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

Samira Ben-Menni Schuler was granted a predoctoral grant (F.P.U. program) from the Spanish Government. Hammadi Hamza was granted by a postdoctoral fellowship (Erasmus Mundus-Al Idrisi II scholarship) from the European Union. The authors thank all those people and institutions that facilitated or helped in the collection of samples (in alphabetical order: angel Banares, Antonio Delgado, Brother Anthony, Government of Ireland, Elizabeth Ojeda, Emer Ni Dhuill, Gobierno de Canarias, Ibai Olariaga-Ibarguren, Inaki Sanz-Azkue, Junta de Andalucia, Kristyna Hanuova, Miguel Perez-Gutierrez, Parque Nacional de Garajonay, Ranger Brian Duffy, Sito Chinea, Yves Krippel). We also thank Ana Garcia-Garcia for technical assistance in SDM analysis. This research was funded by the Regional Andalusian Government, grant number P10-RNM-6198.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.Spanish Government European Commission(Erasmus Mundus-Al Idrisi II scholarship) from the European Union - Regional Andalusian Government P10-RNM-619

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

This study was supported by the Spanish Ministry of Economy and Competitiveness (project CGL2015-70290-P to VS-S) and by the US National Science Foundation (MCB-1817835 to AD). IM-A was supported by a predoctoral grant (F.P.I. program) from the Spanish Government. BK was supported by the Ministry of Education, Youth and Sports of the Czech Republic (project LTC20050) and DH was supported by Czech Science Foundation (project 21-15856S).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 wildtype 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.Spanish Ministry of Economy and Competitiveness CGL2015-70290-PNational Science Foundation (NSF) MCB-1817835Spanish Government European CommissionMinistry of Education, Youth & Sports - Czech Republic LTC20050Grant Agency of the Czech Republic 21-15856

Development of polymorphic microsatellite markers for the Killarney Fern (Vandenboschia speciosa, Hymenophyllaceae)

Premise of the study: We characterize 10 microsatellite loci in the endangered fern Vandenboschia speciosa (Hymenophyllaceae), enabling studies on the genetic population structure of this Macaronesian-European species using DNA hypervariable markers. Methods and Results: Ten primer sets were developed and tested on 47 individuals in a total of two Iberian populations of V. speciosa. The primers amplified di- and hexanucelotide repeats. The number of alleles ranged from two to eight, and the expected heterozygosity ranged from 0.107 to 0.807 among the populations analyzed. Conclusions: The 10 microsatellite markers developed will be useful in characterizing the genetic diversity of V. speciosa and understanding its population structure (including the possible structure between sporophyte and gametophyte phases) and biogeographic history, and will provide important genetic data for the conservation of this species.This study was supported by the Regional Andalusian Government (project P10-RNM-6198). C.G.L. was funded by a postdoctoral grant from the Regional Andalusian Government. S.B.S. and L.T.C. were funded by a Beca-Iniciación a la Investigación grant from the Universidad de Granada during 2012–2013 and 2014–2015, respectively. I.M.A. was funded by a Beca-Colaboración grant (Spanish Government)

Influence of the Quaternary Glacial Cycles and the Mountains on the Reticulations in the Subsection Willkommia of the Genus Centaurea

Late Neogene and Quaternary climatic oscillations have greatly shaped the genetic structure of the Mediterranean Basin flora, with mountain plant species tracking warm interglacials/cold glacials by means of altitudinal shifts instead of broad latitudinal ones. Such dynamics may have enhanced population divergence but also secondary contacts. In this paper, we use a case example of subsection Willkommia of Centaurea (comprising three narrowly distributed endemic species, Centaurea gadorensis, C. pulvinata, and C. sagredoi) to test for reticulate evolution and recurrent hybridizations between nearby populations. For this, we combine analyses of genetic diversity and structuring, gene flow and spatial correlation, and ecological niche modeling. Our results support the contention that the current genetic structure of the three species is the result of historical gene flow at sites of secondary contact during the glacial periods, followed by isolation after the retraction of populations to the middle-upper areas of the mountains during the interglacial periods. The extent and direction of the gene flow was determined largely by the location of the populations on mountainsides oriented toward the same valley or toward different valleys, suggesting the intermountain valleys as the areas where secondary contacts occurred

INP1 involvement in pollen aperture formation is evolutionarily conserved and may require species-specific partners

Pollen wall exine is usually deposited non-uniformly on the pollen surface, with areas of low exine deposition corresponding to pollen apertures. Little is known about how apertures form, with the novel Arabidopsis INP1 (INAPERTURATE POLLEN1) protein currently being the only identified aperture factor. In developing pollen, INP1 localizes to three plasma membrane domains and underlies formation of three apertures. Although INP1 homologs are found across angiosperms, they lack strong sequence conservation. Thus, it has been unclear whether they also act as aperture factors and whether their sequence divergence contributes to interspecies differences in aperture patterns. To explore the functional conservation of INP1 homologs, we used mutant analysis in maize and tested whether homologs from several other species could function in Arabidopsis. Our data suggest that the INP1 involvement in aperture formation is evolutionarily conserved, despite the significant divergence of INP1 sequences and aperture patterns, but that additional species-specific factors are likely to be required to guide INP1 and to provide information for aperture patterning. To determine the regions in INP1 necessary for its localization and function, we used fragment fusions, domain swaps, and interspecific protein chimeras. We demonstrate that the central portion of the protein is particularly important for mediating the species-specific functionality.Funding was provided to AAD by the US National Science Foundation (MCB-1517511) and to VNSS by the Spanish Ministry of Economy and Competitiveness (CGL2015-70290-P). PL was supported by the China Scholarship Council. SB-MS was supported by the University of Granada, Spain (grant Cei BioTic). We thank the Arabidopsis Biological Resource Center (OSU) and the Maize Genetics Cooperation Stock Center (USDA/ ARS) for seed stocks, Priscila Rodriguez Garcia (OSU) for help with characterizing Arabidopsis–tomato INP1 chimeras, and Jay Hollick (OSU) for advice on all things maize

Genetic diversity and phylogeographic pattern of four Tertiary-relict fern species: Vandenboschia speciosa (Willd.) G. Kunkel, Diplazium caudatum (Cav.) Jermy, Pteris incompleta Cav. and Culcita macrocarpa C. Presl.

The authors thank Antonio Delgado and Dr. Gabriel Blanca for help in sample collection and assistance in the field. Special thanks are due to Dr. Carlos García- Verdugo and two anonymous reviewers for valuable comments on the manuscript. S.B.S. was funded by a predoctoral grant (F.P.U. program) from the Spanish Government.One of the main challenges of the evolutionary biology is to know the causes and processes that at level of species have caused the different patterns of diversity, distribution, and diversification of populations and gene lineages. The development and improvement of population genetics and phylogeographic analyses in relation to the advance in molecular biology techniques over the last decades has allowed these challenges to be overcome. However, there is still a large number of groups of organisms in which the phylogeographic and diversity pattern of many of its species has not been studied in depth, such as ferns and lycophytes. Fern and lycophyte species belong to ancestral lineages between the vascular plants, being the ferns the second most abundant group after angiosperms, with about 10,578 species. This doctoral thesis is developed with the objective of elucidating the patterns of genetic diversity and phylogeography of four species of Tertiary relict ferns: Culcita macrocarpa, Diplazium caudatum, Pteris incompleta, and Vandenboschia speciosa. These four ferns have a common history, having hypothetized their past belonging to the Paleotropical geoflora that populated the middle and lower latitudes of Eurasia and the western America from the late Cretaceous to the Upper Miocene. Currently, they share their distribution area and ecological niche, living toghether in most locations, in disjunct areas considered glacial refugia. Various molecular markers, of nuclear and/or chloroplast DNA origin, have been used to accomplish this objective.Uno de los principales desafíos de la biología evolutiva es conocer las causas y procesos que a nivel de especies han causado los diferentes patrones de diversidad, distribución y diversificación de poblaciones y linajes genéticos. El desarrollo y la mejora de la genética de poblaciones y los análisis filogeográficos en relación con el avance de las técnicas de biología molecular en las últimas décadas ha permitido superar estos desafíos. Sin embargo, todavía hay una gran cantidad de grupos de organismos en los que el patrón filogeográfico y de diversidad de muchas de sus especies no se ha estudiado en profundidad, como los helechos y los licófitos. Las especies de helechos y licófitos pertenecen a linajes ancestrales entre las plantas vasculares, siendo los helechos el segundo grupo más abundante después de las angiospermas, con aproximadamente 10.578 especies. Esta tesis doctoral se desarrolla con el objetivo de dilucidar los patrones de diversidad genética y filogeografía de cuatro especies de helechos relictos terciarios: Culcita macrocarpa, Diplazium caudatum, Pteris incompleta y Vandenboschia speciosa. Estos cuatro helechos tienen una historia común, habiendo hipotetizado su pasado perteneciente a la geoflora paleotropical que pobló las latitudes medias y bajas de Eurasia y el oeste de América desde el Cretácico tardío hasta el Mioceno superior. Actualmente, comparten su área de distribución y su nicho ecológico, viviendo juntos en la mayoría de los lugares, en áreas disyuntas consideradas refugios glaciales. Se han utilizado diversos marcadores moleculares, de ADN de origen nuclear y/o cloroplastidial, para lograr este objetivo.Tesis Univ. Granada.This study was supported by the Regional Andalusian Government (project P10-RNM- 6198)

Genetic diversity and phylogeographic pattern of four Tertiary-relict fern species: Vandenboschia speciosa (Willd.) G. Kunkel, Diplazium caudatum (Cav.) Jermy, Pteris incompleta Cav. and Culcita macrocarpa C. Presl.

The authors thank Antonio Delgado and Dr. Gabriel Blanca for help in sample collection and assistance in the field. Special thanks are due to Dr. Carlos García- Verdugo and two anonymous reviewers for valuable comments on the manuscript. S.B.S. was funded by a predoctoral grant (F.P.U. program) from the Spanish Government.One of the main challenges of the evolutionary biology is to know the causes and processes that at level of species have caused the different patterns of diversity, distribution, and diversification of populations and gene lineages. The development and improvement of population genetics and phylogeographic analyses in relation to the advance in molecular biology techniques over the last decades has allowed these challenges to be overcome. However, there is still a large number of groups of organisms in which the phylogeographic and diversity pattern of many of its species has not been studied in depth, such as ferns and lycophytes. Fern and lycophyte species belong to ancestral lineages between the vascular plants, being the ferns the second most abundant group after angiosperms, with about 10,578 species. This doctoral thesis is developed with the objective of elucidating the patterns of genetic diversity and phylogeography of four species of Tertiary relict ferns: Culcita macrocarpa, Diplazium caudatum, Pteris incompleta, and Vandenboschia speciosa. These four ferns have a common history, having hypothetized their past belonging to the Paleotropical geoflora that populated the middle and lower latitudes of Eurasia and the western America from the late Cretaceous to the Upper Miocene. Currently, they share their distribution area and ecological niche, living toghether in most locations, in disjunct areas considered glacial refugia. Various molecular markers, of nuclear and/or chloroplast DNA origin, have been used to accomplish this objective.Uno de los principales desafíos de la biología evolutiva es conocer las causas y procesos que a nivel de especies han causado los diferentes patrones de diversidad, distribución y diversificación de poblaciones y linajes genéticos. El desarrollo y la mejora de la genética de poblaciones y los análisis filogeográficos en relación con el avance de las técnicas de biología molecular en las últimas décadas ha permitido superar estos desafíos. Sin embargo, todavía hay una gran cantidad de grupos de organismos en los que el patrón filogeográfico y de diversidad de muchas de sus especies no se ha estudiado en profundidad, como los helechos y los licófitos. Las especies de helechos y licófitos pertenecen a linajes ancestrales entre las plantas vasculares, siendo los helechos el segundo grupo más abundante después de las angiospermas, con aproximadamente 10.578 especies. Esta tesis doctoral se desarrolla con el objetivo de dilucidar los patrones de diversidad genética y filogeografía de cuatro especies de helechos relictos terciarios: Culcita macrocarpa, Diplazium caudatum, Pteris incompleta y Vandenboschia speciosa. Estos cuatro helechos tienen una historia común, habiendo hipotetizado su pasado perteneciente a la geoflora paleotropical que pobló las latitudes medias y bajas de Eurasia y el oeste de América desde el Cretácico tardío hasta el Mioceno superior. Actualmente, comparten su área de distribución y su nicho ecológico, viviendo juntos en la mayoría de los lugares, en áreas disyuntas consideradas refugios glaciales. Se han utilizado diversos marcadores moleculares, de ADN de origen nuclear y/o cloroplastidial, para lograr este objetivo.Tesis Univ. Granada.This study was supported by the Regional Andalusian Government (project P10-RNM- 6198)

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

Macaronesia Acts as a Museum of Genetic Diversity of Relict Ferns: The Case of <i>Diplazium caudatum</i> (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