Chromosome counts in the genus Festuca section Eskia (Poaceae) in the Iberian Peninsula

Chromosome numbers of taxa of Festuca L. section Eskia Willk. in the Iberian Peninsula are given. The levels of ploidy for five taxa are confirmed. Idiograms and karyotypic formulae of the five taxa are presented for the first time. Two levels of ploidy occur in this section: diploid and tetraploid. One taxon, Festuca elegans ssp. merinoi is tetraploid and two other taxa have diploid and tetraploid populations. The remaining two taxa are solely diploid.  © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society , 2004, 146 , 331–337.     

Karyosystematic study of Potentilla L. subgen. Potentilla (Rosaceae) in the Iberian Peninsula

Chromosome numbers in 80 populations belonging to 18 species of Potentilla L. subgen. Potentilla from the Iberian Peninsula and two of P. maura, a North African endemic taxon, have been counted. The basic number of chromosomes is always x = 7 and these chromosomes are small (between 1 and 2 μm). For three species, the number of chromosomes is reported for the first time and, for another six, this number has been established in Iberian representatives. Moreover, new ploidy levels have been obtained for P. hispanica and P. crantzii with regard to their entire distribution area, and in P. cinerea and P. neumanniana for the Iberian Peninsula. Some taxonomic, phylogenetic and phytogeographic comments are made for several species or groups of species from the West Mediterranean region. In 13 species only one ploidy level has been found, but six species have several ploidy levels. Seven ploidy levels occur in the investigated taxa. The frequency of each ploidy level represented within Iberian Potentilla is analysed and the data are compared with those available for taxa from the rest of the distribution area of the genus.     

The Polyploid Series of the Achillea millefolium Aggregate in the Iberian Peninsula Investigated Using Microsatellites

The Achillea millefolium aggregate is one of the most diverse polyploid complexes of the Northern hemisphere and has its western Eurasian boundary in the Iberian Peninsula. Four ploidy levels have been detected in A. millefolium, three of which have already been found in Iberia (diploid, hexaploid and octoploid), and a fourth (tetraploid) reported during the preparation of this paper. We collected a sample from 26 Iberian populations comprising all ploidy levels, and we used microsatellite markers analyzed as dominant in view of the high ploidy levels. Our goals were to quantify the genetic diversity of A. millefolium in the Iberian Peninsula, to elucidate its genetic structure, to investigate the differences in ploidy levels, and to analyse the dispersal of the species. The lack of spatial genetic structure recovered is linked to both high levels of gene flow between populations and to the fact that most genetic variability occurs within populations. This in turn suggests the existence of a huge panmictic yarrow population in the Iberian Peninsula. This is consistent with the assumption that recent colonization and rapid expansion occurred throughout this area. Likewise, the low levels of genetic variability recovered suggest that bottlenecks and/or founder events may have been involved in this process, and clonal reproduction may have played an important role in maintaining this genetic impoverishment. Indeed, the ecological and phenologic uniformity present in the A. millefolium agg. in Iberia compared to Eurasia and North America may be responsible for the low number of representatives of this complex of species present in the Iberian Peninsula. The low levels of genetic differentiation between ploidy levels recovered in our work suggest the absence of barriers between them.     

Flow cytometric and cytogenetic analyses of Iberian Peninsula Festuca spp.

Festuca L. has an important diversification centre in the Iberian Peninsula. We used chromosome counting, fluorescence (FISH) and genomic in situ hybridization (GISH), and DNA flow cytometry (FCM) to clarify the taxonomic position of several taxa, to search for phylogenetic relationships and to assess the extent and pattern of genome variation in fescues. The chromosome number of Festuca duriotagana var. barbata is determined for the first time and new ploidy level estimations are given for F. rothmaleri and F. summilusitana. In the latter species, besides the reported decaploid level, dodecaploidy was found in some populations, which points to the existence of an unrecognized taxon. Moreover, these differences were confirmed by FCM and a high positive correlation was found with the type of substrate where F. summilusitana was growing. For each section, a decrease of genome size with increase of polyploidy was observed. In general, in situ hybridization techniques failed to reveal phylogenetic relationships among the selected species. In FISH, a variation in the number of rDNA sites was observed in some species. GISH results indicate that F. henriquesii is not a progenitor of the studied polyploid species.     

Molecular characterisation and interpretation of genetic diversity within globally distributed germplasm collections of tall fescue (Festuca arundinacea Schreb.) and meadow fescue (F. pratensis Huds.)

Allohexaploid tall fescue (Festuca arundinacea Schreb. syn. Lolium arundinaceum [Schreb.] Darbysh.) is an agriculturally important grass cultivated for pasture and turf world-wide. Genetic improvement of tall fescue could benefit from the use of non-domesticated germplasm to diversify breeding populations through the incorporation of novel and superior allele content. However, such potential germplasm must first be characterised, as three major morphotypes (Continental, Mediterranean and rhizomatous) with varying degrees of hybrid interfertility are commonly described within this species. As hexaploid tall fescue is also a member of a polyploid species complex that contains tetraploid, octoploid and decaploid taxa, it is also possible that germplasm collections may have inadvertently sampled some of these sub-species. In this study, 1,040 accessions from the publicly available United States Department of Agriculture tall fescue and meadow fescue germplasm collections were investigated. Sequence of the chloroplast genome-located matK gene and the nuclear ribosomal DNA internal transcribed spacer (rDNA ITS) permitted attribution of accessions to the three previously known morphotypes and also revealed the presence of tall fescue sub-species of varying ploidy levels, as well as other closely related species. The majority of accessions were, however, identified as Continental hexaploid tall fescue. Analysis using 34 simple sequence repeat markers was able to further investigate the level of genetic diversity within each hexaploid tall fescue morphotype group. At least two genetically distinct sub-groups of Continental hexaploid tall fescue were identified which are probably associated with palaeogeographic range expansion of this morphotype. This work has comprehensively characterised a large and complex germplasm collection and has identified genetically diverse accessions which may potentially contribute valuable alleles at agronomic loci for tall fescue cultivar improvement programs.     

Genetic relationships within and among Iberian fescues (Festuca L.) based on PCR-amplified markers.

The genus Festuca comprises approximately 450 species and is widely distributed around the world. The Iberian Penninsula, with more than 100 taxa colonizing very diverse habitats, is one of its main centers of diversification. This study was conducted to assess molecular genetic variation and genetic relatedness among 91 populations of 31 taxa of Iberian fescues, based on several molecular markers (random amplified polymorphic DNA, amplified fragment length polymorphisms, and trnL sequences). The analyses showed the paraphyletic origin of the broad-leaved (subgenus Festuca, sections Scariosae and Subbulbosae, and subgenus Schedonorus) and the fine-leaved fescues (subgenus Festuca, sections Aulaxyper, Eskia, and Festuca). Schedonorus showed a weak relationship with Lolium rigidum and appeared to be the most recent of the broad-leaved clade. Section Eskia was the most ancient and Festuca the most recent of the fine-leaved clade. Festuca and Aulaxyper were the most related sections, in concordance with their taxonomic affinities. All taxa grouped into their sections, except F. ampla and F. capillifolia (section Festuca), which appeared to be more closely related to Aulaxyper and to a new independent section, respectively. Most populations clustered at the species level, but some subspecies and varieties mixed their populations. This study demonstrated the value in combining different molecular markers to uncover hidden genetic relationships between populations of Festuca.     

Decaploidy in Fragaria iturupensis (Rosaceae)

The strawberry genus, Fragaria (Rosaceae), has a base chromosome number of x = 7. Cultivated strawberries (F. ×ananassa nothosubsp. ananassa) are octoploid (2n = 8x = 56) and first hybridized from F. chiloensis subsp. chiloensis forma chiloensis × F. virginiana subsp. virginiana. Europe has no known native octoploid species, and only one Asian octoploid species has been reported: F. iturupensis, from Iturup Island. Our objective was to examine the chromosomes of F. iturupensis. Ploidy levels of wild strawberry species, include diploid (2n = 2x = 14), tetraploid (2n = 4x = 28), pentaploid (2n = 5x = 35), hexaploid (2n = 6x = 42), octoploid (2n = 8x = 56), and nonaploid (2n = 9x = 63). Artificial triploid (2n = 3x = 21), tetraploid, pentaploid, octoploid, decaploid (2n = 10x = 70), 16-ploid, and 32-ploid plants have been constructed and cultivated. Surprisingly, chromosome counts and flow cytometry revealed that F. iturupensis includes natural decaploid genotypes with 2n = 10x = 70 chromosomes. This report is the first of a naturally occurring decaploid strawberry species. Further research on F. iturupensis and exploration on northern Pacific islands is warranted to ascertain the phylogeny and development of American octoploid species.     

Phylogenetic position of Mediterranean Astereae and character evolution of daisies (Bellis,Asteraceae) inferred from nrDNA ITS sequences

Phylogenetic analyses of nrITS sequences of Asteraceae revealed that the Bellis group is a natural assemblage comprising all the species of Bellis and Bellium, but not Rhynchospermum. In contrast, we propose to include the genera Bellis, Bellium, and Bellidastrum in the subtribe Bellidinae in the interest of circumscribing natural groups. Our results also suggest an early diversification in the western Mediterranean Basin of two monophyletic lineages, Bellis and Bellium. Three major groups can be distinguished within BELLIS: (1) the B. perennis group, containing five annual and perennial species with three ploidy levels (diploid, octoploid, and decaploid), which are distributed throughout the Mediterranean Basin despite lack of pappus; (2) the Bellis sylvestris group, with five annual and perennial species primarily from the western Mediterranean, in which there are five ploidy levels (diploid, tetraploid, hexaploid, octoploid, and decaploid); and (3) a basal grade consisting of three diploid, perennial species which displays remarkable diversification of morphologies. Striking characteristics, such as an annual life form, polyploidy, and loss of pappus, seem to have occurred in parallel several times and in different geographical areas during the early diversification of Bellis species in the western Mediterranean. Character evolution reconstructions allow us to describe a putative ancestor of the genus Bellis (proto-Bellis).     

Hybridization in the genera Vulpia and Festuca (Poaceae): meiotic behaviour of artificial hybrids

The course of meiosis, including an analysis of chromosome configurations, is described for five diploid × diploid Vulpia crosses, five tetraploid × diploid Vulpia crosses, one hexaploid × diploid Festuca × Vulpia cross, one tetraploid × hexaploid Vulpia × Festuca cross, and one hexaploid × hexaploid Vulpia × Festuca cross. In most cases there was 97.5% or more pollen sterility, but two heptaploid plants obtained (presumably by non-reduction) from a hexaploid × diploid cross had about 60% stainable pollen. In the diploid hybrids pairing was quite extensive, and in V. ligustica × V. geniculata it was more or less as in the parent species (mode 7 bivalents, with regular separation). In the triploid hybrids the modal situation was 7 bivalents + 7 univalents, but evidence concerning the genomes which were pairing was equivocal. Evidence from the crosses at higher ploidy levels shows that both homogenetic and heterogenetic pairing does occur, although the relative amounts are uncertain. The results in general support the current classsification of Vulpia , except that they suggest the removal of V. alopecuros from section Loretia.     

Nuclear DNA content variation among Central European Koeleria taxa

BACKGROUND AND AIMS: Polyploidization plays an important role in the evolution of many plant genera, including Koeleria. The knowledge of ploidy, chromosome number and genome size may enable correct taxonomic treatment when other features are insufficient as in Koeleria. Therefore, these characteristics and their variability were determined for populations of six central European Koeleria taxa. METHODS: Chromosome number analysis was performed by squashing root meristems, and ploidy and 2C nuclear DNA content were estimated by flow cytometry. KEY RESULTS: Three diploids (K. glauca, K. macrantha var. macrantha and var. pseudoglauca), one tetraploid (K. macrantha var. majoriflora), one decaploid (K. pyramidata) and one dodecaploid (K. tristis) were found. The 2C nuclear DNA content of the diploids ranged from 4.85 to 5.20 pg. The 2C DNA contents of tetraploid, decaploid and dodecaploid taxa were 9.31 pg, 22.89 pg and 29.23 pg, respectively. The DNA content of polyploids within the K. macrantha aggregate (i.e. within K. macrantha and K. pyramidata) was smaller than the expected multiple of the diploid genome (K. macrantha var. macrantha). Geography-correlated variation of DNA content was found for some taxa. Czech populations of K. macrantha var. majoriflora had a 5.06% smaller genome than the Slovak ones. An isolated eastern Slovakian population of K. tristis revealed 8.04% less DNA than populations from central Slovakia. In central and north-west Bohemia, diploid and tetraploid cytotypes of K. macrantha were sympatric; east from this region diploid populations, and towards the west tetraploid populations were dominant. CONCLUSIONS: Remarkable intra-specific inter-population differences in nuclear DNA content were found between Bohemian and Pannonian populations of Koeleria macrantha var. majoriflora and between geographically isolated central and eastern Slovakian populations of K. tristis. These differences occur over a relatively small geographical scale.     

Geographical Distribution of Diploid and Tetraploid Cytotypes of Thymus sect. Mastichina (Lamiaceae) in the Iberian Peninsula, Genome Size and Evolutionary Implications

The occurrence of diploid and tetraploid cytotypes in Thymus section Mastichina (Lamiaceae) was investigated by chromosome counting and flow cytometric measurements of DNA content. To cover the entire native distribution of the section across the Iberian Peninsula, plant material was sampled from 29 populations, representing all three taxa (Thymus albicans, T. mastichina subsp. mastichina and T. mastichina subsp. donyanae). We provide first estimates of genome size for taxa of this section. Analyses revealed the existence of new cytotypes in T. mastichina subsp. mastichina (2n?=?2x?=?28, 30) and confirmed the existence of previous ones (2n?=?4x?=?56, 58, 60). We also confirmed the presence of exclusively diploid cytotypes (2n?=?2x?=?30) in the southwestern Iberian endemics T. albicans and T. mastichina subsp. donyanae. We conclude that the southwestern Iberian Peninsula acted as a Pleistocene glacial refugium that may have permitted speciation processes within this section. Several hypotheses concerning these processes and the origin and distribution of the studied taxa are discussed.     

One plus one makes seven: Intricate phylogeographic patterns in Odontites vernus (Orobanchaceae: Rhinantheae) in the Iberian Peninsula

The Odontites vernus group is the most widespread of the genus Odontites, occupying the temperate regions of Eurasia and northern Morocco. The group contains three species, all inhabiting the Iberian Peninsula, where O. vernus s.l. (sensu lato) exhibits remarkable morphological variability and includes diploid and tetraploid individuals corresponding to the two subspecies that occur there. We collected 301 individuals from 100 sampling sites covering the entire distribution of O. vernus in the Iberian Peninsula and genotyped them using 12 simple sequence repeat (SSR) markers. Their ploidy level was estimated by flow cytometry, and two cpDNA regions (rps16 intron and trnK-rps16) were sequenced. We found 129 diploids and 172 tetraploids distributed following a mosaic parapatry model, while only two mixed-ploidy populations were discovered. The 20 haplotypes found fit two well-defined haplogroups, to some extent correlated with estimated ploidy levels. The frequencies of the SSR alleles shared by both cytotypes, as well as those of the private alleles corresponding to the tetraploid cytotype, indicate that tetraploids likely originated at least twice through autopolyploidy. Additionally, the results from SSR markers were structured in a higher number of groups than did the cpDNA sequences. Thus, the genetic distance analysis detected four groups, but the Bayesian analysis of population structure identified seven, with only low levels of gene flow detected among groups. The distributions of the seven genetic groups coincide with well-known refugium areas within the Iberian Peninsula during the climatic oscillations of the Quaternary. Thus, the results give additional support to the “refugia within refugia” hypothesis.     

Contributions to the karyological study of the genus Ranunculus L. subgenus Batrachium (DC.) A Gray from the Iberian Peninsula

DIOSDADO. J. C, PASTOR, J. E. & VALDÉS, B. 1993. Contributions to the karyological study of the genus Ranunculus L. subgenus Batrachium (DC.) A. Gray from the Iberian Peninsula. The somatic and gametic chromosome numbers and detailed chromosome morphology are presented for eight taxa of aquatic ranunculi from the Iberian Peninsula. Within the subgenus Batrachium diploid and tetraploid levels have been observed (2 n = 16, 32) the commoner of which is the diploid. From the karyological data relationships have been established between the subgenus Batrachium and subgenus Ranunculus.     

Hybridisation in the genera Vulpia and Festuca: the production of artificial F1 plants

The results of hybridisation experiments involving 24 strains of diploid, tetraploid and hexaploid Vulpia , representing nine species in three sections, and 22 strains of hexaploid and octoploid Festuca rubra agg., are presented, and the taxonomic implications discussed. 7848 pollinations produced 741 hybrid caryopses, from which 137 mature hybrid plants were raised (1.75% success rate) by embryo culture. Festuca x Festuca crosses were relatively easily achieved, and Festuca x Vulpia crosses were as successful as Vulpia x Vulpia crosses, indicating a close relationship between these two genera. Hybrids with F. rubra agg. were raised from females of species from all three sections of Vulpia. Within Vulpia , the most successful crosses involved V. geniculata, V. ligustica, V. fasciculata and V. pyramidata , whereas V. alopecuros was the least successful parent of all, producing only one mature hybrid from 2595 pollinations.     

Systemic fungal endophytes and ploidy level in Festuca vivipara populations in North European Islands

Exploring the regional pattern of variation in traits driven by symbiotic interactions may provide insights to understand the evolutionary processes that operate over plant populations. Polyploidy, which is associated with fitness improvement, is expected to increase with latitude and altitude. However, it has never been explored in relation with the occurrence of epichloid fungal endophytes in plants. Both, variation in ploidy level and in the incidence of fungal endophytes, are known to occur in species of fine fescues. Here, we surveyed the occurrence of systemic fungal endophytes in natural Festuca vivipara populations in North European islands. In addition, we identified the fungal species associated with this grass and determined the predominant ploidy level for each population. Endophytes were found in four of six, two of three, and one of three populations for Faroe Islands, Iceland and Great Britain, respectively. With an average low incidence level of 15 % in infected populations, there was no relationship between infection level and either latitude or altitude. The phylogenetic analysis based on sequences ITS and the tub2 genes, supports that the endophytic species is Epichloë festucae, the same as in other fine fescues. We found no variation in ploidy level as all the plants were tetraploid (4X) with 28 chromosomes, a pattern which contrasts with the variation reported in previous antecedents. Our results suggest that apart from low and variable benefits of the endophyte to the plants, there would be a complex dynamics between epichloid endophytes and species of the fine fescue complex which merits further studies.     

Phylogeny and evolution of the genus Barbus in the Iberian Peninsula as revealed by allozyme electrophoresis

Variation of 14 enzyme systems encoded by 31 presumptive loci in different barbel species was studied using starch gel electrophoresis. Eighteen samples representing 11 Barbus tetraploid taxa were taken, including 10 populations from the Iberian Peninsula, six from other parts of western and southern Europe, one from northern Africa and one diploid species as outgroup from Asia Minor. The genetic analysis reassessed of the taxonomic status of the Iberian barbels into two distinct species groups. The first group included B. bocagei, B. comiza, B. graellsii, B. gulraonis, B. microcephalus , and B. sclateri , that aligned with B. callensis from northern Africa and with B. apoensis from Asia Minor; the other group included B. haasi and B. merldlonahs that was related to the European species, B. barbus, B. plebejus and B. peloponnesius . These groups are probably not monophyletic. It is suggested that the isolation of the Iberian Peninsula from Europe since the Oligocene-Miocene may explain the genetic affinities of the Iberian barbels with those of North African rather than with the European group.     

Novelties in the Festuca valesiaca group (Poaceae) from the central Alps

Due to their ploidy levels, their morphological and anatomical characters and their distribution the central alpine Festuca stricta subsp. bauzanina (2n = 8x = 56) and Festuca ovina var. guinochetii (2n = 10x = 70), both belonging to the Festuca valesiaca group, are raised to the specific level. As a new subspecies Festuca bauzanina subsp. rhaetica is described here. The taxa of the polyploid Festuca valesiaca group are morphologically, anatomically and chorologically characterised and an identification key for these taxa in the central Alps is presented.