Genetic Diversity and Phylogeography of the Relict Tree Fern Culcita macrocarpa: Influence of Clonality and Breeding System on Genetic Variation

Abstract

The tree fern Culcita macrocarpa, a threatened Iberian-Macaronesian endemism, represents the sole European species of the order Cyatheales. Considered a Tertiary relict of European Palaeotropical flora, its evolutionary history and genetic diversity, potentially influenced by presumed high clonal propagation, remain largely unknown. This study elucidates the phylogeographic history of C. macrocarpa, assessing the impact of vegetative reproduction on population dynamics and genetic variability. We provide genetic data from eight newly identified nuclear microsatellite loci and one plastid DNA region for 17 populations spanning the species' range, together with species distribution modeling data. Microsatellites reveal pervasive clonality in C. macrocarpa, which has varied among populations. We assess the impact of clonality on genetic diversity and evaluate how estimates of intra-population genetic diversity indices and genetic structuring are affected by the chosen definition of "individual" (focusing exclusively on genetically distinct individuals, genets, as opposed to considering all independent clonal replicates, ramets). We identify two main population groups, one in the northern Iberian Peninsula and the other in the Macaronesian archipelagos and southern Iberian Peninsula. Within each group, we found relict populations (in the Azores and the Cantabrian Cornice) as well as recent originated populations. This population structure suggests colonization dynamics in which recent populations originated from one or a few genets of relict populations and became established through intra-gametophytic self-fertilization and vegetative expansion. DAPC analysis facilitated the identification of alleles that most significantly contributed to the observed population structure. The current Andalusian populations appear to have originated from colonization events from the Azores and the Cantabrian Cornice. Our findings suggest that C. macrocarpa persisted through the Last Glacial Maximum in two refugia: the Azores and the Cantabrian Cornice. Colonization into new areas occurred presumably from these refuges, generating two large population groups with structured genetic diversity. This study underscores the significance of clonality in establishing new populations and shaping genetic structure.

Keywords: Culcita macrocarpa; breeding system; clonality; fern phylogeography; genetic diversity; glacial refugia; microsatellites; palaeotropical flora; plastid DNA; species distribution modeling.

Figure 1

Culcita macrocarpa. Individual from the Almoraima population in Cádiz province (Andalusia, Spain), exhibiting the subarborescent habit. Inset (upper right), detail of a frond with sori. Photos: Gabriel Blanca.

Figure 2

Map of the Culcita macrocarpa sampling locations. Frequency of each multilocus lineage (MLL) based on microsatellite data is represented as pie charts per location. Colors represent the different MLLs that are shared among sites, and yellow represents private MLLs that only appear in one site. See Table 1 for the full name of locations. Note that the pie chart size is the same for all locations and does not represent the number of individuals.

Figure 3

Estimated genetic structure based on microsatellite data using the Bayesian approach implemented in STRUCTURE v2.3.4. Histograms of individual assignments to clusters show the two most probable structures, K = 3 and K = 11, for all sampling units (ramet-based analyses), and K = 2 and K = 7 for analyses including only one individual per multilocus lineage per population (MLLs; genet-based analyses). The colors represent each of the genetic clusters identified. See Table 1 for full name of locations.

Figure 4

Result of the discriminant analysis of principal components (DAPC) using microsatellites. Scatterplot showing the first and second principal components. See Table 1 for full name of locations.

Figure 5

ptDNA information of the populations sampled. (A) Distribution of the ptDNA haplotypes (pie charts represent the frequency of each haplotype per location); (B) inferred ptDNA network, following the statistical parsimony method, with TCS software v1.21. Note that, in (A), the pie chart size is the same for all locations and does not represent the number of individuals. In (B), Roman numerals are the designation provided to haplotypes and circle sizes are proportional to haplotype frequencies. The color assigned to each haplotype is the same in (A,B). See Table 1 for full name of locations.

Figure 6

Potential distribution of Culcita macrocarpa drawn with MAXENT v3.4.1. (Top left), at the present time (Current); (top right), at the Last Interglacial (LIG, ca. 120,000 years BP); (bottom left), at the Last Glacial Maximum (LGM, ca. 21,000 years BP) using the Community Climate System Model (CCSM); (bottom right), prediction for the year 2080 under RCP 8.5 conditions.