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.

Identification of Candidate Genes Involved in the Determinism of Pollen Grain Aperture Morphology by Comparative Transcriptome Analysis in Papaveraceae.

In the last decade, certain genes involved in pollen aperture formation have been discovered. However, those involved in pollen aperture shape remain largely unknown. In Arabidopsis, the interaction during the tetrad development stage of one member of the ELMOD protein family, ELMOD_E, with two others, MCR/ELMOD_B and ELMOD_A, can change the morphology of apertures from colpus (elongated) to pore (round). Here, comparative transcriptome analysis is used to identify candidate genes involved in the determination of pollen aperture morphology in Papaveraceae (order Ranunculales). Furthermore, the role of ELMOD genes in the genetic determinism of aperture shape was tested by comparative analysis of their expression levels using RNA-seq data and RT-qPCR. Two pairs of species belonging to two different subfamilies were used. Within each pair, one species has colpate pollen and the other porate (Fumarioideae-Dactylicapnos torulosa, 6-colpate, and Fumaria bracteosa, pantoporate; Papaveroideae-Eschsholzia californica, 5-7 colpate, and Roemeria refracta, 6-porate). The transcriptomes were obtained at the tetrad stage of pollen development. A total of 531 DEGs were found between the colpate and porate pollen species groups. The results from RNA-seq and RT-qPCR indicate that pollen aperture shape is not determined by the relative expression levels of ELMOD family genes in Papaveraceae. However, genes related to callose wall formation or cytoskeleton organisation were found, these processes being involved in pollen aperture formation. In addition, transcriptomes from anthers with pollen during the tetrad stage of three species (D. torulosa, R. refracta, and F. bracteosa) were obtained for the first time. These data will be available for further studies in the field of floral evolution and development.

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.

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.

Comparative exine development from the post-tetrad stage in the early-divergent lineages of Ranunculales: the genera Euptelea and Pteridophyllum.

Studies of pollen wall development produce a great deal of morphological data that supplies useful information regarding taxonomy and systematics. We present the exine development of Euptelea and Pteridophyllum, two taxa whose pollen wall development has never previously been studied using transmission electron microscopy. Both genera are representatives of the two earliest-diverging families of the order Ranunculales and their pollen data are important for the diagnosis of the ancestral pollen features in eudicots. Our observations show these genera are defined by having microechinate microreticulate exine ornamentation, perforate tectum, columellate morphology of the infratectum and the existence of a foot layer and endexine. The presence of lamellations is detected during the early stages of development in the nexine of both genera, especially in the apertures. Euptelea presents remains of the primexine layer during the whole maturation process, a very thin foot layer, and a laminate exinous oncus in the apertural region formed by ectexine and endexine elements. Pteridophyllum has a thicker tectum than Euptelea, a continuous foot layer and a thicker endexine. In the apertures, the exinous oncus is formed by islets and granules of endexine, in contrast to the Euptelea apertures. The secretory tapetum produces orbicules in both genera, but they have different morphology and electron-density. Comparisons with pollen data from related orders and families confirm the ancestral states for the pollen of eudicots proposed in previous studies: reticulate and echinate surfaces, columellate infractectum and a thin foot layer relative to the thickness of the ectexine. According to our observations, we propose considering the possibility of a polymorphic state for the aperture number in the ancestor of Ranunculales, and suggest the development of orbicules as the ancestral state in this order.

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.

Evolutionary history of fumitories (subfamily Fumarioideae, Papaveraceae): An old story shaped by the main geological and climatic events in the Northern Hemisphere.

Fumitories (subfamily Fumarioideae, Papaveraceae) represent, by their wide mainly northern temperate distribution (also present in South Africa) a suitable plant group to use as a model system for studying biogeographical links between floristic regions of the Northern Hemisphere and also the Southern Hemisphere Cape region. However, the phylogeny of the entire Fumarioideae subfamily is not totally known. In this work, we infer a molecular phylogeny of Fumarioideae, which we use to interpret the biogeographical patterns in the subfamily and to establish biogeographical links between floristic regions, such as those suggested by its different inter- and intra-continental disjunctions. The tribe Hypecoeae is the sister group of tribe Fumarieae, this latter holding a basal grade of monotypic or few-species genera with bisymmetric flowers, and a core group, Core Fumarieae, of more specious rich genera with zygomorphic flowers. The biogeographical analysis shows a subfamily that originated in East Asia at the end of the Early Cretaceous. From here, ancestral range expansions followed three different directions, one at the beginning of the Late Cretaceous by the ancestor of tribe Hypecoeae towards central Asia, and two during the Cretaceous-Palaeogene transition towards western North America and Indochina by the ancestor of the tribe Fumarieae. The ancestor of Core Fumarieae expanded its range from East Asia into the Himalayas before to the middle Eocene. The uplifts of the Qinghai-Tibetan Plateau together with the zonal climate pattern of the Palaeogene are suggested to be responsible both for the accelerated diversification rate resulting in the origin of the basal lineages of Core Fumarieae as well as for the westward migration of the ancestor of Fumarieae s.str. into the Irano-Turanian region. From here, this latter group reached South Africa during late Eocene and Mediterranean basin during Oligocene. There were two colonization waves of the Mediterranean following two different routes: a northern route during the early Oligocene by the subtribe Sarcocapninae, probably facilitated by the land bridge resulting of the Mediterranean microplate accretion; and a southern route into North Africa, through the Gomphotherium land bridge, taken by the subtribe Fumariinae between late Oligocene and middle Miocene.

Multiple origins and chromosomal novelty in the allotetraploid Tragopogon castellanus (Asteraceae).

Tragopogon includes two classic examples of recently formed allopolyploid species in North America: T. mirus and T. miscellus. Older Tragopogon allotetraploids from Eurasia offer ideal taxa for comparing the longer term outcomes of allopolyploidy. To help resolve the ancestry of one of these older polyploids, phylogenetic analyses of multiple populations of the allotetraploid T. castellanus (2n = 24) and its putative diploid parents, T. crocifolius and T. lamottei, were conducted using sequences from nuclear (internal transcribed spacer, ITS; and alcohol dehydrogenase 1A, Adh) and plastid (trnT-trnL spacer, trnL intron, trnL-trnF spacer and rpl16 intron) loci. Fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) were used to investigate the chromosomal constitution of T. castellanus. Our data confirm that the widely distributed T. crocifolius and the Iberian endemic, T. lamottei, are the diploid parents of T. castellanus, and that this polyploid formed at least three times. One group of populations of T. castellanus is distinct in exhibiting two pairs of rearranged chromosomes. These data suggest that some of the chromosomal variants that originate in young polyploids (here, an intergenomic translocation) may become fixed in populations, contributing to novelty in older polyploid lineages. The geographical distributions of the allopolyploids and parents are also complex, with allotetraploid populations being disjunct from one or both of the most closely related diploid parental populations.

Satellite-DNA diversification and the evolution of major lineages in Cardueae (Carduoideae Asteraceae).

In a previous work, we characterized the HinfI satellite DNA family in the subtribe Centaureinae (Cardueae) demonstrating that a "library" of eight HinfI subfamilies would exist in the common ancestor of all Centaureinae, which were differentially amplified in different lineages. Now, we extend our study by analyzing a total of 219 additional repeats from fifteen species belonging to Carlininae, Echinopsinae and Carduinae, and comparing them to those of Centaureinae. Most HinfI sequences belonged to the subfamily II, although a few sequences of other subfamilies were detected in some species. Additionally, a new subfamily characteristic of several Carduinae species was discovered. Although phylogenetic trees grouped sequences by subfamily affinity instead of species provenance, when comparing repeats of the same subfamily, the degree of divergence between any pair of sequences was related to the evolutionary distance between the species compared in most cases. Exceptions were in comparisons between sequences of some Centaureinae species, and between sequences of some Carduinae species and those of Centaureinae. Our results demonstrate that: (1) At least nine HinfI subfamilies would exist in the common ancestor of Cardueae, each one differentially amplified in different lineages; (2) After differential spreading, sequences of each subfamily evolved concertedly through molecular drive, resulting in the gradual divergence of repeats between different species; (3) The rate to which concerted evolution occurred was different between lineages according to the evolutionary history of each one.

Phylogeny of the Centaurea group (Centaurea, Compositae) - geography is a better predictor than morphology.

The Centaurea group is part of the Circum-Mediterranean Clade (CMC) of genus Centaurea subgenus Centaurea, a mainly Mediterranean plant group with more than 200 described species. The group is traditionally split on morphological basis into three sections: Centaurea, Phalolepis and Willkommia. This division, however, is doubtful, especially in light of molecular approaches. In this study we try to resolve this phylogenetic problem and to consolidate the circumscription and delimitation of the entire group against other closely related groups. We analyzed nuclear (internal transcribed spacer of the ribosomal genes) and chloroplast (rpl32-trnL intergenic spacer) DNA regions for most of the described species of the Centaurea group using phylogenetic and network approaches, and we checked the data for recombination. Phylogeny was used to reconstruct the evolution of the lacerate-membranaceous bract appendages using parsimony. The magnitude of incomplete lineage sorting was tested estimating the effective population sizes. Molecular dating was performed using a Bayesian approach, and the ancestral area reconstruction was conducted using the Dispersal-Extinction-Cladogenesis method. Monophyly of the Centaurea group is confirmed if a few species are removed. Our results do not support the traditional sectional division. There is a high incongruence between the two markers and between genetic data and morphology. However, there is a clear relation between geography and the structure of the molecular data. Diversification in the Centaurea group mainly took place during the Pliocene and Pleistocene. The ancestral area infered for the Circum-Mediterranean Clade of Centaurea is the Eastern Mediterranean, whereas for the Centaurea group it is most likely NW-Africa. The large incongruencies, which hamper phylogenetic reconstruction, are probably the result of introgression, even though the presence of incomplete lineage sorting as an additional factor cannot be ruled out. Convergent evolution of morphological traits may have led to incongruence between morphology-based, traditional systematics and molecular results. Our results also cast major doubts about current species delimitation.

Differential spreading of HinfI satellite DNA variants during radiation in Centaureinae.

BACKGROUND AND AIMS: Subtribe Centaureinae appears to be an excellent model group in which to analyse satellite DNA and assess the influence that the biology and/or the evolution of different lineages have had on the evolution of this class of repetitive DNA. Phylogenetic analyses of Centaureinae support two main phases of radiation, leading to two major groups of genera of different ages. Furthermore, different modes of evolution are observed in different lineages, reflected by morphology and DNA sequences. METHODS: The sequences of 502 repeat units of the HinfI satellite DNA family from 38 species belonging to ten genera of Centaureinae were isolated and compared. A phylogenetic reconstruction was carried out by maximum likelihood and Bayesian inference. KEY RESULTS: Up to eight different HinfI subfamilies were found, based on the presence of a set of diagnostic positions given by a specific mutation shared by all the sequences of one group. Subfamilies V-VIII were mostly found in older genera (first phase of radiation in the subtribe, late Oligocene-Miocene), although some copies of these types of repeats were also found in some species of the derived genera. Subfamilies I-IV spread mostly in species of the derived clade (second phase of radiation, Pliocene to Pleistocene), although repeats of these subfamilies exist in older species. Phylogenetic trees did not group the repeats by taxonomic affinity, but sequences were grouped by subfamily provenance. Concerted evolution was observed in HinfI subfamilies spread in older genera, whereas no genetic differentiation was found between species, and several subfamilies even coexist within the same species, in recently radiated groups or in groups with a history of recurrent hybridization of lineages. CONCLUSIONS: The results suggest that the eight HinfI subfamilies were present in the common ancestor of Centaureinae and that each spread differentially in different genera during the two main phases of radiation following the library model of satellite DNA evolution. Additionally, differential speciation pathways gave rise to differential patterns of sequence evolution in different lineages. Thus, the evolutionary history of each group of Centaureinae is reflected in HinfI satellite DNA evolution. The data reinforce the value of satellite DNA sequences as markers of evolutionary processes.

Phylogeny of the tribe Fumarieae (Papaveraceae s.l.) based on chloroplast and nuclear DNA sequences: evolutionary and biogeographic implications.

PREMISE OF THE STUDY: Little research has been done at the molecular level on the tribe Fumarieae (Papaveraceae). Papaveraceae is a model plant group for studying evolutionary patterns despite the lack of a reference phylogeny for this tribe. We investigated the phylogenetic relationships within the tribe to complete the molecular data for this family in order to help understand its character evolution and biogeographic pattern. METHODS: We used maximum-parsimony and Bayesian approaches to analyze five DNA regions for 25 species representing 10 of the 11 Fumarieae genera and five outgroups. Evolutionary pathways of four characters (habit, life span, type of fruit, and number of seeds per fruit) were inferred on the phylogeny using parsimony. The ancestral distribution areas were reconstructed using dispersal-vicariance analysis. KEY RESULTS: Fumarieae is monophyletic and includes three groups that agree with the morphology-based subtribes: Discocapninae, Fumariinae, and Sarcocapninae. Within subtribes, the relationships among genera were different from those obtained with morphological data. Annual life span, nonchasmophytic habit, and a several-seeded capsule were the basal character states for the tribe. The ancestor occupied a continuous area between West Eurasia and Africa. Vicariances explain the divergence between lineages Discocapninae (South Africa) and Fumariinae-Sarcocapninae (Mediterranean), and the disjunction of Fumariinae (Mediterranean-Central Asia). CONCLUSIONS: Molecular phylogeny confirms the subtribal classification of Fumarieae based on morphology. However it provides different results regarding the relationships among genera within each subtribe, which affects the inference of the evolutionary pathway followed by the four selected characters. The disjunct distribution of the tribe is explained by different vicariance scenarios.

Concerted evolution of satellite DNA in Sarcocapnos: a matter of time.

SarkOne is a genus-specific satellite-DNA family, isolated from the genomes of the species of the genus Sarcocapnos. This satellite DNA is composed of repeats with a consensus length of 855 bp and a mean G+C content of 52.5%. We have sequenced a total of 189 SarkOne monomeric repeats belonging to a total of seven species of the genus Sarcocapnos. The comparative analysis of these sequences both at the intraspecific and the interspecific levels have revealed divergence patterns between species are proportional to between-species divergence according to the phylogeny of the genus. Our study demonstrates that the molecular drive leading to the concerted-evolution pattern of this satellite DNA is a time-dependent process by which new mutations are spreading through genomes and populations at a gradual pace. However, time is a limiting factor in the observation of concerted evolution in some pairwise comparisons. Thus, pairwise comparisons of species sharing a recent common ancestor did not reveal nucleotide sites in transitional stages higher than stage III according to the Strachan's model. By contrast, there was a gradation in the percentage of upper transition stages (IV, V, VI) the more phylogenetically distant the species were. In addition, closely related species shared a high number of polymorphic sites, but these types of sites were not common when comparing more distant species. All these data are discussed in the light of current life-cycle models of satellite-DNA evolution.

Study on Cortinarius subgenus Telamonia section Hydrocybe in Europe, with especial emphasis on Mediterranean taxa.

In this paper we have attempted to clarify the taxonomy and nomenclature of thirteen taxa of the genus Cortinarius subgenus Telamonia (sections Hydrocybe, Fraternii) well represented in the southwestern Mediterranean area of Europe (C. atrocoeruleus, C. bombycinus, C casimiri, C. contrarius, C. decipiens, C. fraternus, C. gallurae, C. hoffmannii, C. petroselineus, C. sertipes, C. subturibulosus, C. urdaibaiensis and C. vernus). To this end we have performed a combined study of morphological and molecular data (rDNA ITS sequences). The morphological analysis was carried out on 114 collections and the molecular analysis involved 31 of the 114 collections, including 11 type collections (types for C. casimiri and C. fraternus were not available). In addition, a study of spores under field emission scanning electron microscopy (FESEM) was conducted. The results of the combined analysis allowed us to asign the studied material to five species (C. casimiri s.l., C. decipiens s.l., C. gallurae, C. subturibulosus s.l. and C. vernus s.l.). Thus, all collections from more continental areas, which were originally identified as six different taxa (C. atrocoeruleus, C. contrarius, C. decipiens, C. fraternus, C. sertipes, C. flexipes fo. sertipes) corresponded to C. decipiens sensu lato, a widely distributed, genetically and morphologically variable species. Cortinarius casimiri is also found in such habitats, but it is confirmed as distinct taxon. Collections from Mediterranean sclerophyllous communities correspond to C. gallurae, C. vernus sensu lato and C. subturibulosus sensu lato. Due to close phylogenetic relationships we propose the new combinations C. casimiri var. hoffmannii (=C. decipiens var. hoffmannii non C. hoffmannii) and C. subturibulosus var. bombycinus (=C. bombycinus), and the new variety C. vernus var. nevadavernus (=C. vernus H. Lindstr. & Melot sensu auct.).

Eight polymorphic microsatellite markers in the Andalusian belladonna (Atropa baetica, Solanaceae).

We report on the isolation and characterization of eight microsatellite markers from enriched libraries for the critically endangered Atropa baetica. These are the first microsatellite loci reported for Atropa species. The total number of alleles found was 18, the expected heterozygosity ranged from 0.198 to 0.505. These markers will be useful to establish the real census of individuals and the genetic diversity both within and among the different populations of A. baetica.

Satellite-DNA evolutionary patterns under a complex evolutionary scenario: the case of Acrolophus subgroup (Centaurea L., Compositae) from the western Mediterranean.

Within the genus Centaurea (subtribe Centaureinae, tribe Cardueae, Compositae) hybridizations and reticulate-evolution phenomena have widely been recognized. This is especially true in the taxa included in the subgroup Acrolophus from the western Mediterranean area, in which recurrent hybridizations of parapatric ("microallopatric") lineages within the geographical range of a primary radiation have been suggested. The subgroup Acrolophus includes taxa from three sections (i.e. Acrolophus, Phalolepis and Willkommia), and, together with other subgroups, forms the named Jacea group (one of the three main groups into which Centaurea is divided). In this paper, we have studied the influence that the complex evolutionary scenario described for the Acrolophus subgroup from the western Mediterranean exerts on the evolutionary pattern of a satellite-DNA family, the HinfI family, which exists within the genomes of these taxa. To this end, we have analyzed the evolution of this satellite-DNA family in taxa from different taxonomic comparative levels: i) seven subspecies of the C. boissieri complex (one of which with two varieties) of the sect. Willkommia; ii) species of the sections Willkommia (10 species, 19 taxa), Acrolophus (two species), and Phalolepis (two species), all in the Acrolophus subgroup; iii) one external species to the Jacea group, C. granatensis from the group Acrocentron; iv) and species from other related genera from the Centaureinae subtribe (Phonus and Carthamus, both belonging to the Carthamus group). The influence of the suggested model for the origin and diversification of the Acrolophus subgroup is evidenced by the existence of three different HinfI satellite-DNA subfamilies coexisting in some genomes, and by the analysis that we have made by comparing site-by-site the transition stages in the process of concerted evolution between the sequences of the each subfamily. From this analysis, we can deduce that the HinfI repeated subfamilies evolved in a gradual manner, and that the different stages of concerted evolution fit quite well with the combined nuclear-chloroplast-DNA-deduced divergences and phylogeny of the subtribe Centaureinae. The HinfI satellite-DNA from the Carthamus species group (genera Carthamus and Phonus) and from the Acrocentron group (Centaurea granatensis) shows a high intraspecific conservation of the repeats, suggesting that the mechanisms producing concerted evolution have been efficient in these taxa. In addition, the comparison of individual nucleotide positions between related species shows a paucity in the spreading of variants in each subfamily with satellite-DNA divergence, an indication of a constant rate of homogenization of the repeated cluster. On the contrary, this trend is absent in the comparisons of the HinfI sequences from taxa of the subgroup Acrolophus. In this subgroup, we have found in this repetitive family similar representative average sequences for each taxon analyzed, polymorphic sites in each taxon being scant, most of them autapomorphic, representing early stages of genetic differentiation between taxa in the process of concerted evolution. The absence of concerted evolution was visualized by similar levels of intraspecific variation and interspecific divergence and by the lack of fixed species-diagnostic nucleotide sites. These facts might reflect the reticulate mode of evolution of Acrolophus.

Reticulate evolution in the Acrolophus subgroup (Centaurea L., Compositae) from the western Mediterranean: origin and diversification of section Willkommia Blanca.

Section Willkommia (Centaurea, Compositae) is endemic to the east-central portion of the Iberian Peninsula and northwestern Africa. The section has been included with sections Acrolophus and Phalolepis in the informal subgroup Acrolophus. We have used a molecular phylogenetic approach to test the hypothesis proposed by earlier authors that the diversification of section Willkommia involved a schizoendemic process from an ancestral syngameon. Comparative analysis of the transcribed spacer sequences of the nuclear ribosomal DNA (ITS and 3'ETS) regions reveals the presence of three different types of ribosomal sequences in the Acrolophus subgroup (referred to here as the Willkommia, Acrolophus-Phalolepis and Simulans ribotypes) which show a sectional-independent geographic structure. This evidence, together with the presence of additive polymorphic sites in the Willkommia sequences which agree with the geographic distribution of the taxa, suggests that members of section Willkommia and the western Mediterranean taxa of sections Acrolophus and Phalolepis fit a reticulate evolution model.