Background and Aims Inferring the evolutionary relationships of species and their boundaries is critical in order to understand patterns of diversification and their historical drivers. Despite Abies (Pinaceae) being the second most diverse group of conifers, the evolutionary history of Circum-Mediterranean firs (CMFs) remains under debate. Methods We used restriction site-associated DNA sequencing (RAD-seq) on all proposed CMF taxa to investigate their phylogenetic relationships and taxonomic status. Key Results Based on thousands of genome-wide single nucleotide polymorphisms (SNPs), we present here the first formal test of species delimitation, and the first fully resolved, complete species tree for CMFs. We discovered that all previously recognized taxa in the Mediterranean should be treated as independent species, with the exception of Abies tazaotana and Abies marocana. An unexpectedly early pulse of speciation in the Oligocene–Miocene boundary is here documented for the group, pre-dating previous hypotheses by millions of years, revealing a complex evolutionary history encompassing both ancient and recent gene flow between distant lineages. Conclusions Our phylogenomic results contribute to shed light on conifers’ diversification. Our efforts to resolve the CMF phylogenetic relationships help refine their taxonomy and our knowledge of their evolution.

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Twelve nuclear microsatellite primers (nSSR) were developed for the endangered species Abies pinsapo Boiss. to enable the study of gene flow and genetic structure in the remaining distribution areas. Microsatellite primers were developed using next-generation sequencing (454) data from a single Abies pinsapo individual. Primers were applied to thirty individuals from the three extant localities. The number of alleles per locus ranged from one to four. Cross-amplification was tested for other Abies species from the Mediterranean Basin, and most of the loci showed higher polymorphisms in the Mediterranean species than in A. pinsapo. These microsatellite markers provide tools for conservation genetic studies in Abies pinsapo as well other Abies species from the Mediterranean Basin.

Plant architecture is crucial to pollination and mating in wind-pollinated species. We investigated the effect of crown architecture on pollen dispersal, mating system and offspring quality, combining phenotypic and genotypic analyses in a low-density population of the endangered species Abies pinsapo. A total of 598 embryos from three relative crown height levels (bottom, middle and top) in five mother plants were genotyped using eleven nuclear microsatellite markers (nSSRs). Paternity analysis and mating system models were used to infer mating and pollen dispersal parameters. In addition, seeds were weighed (N = 16 110) and germinated (N = 736), and seedling vigour was measured to assess inbreeding depression. Overall, A. pinsapo shows a fat-tailed dispersal kernel, with an average pollen dispersal distance of 113-227 m, an immigration rate of 0.84-26.92%, and a number of effective pollen donors (Nep ) ranging between 3.5 and 11.9. We found an effect of tree height and relative crown height levels on mating parameters. A higher proportion of seeds with embryo (about 50%) and a higher rate of self-fertilization (about 60%) were found at the bottom level in comparison with the top level. Seed weight and seedling vigour are positively related. Nevertheless, no differences were found in seed weight or in seedling-related variables such as weight and length of aerial and subterranean parts among the different relative crown height levels, suggesting that seeds from the more strongly inbred bottom level are not affected by inbreeding depression. Our results point to vertical isotropy for outcross-pollen and they suggest that self-pollen may ensure fertilization when outcross-pollen is not available in low-density population.

A better understanding of long-term effects of climate and historical anthropogenic changes is needed to define effective conservation measures of endangered forest inhabiting managed landscapes. Diversification and distribution of Mediterranean firs are attributed to the global climate change during the Miocene and Quaternary as well as to the effects of human-driven deforestation. We evaluated the impact of climate change and anthropogenic activities in shaping the genetic diversity and structure of Abies pinsapo Boiss. (Pinaceae), a relict fir endemic from SW Spain. We genotyped a total of 440 individuals from 44 populations by using two different molecular markers (cpSSRs and nSSRs). Overall, low genetic structure was found; however, incipient differentiation appeared within mountain ranges. Analyses suggest that the effects of isolation by distance and lithological or topographical diversity were not enough to structure the populations of the different mountain ranges. The combined role of genetic drift in the small populations and the anthropogenic action associated with forest management has shaped the current genetic pattern of this fir species in the study area. Demographic inference analyses pointed to a very recent synchronic divergence (eleventh–sixteenth century) of the ancestral A. pinsapo population into its current scattered distribution range. Although population bottlenecks were supported by several analyses, the conservation of this endangered species seems not to be limited by lacking genetic diversity, while threats of current climate change and habitat loss must be regarded.