Alfredo García-Fernández

Unravelling genetics at the top: mountain islands or isolated belts?

BACKGROUND AND AIMS In mountain plant populations, local adaptation has been described as one of the main responses to climate warming, allowing plants to persist under stressful conditions. This is especially the case for marginal populations at their lowest elevation, as they are highly vulnerable. Adequate levels of genetic diversity are required for selection to take place, while high levels of altitudinal gene flow are seen as a major limiting factor potentially precluding local adaptation processes. Thus, a compromise between genetic diversity and gene flow seems necessary to guarantee persistence under oncoming conditions. It is therefore critical to determine if gene flow occurs preferentially between mountains at similar altitudinal belts, promoting local adaptation at the lowest populations, or conversely along altitude within each mountain. METHODS Microsatellite markers were used to unravel genetic diversity and population structure, inbreeding and gene flow of populations at two nearby altitudinal gradients of Silene ciliata, a Mediterranean high-mountain cushion plant. KEY RESULTS Genetic diversity and inbreeding coefficients were similar in all populations. Substantial gene flow was found both along altitudinal gradients and horizontally within each elevation belt, although greater values were obtained along altitudinal gradients. Gene flow may be responsible for the homogeneous levels of genetic diversity found among populations. Bayesian cluster analyses also suggested that shifts along altitudinal gradients are the most plausible scenario. CONCLUSIONS Past population shifts associated with glaciations and interglacial periods in temperate mountains may partially explain current distributions of genetic diversity and population structure. In spite of the predominance of gene flow along the altitudinal gradients, local genetic differentiation of one of the lower populations together with the detection of one outlier locus might support the existence of different selection forces at low altitudes.

Isolation and characterization of 10 microsatellite loci in Cneorum tricoccon (Cneoraceae), a Mediterranean relict plant

PREMISE OF THE STUDY The main aim of this study was to isolate and characterize microsatellite loci in Cneorum tricoccon (Cneoraceae), a Mediterranean shrub relict of the early Tertiary, which inhabits western Mediterranean islands and coasts. Microsatellites will be useful for investigating biogeography and landscape genetics across the species distribution range, including current or past gene flow. METHODS AND RESULTS Seventeen microsatellite loci were characterized, of which 10 were polymorphic and amplified for a total of 56 alleles in three populations of C. tricoccon. The markers revealed average coefficients of expected heterozygosity (H(e) = 0.425), observed heterozygosity (H(o) = 0.282), and inbreeding coefficient value per population (F(IS) = 0.408). CONCLUSIONS These microsatellite primers will potentially be useful in the study of population and landscape genetics, conservation status of isolated populations, island-continental distribution, current or historical movements between populations, and in the investigation of the consequences of dispersal mechanisms of these plants.

Habitat Fragmentation Differentially Affects Genetic Variation, Phenotypic Plasticity and Survival in Populations of a Gypsum Endemic

Habitat fragmentation, i.e., fragment size and isolation, can differentially alter patterns of neutral and quantitative genetic variation, fitness and phenotypic plasticity of plant populations, but their effects have rarely been tested simultaneously. We assessed the combined effects of size and connectivity on these aspects of genetic and phenotypic variation in populations of Centaurea hyssopifolia, a narrow endemic gypsophile that previously showed performance differences associated with fragmentation. We grew 111 maternal families sampled from 10 populations that differed in their fragment size and connectivity in a common garden, and characterized quantitative genetic variation, phenotypic plasticity to drought for key functional traits, and plant survival, as a measure of population fitness. We also assessed neutral genetic variation within and among populations using eight microsatellite markers. Although C. hyssopifolia is a narrow endemic gypsophile, we found substantial neutral genetic variation and quantitative variation for key functional traits. The partition of genetic variance indicated that a higher proportion of variation was found within populations, which is also consistent with low population differentiation in molecular markers, functional traits and their plasticity. This, combined with the generally small effect of habitat fragmentation suggests that gene flow among populations is not restricted, despite large differences in fragment size and isolation. Importantly, population’s similarities in genetic variation and plasticity did not reflect the lower survival observed in isolated populations. Overall, our results indicate that, although the species consists of genetically variable populations able to express functional plasticity, such aspects of adaptive potential may not always reflect populations’ survival. Given the differential effects of habitat connectivity on functional traits, genetic variation and fitness, our study highlights the need to shift the focus of fragmentation studies to the mechanisms that regulate connectivity effects, and call for caution on the use of genetic variation and plasticity to forecast population performance.

Identification and development of eight microsatellite markers for the gypsophyte Helianthemum squamatum and cross-species amplification

AbstractHelianthemum squamatum (L.) Dum. Cours. (Cistaceae) is an endemic specialist plant that inhabits the gypsum soils of the semi-arid Iberian Peninsula, one of the ecosystems most vulnerable to climate change. In this study, eight microsatellite markers were isolated and developed using next-generation sequencing data in order to provide a validated tool for genetic studies of this species. Their polymorphisms were tested in two populations. The number of alleles and the expected heterozygosity ranged from 2 to 8 and from 0.29 to 0.82 respectively. Cross-amplification was carried out in the related congeners Helianthemum hirtum and Helianthemum cinereum.

Characterization of microsatellites in the mountain plant Armeria caespitosa (Plumbaginaceae) and transferability to congeners

PREMISE OF THE STUDY The focus of this study is to develop microsatellite markers in Armeria caespitosa, a narrow endemic of central Spain. Microsatellite loci are sought to clarify population structure and estimate gene flux among populations. METHODS AND RESULTS Enriched microsatellite genomic libraries were used for microsatellite isolation. Sixteen microsatellite loci were characterized, eight of which can be used for successful genotyping. Allele number ranged from two to seven per locus. Observed and expected heterozygosity ranged from 0.300 to 0.800 and from 0.296 to 0.733, respectively. Cross-amplification of seven and six loci was successful for A. maritima and A. cantabrica, respectively. CONCLUSIONS These microsatellites are suitable in the study of population genetics and gene flow among A. caespitosa populations. The information provided by these markers may be useful in the study of this plants response to global warming.

A glacial survivor of the alpine Mediterranean region: phylogenetic and phylogeographic insights into Silene ciliata Pourr. (Caryophyllaceae)

Silene ciliata Pourr. (Caryophyllaceae) is a species with a highly disjunct distribution which inhabits the alpine mountains of the Mediterranean Basin. We investigated the phylogeny and phylogeography of the species to (a) clarify the long-suggested division of S. ciliata into two subspecies, (b) evaluate its phylogenetic origin and (c) assess whether the species’ diversification patterns were affected by the Mediterranean relief. For this purpose, we collected DNA from 25 populations of the species that inhabit the mountains of Portugal, Spain, France, Italy, former Yugoslav Republic of Macedonia, Bulgaria and Greece and studied the plastid regions rbcL, rps16 and trnL. Major intraspecific variation was supported by all analyses, while the possibility of the existence of more varieties or subspecies was not favoured. Plastid DNA (cpDNA) evidence was in accordance with the division of S. ciliata into the two subspecies, one spreading west (Iberian Peninsula and Central Massif) and the other east of the Alps region (Italian and Balkan Peninsula). This study proposes that the species’ geographically disconnected distribution has probably derived from vicariance processes and from the Alps acting as a barrier to the species’ dispersal. The monophyletic origin of the species is highly supported. cpDNA patterns were shown independent of the chromosome evolution in the populations and could have resulted from a combination of geographic factors providing links and barriers, climatic adversities and evolutionary processes that took place during Quaternary glaciations.

Population size, center-periphery, and seed dispersers’ effects on the genetic diversity and population structure of the Mediterranean relict shrub Cneorum tricoccon

Abstract The effect of population size on population genetic diversity and structure has rarely been studied jointly with other factors such as the position of a population within the species’ distribution range or the presence of mutualistic partners influencing dispersal. Understanding these determining factors for genetic variation is critical for conservation of relict plants that are generally suffering from genetic deterioration. Working with 16 populations of the vulnerable relict shrub Cneorum tricoccon throughout the majority of its western Mediterranean distribution range, and using nine polymorphic microsatellite markers, we examined the effects of periphery (peripheral vs. central), population size (large vs. small), and seed disperser (introduced carnivores vs. endemic lizards) on the genetic diversity and population structure of the species. Contrasting genetic variation (H E: 0.04–0.476) was found across populations. Peripheral populations showed lower genetic diversity, but this was dependent on population size. Large peripheral populations showed high levels of genetic diversity, whereas small central populations were less diverse. Significant isolation by distance was detected, indicating that the effect of long‐distance gene flow is limited relative to that of genetic drift, probably due to high selfing rates (FIS = 0.155–0.887), restricted pollen flow, and ineffective seed dispersal. Bayesian clustering also supported the strong population differentiation and highly fragmented structure. Contrary to expectations, the type of disperser showed no significant effect on either population genetic diversity or structure. Our results challenge the idea of an effect of periphery per se that can be mainly explained by population size, drawing attention to the need of integrative approaches considering different determinants of genetic variation. Furthermore, the very low genetic diversity observed in several small populations and the strong among‐population differentiation highlight the conservation value of large populations throughout the species’ range, particularly in light of climate change and direct human threats.