fungi significantly improved uptake of minerals by C. atlantica in both sterilized and non-sterilized soils independently of water regimes. We found that mycorhized plants maintained higher relative water content (RWC) and water potential compared with non-mycorhized plants that were subjected to drought-stress regimes (50% and 25% of soil retention capacity). The contents of proline and of soluble sugars showed that their concentrations decreased in non-mycorhized plants subjected to DS. Superoxide dismutase (SOD) and catalase (CAT) activities also decreased in non-mycorhized plants submitted to DS compared to mycorhized plants. The same pattern was observed by measuring peroxidase (POD) enzyme activity. The results demonstrated that AM fungal inoculation promoted the growth and tolerance of C. atlantica against DS in pot cultures. Therefore, mycorrhizal inoculation could be a potential solution for the conservation and reestablishment of C. atlantica in its natural ecosystem.

Aims: The study aimed to determine whether inoculation with native arbuscular mycorrhizal (AM) fungi could improve survival and growth of seedlings in degraded soils of Morocco. Methods and Results: Soil samples were collected from the rhizosphere of Cupressus atlantica trees in the N’Fis valley (Haut Atlas, Morocco). AM spores were extracted from the soil, identified and this mixture of native AM fungi was propagated on maize for 12 weeks on a sterilized soil to enrich the fungal inoculum. Then C. atlantica seedlings were inoculated with and without (control) mycorrhizal maize roots, cultured in glasshouse conditions and further, transplanted into the field. The experiment was a randomized block design with one factor and three replication blocks. The results showed that a high AM fungal diversity was associated with C. atlantica; native AM fungi inoculation was very effective on the growth of C. atlantica seedlings in glasshouse conditions and this plant growth stimulation was maintained for 1 year after outplanting. Conclusions: Inoculation of C. atlantica with AM fungi increased growth and survival in greenhouse and field. Significance and Impact of the Study: The data indicate that use of native species of AM fungi may accelerate reforestation of degraded soils. Further studies have to be performed to determine the persistence of these mycorrhizae for a longer period of plantation and to measure the effects of this microbial inoculation on soil biofunctioning.