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

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.

Phylogeographical Analyses of a Relict Fern of Palaeotropical Flora (Vandenboschia speciosa): Distribution and Diversity Model in Relation to the Geological and Climate Events of the Late Miocene and Early Pliocene.

Fern phylogeographic studies have mostly focused on the influence of the Pleistocene climate on fern distributions and the prevalence of long-distance dispersal. The effect of pre-Pleistocene events on the distributions of fern species is largely unexplored. Here, we elucidate a hypothetical scenario for the evolutionary history of Vandenboschia speciosa, hypothesised to be of Tertiary palaeotropical flora with a peculiar perennial gametophyte. We sequenced 40 populations across the species range in one plastid region and two variants of the nuclear gapCp gene and conducted time-calibrated phylogenetic, phylogeographical, and species distribution modelling analyses. Vandenboschia speciosa is an allopolyploid and had a Tertiary origin. Late Miocene aridification possibly caused the long persistence in independent refugia on the Eurosiberian Atlantic and Mediterranean coasts, with the independent evolution of gene pools resulting in two evolutionary units. The Cantabrian Cornice, a major refugium, could also be a secondary contact zone during Quaternary glacial cycles. Central European populations resulted from multiple post-glacial, long-distance dispersals. Vandenboschia speciosa reached Macaronesia during the Pliocene-Pleistocene, with a phylogeographical link between the Canary Islands, Madeira, and southern Iberia, and between the Azores and northwestern Europe. Our results support the idea that the geological and climate events of the Late Miocene/Early Pliocene shifted Tertiary fern distribution patterns in Europe.

Macaronesia Acts as a Museum of Genetic Diversity of Relict Ferns: The Case of Diplazium caudatum (Athyriaceae).

Macaronesia has been considered a refuge region of the formerly widespread subtropical lauroid flora that lived in Southern Europe during the Tertiary. The study of relict angiosperms has shown that Macaronesian relict taxa preserve genetic variation and revealed general patterns of colonization and dispersal. However, information on the conservation of genetic diversity and range dynamics rapidly diminishes when referring to pteridophytes, despite their dominance of the herbaceous stratum in the European tropical palaeoflora. Here we aim to elucidate the pattern of genetic diversity and phylogeography of Diplazium caudatum, a hypothesized species of the Tertiary Palaeotropical flora and currently with its populations restricted across Macaronesia and disjunctly in the Sierras de Algeciras (Andalusia, southern Iberian Peninsula). We analysed 12 populations across the species range using eight microsatellite loci, sequences of a region of plastid DNA, and carry out species-distribution modelling analyses. Our dating results confirm the Tertiary origin of this species. The Macaronesian archipelagos served as a refuge during at least the Quaternary glacial cycles, where populations of D. caudatum preserved higher levels of genetic variation than mainland populations. Our data suggest the disappearance of the species in the continent and the subsequent recolonization from Macaronesia. The results of the AMOVA analysis and the indices of clonal diversity and linkage disequilibrium suggest that D. caudatum is a species in which inter-gametophytic outcrossing predominates, and that in the Andalusian populations there was a shift in mating system toward increased inbreeding and/or clonality. The model that best explains the genetic diversity distribution pattern observed in Macaronesia is, the initial and recurrent colonization between islands and archipelagos and the relatively recent diversification of restricted area lineages, probably due to the decrease of favorable habitats and competition with lineages previously established. This study extends to ferns the concept of Macaronesia archipelagos as refugia for genetic variation.

The Role of INAPERTURATE POLLEN1 as a Pollen Aperture Factor Is Conserved in the Basal Eudicot Eschscholzia californica (Papaveraceae).

Pollen grains show an enormous variety of aperture systems. What genes are involved in the aperture formation pathway and how conserved this pathway is in angiosperms remains largely unknown. INAPERTURATE POLLEN1 (INP1) encodes a protein of unknown function, essential for aperture formation in Arabidopsis, rice and maize. Yet, because INP1 sequences are quite divergent, it is unclear if their function is conserved across angiosperms. Here, we conducted a functional study of the INP1 ortholog from the basal eudicot Eschscholzia californica (EcINP1) using expression analyses, virus-induced gene silencing, pollen germination assay, and transcriptomics. We found that EcINP1 expression peaks at the tetrad stage of pollen development, consistent with its role in aperture formation, which occurs at that stage, and showed, via gene silencing, that the role of INP1 as an important aperture factor extends to basal eudicots. Using germination assays, we demonstrated that, in Eschscholzia, apertures are dispensable for pollen germination. Our comparative transcriptome analysis of wild-type and silenced plants identified over 900 differentially expressed genes, many of them potential candidates for the aperture pathway. Our study substantiates the importance of INP1 homologs for aperture formation across angiosperms and opens up new avenues for functional studies of other aperture candidate genes.