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Vandenboschia speciosa is an endangered tetraploid fern species with a large genome (10.5 Gb). Its geographical distribution is characterized by disjoined tertiary flora refuges, with relict populations that survived past climate crises. Here, we analyzed the transposable elements (TEs) and found that they comprise approximately 76% of the V. speciosa genome, thus being the most abundant type of DNA sequence in this gigantic genome. The V. speciosa genome is composed of 51% and 5.6% of Class I and Class II elements, respectively. LTR retrotransposons were the most abundant TEs in this species (at least 42% of the genome), followed by non-LTR retrotransposons, which constituted at least 8.7% of the genome of this species. We introduce an additional analysis to identify the nature of non-annotated elements (19% of the genome). A BLAST search of the non-annotated contigs against the V. speciosa TE database allowed for the identification of almost half of them, which were most likely diverged sequence variants of the annotated TEs. In general, the TE composition in V. speciosa resembles the TE composition in seed plants. In addition, repeat landscapes revealed three episodes of amplification for all TEs, most likely due to demographic changes associated with past climate crises.

This research was financed by the Spanish Ministerio de Economia y Competitividad and FEDER funds, grant: CGL20210-14856 (subprograma BOS). F.J. Ruiz-Ruano was supported by a Junta de Andalucia fellowship (Spain), a postdoctoral fellowship from Sven och Lilly Lawskis fond (Sweden), and a Marie Sklodowska-Curie Individual Fellowship grant agreement 875732 (EU). The Direccion General de Gestion del Medio Natural y Espacios Protegidos of the Consejeria de Medio Ambiente y Ordenacion del Territorio de la Junta de Andalucia authorized and facilitated the sampling of the material. We are highly indebted to Carmen Rodriguez Hiraldo and Jaime Perena Ortiz, who, together with the team of Agentes de Medio Ambiente of the Consejeria, helped us with the sampling procedure., Vandenboschia speciosa is an endangered tetraploid fern species with a large genome (10.5 Gb). Its geographical distribution is characterized by disjoined tertiary flora refuges, with relict populations that survived past climate crises. Here, we analyzed the transposable elements (TEs) and found that they comprise approximately 76% of the V. speciosa genome, thus being the most abundant type of DNA sequence in this gigantic genome. The V. speciosa genome is composed of 51% and 5.6% of Class I and Class II elements, respectively. LTR retrotransposons were the most abundant TEs in this species (at least 42% of the genome), followed by non-LTR retrotransposons, which constituted at least 8.7% of the genome of this species. We introduce an additional analysis to identify the nature of non-annotated elements (19% of the genome). A BLAST search of the non-annotated contigs against the V. speciosa TE database allowed for the identification of almost half of them, which were most likely diverged sequence variants of the annotated TEs. In general, the TE composition in V. speciosa resembles the TE composition in seed plants. In addition, repeat landscapes revealed three episodes of amplification for all TEs, most likely due to demographic changes associated with past climate crises., Spanish Ministerio de Economia y Competitividad and FEDER funds CGL20210-14856, Junta de Andalucia, Sven och Lilly Lawskis fond (Sweden), Marie Sklodowska-Curie Individual Fellowship 875732

We sequenced the sporophyte transcriptome of Killarney fern (Vandenboschia speciosa (Willd.) G. Kunkel). In addition to being a rare endangered Macaronesian-European endemism, this species has a huge genome (10.52 Gb) as well as particular biological features and extreme ecological requirements. These characteristics, together with the systematic position of ferns among vascular plants, make it of high interest for evolutionary, conservation and functional genomics studies. The transcriptome was constructed de novo and contained 36,430 transcripts, of which 17,706 had valid BLAST hits. A total of 19,539 transcripts showed at least one of the 7362 GO terms assigned to the transcriptome, whereas 6547 transcripts showed at least one of the 1359 KEGG assigned terms. A prospective analysis of functional annotation results provided relevant insights on genes involved in important functions such as growth and development as well as physiological adaptations. In this context, a catalogue of genes involved in the genetic control of plant development, during the vegetative to reproductive transition, in stress response as well as genes coding for transcription factors is given. Altogether, this study provides a first step towards understanding the gene expression of a significant fern species and the in silico functional and comparative analyses reported here provide important data and insights for further comparative evolutionary studies in ferns and land plants in general., Spanish Ministerio de Economía y Competitividad and FEDER founds, grant: CGL2010-14856 (subprograma BOS).

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-6198

espanolSe presenta informacion de diverso interes sobre seis helechos recolectados en Galicia [Adiantum capillus-veneris L., Hymenophyllum tunbrigense (L.) Sm., Isoetes fluitans M.I. Romero, Oeosporangium hispanicum (Mett.) Fraser-Jenk. & Pariyar, Vandenboschia speciosa (Willd.) G. Kunkel y Woodwardia radicans (L.) Sm.], la mayor parte de ellos amenazados y protegidos por diferentes normativas legales. EnglishInformation of interest on six ferns collected in Galicia [Adiantum capillus-veneris L., Hymenophyllum tunbrigense (L.) Sm., Isoetes fluitans M.I. Romero, Oeosporangium hispanicum (Mett.) Fraser-Jenk. & Pariyar, Vandenboschia speciosa (Willd.) G. Kunkel, and Woodwardia radicans (L.) Sm.] is provided, most of them endangered and protected by different legal regulations.

We report and discuss the results of a quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of the expression patterns of seven three amino acid loop extension (TALE) homeobox genes (four KNOTTED-like homeobox (KNOX) and three BEL1-like homeobox (BELL) genes) identified after next generation sequencing (NGS) and assembly of the sporophyte and gametophyte transcriptomes of the endangered fern species Vandenboschia speciosa. Among the four KNOX genes, two belonged to the KNOX1 class and the other two belonged to the KNOX2 class. Analysis of the deduced amino acid sequences supported the typical domain structure of both types of TALE proteins, and the homology to TALE proteins of mosses, lycophytes, and seed plant species. The expression analyses demonstrate that these homeodomain proteins appear to have a key role in the establishment and development of the gametophyte and sporophyte phases of V. speciosa lifecycle, as well as in the control of the transition between both phases. Vandenboschia speciosa VsKNAT3 (a KNOX2 class protein) as well as VsBELL4 and VsBELL10 proteins have higher expression levels during the sporophyte program. On the contrary, one V. speciosa KNOX1 protein (VsKNAT6) and one KNOX2 protein (VsKNAT4) seem important during the development of the gametophyte phase. TALE homeobox genes might be among the key regulators in the gametophyte-to-sporophyte developmental transition in regular populations that show alternation of generations, since some of the genes analyzed here (VsKNAT3, VsKNAT6, VsBELL4, and VsBELL6) are upregulated in a non-alternating population in which only independent gametophytes are found (they grow by vegetative reproduction outside of the range of sporophyte distribution). Thus, these four genes might trigger the vegetative propagation of the gametophyte and the repression of the sexual development in populations composed of independent gametophytes. This study represents a comprehensive identification and characterization of TALE homeobox genes in V. speciosa, and gives novel insights about the role of these genes in fern development., This research has been financed by the Spanish Ministerio de Economía y Competitividad and FEDER founds, grant: CGL2010-14856 (subprograma BOS)., M. Bakkali wishes to thank the Spanish Ministerio de Economía y Competitividad for granting him with a Ramón y Cajal Fellowship, the BFU2010-16438 grant, and an FPI studentship to R. Martín-Blázquez.

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.

Neste traballo avalíase a importancia botánica para a conservación da contorna da Fraga de Sto. Estevo do Ermo, situada no concello de Barreiros (Lugo, NW España). Dentro das 331 ha que comprende este espazo, identificáronse un total de total de 219 taxons de plantas vasculares, das que 19 (8,6% do total) son Pteridófitas. Seis taxóns, entre eles os fentos Dryopteris aemula, Vandenboschia speciosa e Woodwardia radicans, son especies protexidas cuxas poboacións locais están asociadas ás 15,2 ha que ocupan os 6 tipos de hábitat do Anexo I da DC 92/43/CEE (dous deles de carácter prioritario) recoñecidos na área de estudo. A pesar da reducida superficie que ocupan estes ambientes ecolóxicos, este enclave pode cualificarse como un lugar de gran relevancia para a conservación das pteridófitas a nivel rexional This study assesses the importance of the Fraga de Sto Estevo do Hermo and its surroundings (Barreiros council, Lugo, NW Spain) as a botanical conservation site. A total of 219 taxa of vascular plants were identified, 19 of them (8,6%) being Pteridophytes. Six species (Ilex aquifolium, Ruscus aculeatus, Narcissus triandrus, and the ferns Dryopteris aemula, Vandenboschia speciosa and Woodwardia radicans) are protected species. They were associated to the 6 habitat types of the Annex I of the CD 92/43EEC that exist in the area, which occupy only 15,2 out of the 331 ha of the whole study area. Despite the small area covered by these habitats, this site can be qualified as very important for pteridophyte conservation at the regional level SI

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).