Genomic relationships among diploid and polyploid species of the genus Eryngium L. using genomic in situ hybridization

Eryngium L. (Umbelliferae) is a large genus including more than 250 species worldwide. The large morphological variability in this genus makes it difficult to delimit the species or to establish phylogenetic relationships. The occurrence of different ploidy levels within the genus might indicate a hybrid origin of the polyploid species. In the present study, the chromosome number and karyotype of E. regnellii are reportedfor the first time and the ploidy level of a population of E. paniculatum is confirmed. We compare the genomes of the diploids E. horridum and E. eburneum, the tetraploids E. megapotamicum and E. regnellii, and the hexaploids E. pandanifolium (as a representative of the whole pandanifolium complex) and E. paniculatum using genomic in situ hybridization (GISH). Although it was not possible to identify the parental species of the polyploid taxa analyzed, the GISH technique allowed us to postulate some hypotheses about their origin. Eryngium horridum and E. eburneum do not seem to be the direct progenitors of the polyploids analyzed. On the other hand, it seems that other diploid species unrelated to E. horridum and E. eburneum are involved in their origin. Our results are consistent with morphological and phylogenetic studies, indicating a close relationship between the species of the series Latifolia.     

Genetic variation of Eryngium campestre L. (Apiaceae) in Central Europe

In Germany, Eryngium campestre is restricted to dry habitats along the rivers Rhine and Elbe and to a few areas in Central Germany. This distribution pattern is usually regarded as a typical pattern of postglacial immigration. In the present study, we investigated whether these two geographically distinct distribution areas are genetically differentiated and whether conclusions can be drawn regarding colonization history. To analyse the phylogeographic structure of E. campestre in Central Europe, 278 individuals from 29 populations within Germany and from further reference populations within Europe were analysed. We applied amplified fragment length polymorphisms to examine their genetic relatedness. Our analyses revealed three groups: a Mediterranean group additionally including two Rhine populations; a Rhine–Main group which further includes the westernmost population from the central German dry area; and one group which includes all eastern populations. Our results show that the two geographically distinct areas are genetically differentiated. As genetic diversity within the Elbe populations is very low, we conclude that this area, which was strongly affected through the late glacial maximum, was colonized relatively recently. High genetic diversity in the Rhine populations indicates a contact zone where lineages of different origin met. This would imply that today's patterns of genetic variation were caused through glacial range contractions and expansions. The present study is one of the first studies that deal with the postglacial distribution pattern of a dry grassland plant species in Central Europe and the results suggest that a survival of E. campestre at least during the Dryas cold stage might be possible.     

Low selfing in a mass-flowering, endangered perennial, Eryngium alpinum L. (Apiaceae)

We investigated the reproductive ecology of an endangered alpine species, Eryngium alpinum L., to determine its selfing rate and to propose possible mechanisms that may shape its breeding system. Whereas pollinators' foraging behavior suggested a high potential for geitonogamy (70% of the flights occur within plants), microsatellite analyses of seed progenies demonstrated that plants are primarily outcrossing (outcrossing rate [tm] = 0.65, 0.96, and 1 in three populations). Given the relatively long pollen viability (at least 4-5 d) and the high number of simultaneously opened flowers on each plant, protandry is not sufficient to eliminate selfing. Second, controlled crosses demonstrated not only auto-fertility, but also partial self-incompatibility. Partial self-incompatibility is probably due to the competitive advantage of cross vs. self-pollen, and, together with protandry, could lead the species to selfing as a reproductive assurance. These results are encouraging for the maintenance of large populations. However, higher selfing was observed in a small population that could suffer inbreeding depression, as observed on experimentally selfed seeds. Thus, these populations should be carefully monitored. Finally, this study shows how molecular markers and field experiments may complement each other in our reaching a global understanding of mating patterns.     

Further studies on polyploid amphibians (Ceratophrydidae)

The comparative DNA values were measured in three species of South American frogs belonging to the family Ceratophrydidae: Odontophrynus cultripes (2n=22), O. americanus (2n=44) and Ceratophrys dorsata (2n=104). Nuclei of erythrocytes, liver, kidney, pancreas and testis were used for measurements. The results obtained confirmed polyploid evolution in the family Ceratophrydidae. The relative DNA values of these three species conformed to the expected 124 ratio. — In general, the proportional increase in nuclear volume was observed in corresponding tissues of tetraploid and octoploid species.Supported by a grant (GM-14577-01) from National Institute of General Medical Sciences — U.S. Public Health Service.Supported by grants from CNPq, UFMG and Rockefeller Foundation.     

Reproductive ecology of the endangered Alpine species Eryngium alpinum L. (Apiaceae): phenology, gene dispersal and reproductive success

BACKGROUND AND AIMS: Eryngium alpinum (Apiaceae) is an endangered perennial, characteristic of the Alpine flora. Because the breeding system influences both demographic (reproductive success) and genetic (inbreeding depression, evolutionary potential) parameters that are crucial for population maintenance, the reproductive ecology of E. alpinum was investigated. Specifically, the aims of the study were (1) to determine the factors (resources and/or pollen) limiting plant fitness; and (2) to assess the potential for gene flow within a plant, within a patch of plants, and across a whole valley where the species is abundant. METHODS: Field experiments were performed at two sites in the Fournel valley, France, over three consecutive years. Studies included a phenological survey, observations of pollinators (visitation rates and flight distances), dispersal of a fluorescent powder used as a pollen analogue, the use of seed traps, determination of the pollen/ovule ratio, and an experiment to test whether seed production is limited by pollen and/or by resources. KEY RESULTS: E. alpinum is pollinated by generalist pollinators, visitation rates are very high and seed set is resource- rather than pollen-limited. The short flights of honeybees indicate a high potential for geitonogamy, and low pollen and seed dispersals suggest strong genetic structure over short distances. These results are interpreted in the light of previous molecular markers studies, which, in contrast, showed complete outcrossing and high genetic homogeneity. CONCLUSION:S. The study highlights the usefulness of adopting several complementary approaches to understanding the dynamic processes at work in natural populations, and the conservation implications for E. alpinum are emphasized. Although the studied populations do not seem threatened in the near future, long-term monitoring appears necessary to assess the impact of habitat fragmentation. Moreover, this study provides useful baseline data for future investigations in smaller and more isolated populations.     

Further studies on polyploid amphibians (Ceratophrydidawe)

Odontophrynus cultripesReinhardt and Lutken, 1862 has 22 chromosomes in its diploid complement. Spermatocyte I contained 11 ring bivalents and metaphase II exhibited 11 chromosomes. Odontophrynus americanus (Duméril and Bibron) 1882 has 44 chromosomes in somatic as well as germ cells, these can be sorted into 11 groups of homologues. Metaphase I showed varying numbers of quadrivalents and metaphase II exhibited 22 dyads. Ceratophrys dorsataWied., 1824 has 104 chromosomes in somatic and germ cells; these 104 chromosomes comprise 8 each of 13 kinds of homologues. The spermatocyte I contained ring octovalents and other multivalents, and metaphase II 52 chromosomes. The above findings indicate that evolution by polyploidization occurred in South American frogs belonging to the family Ceratophrydidae.This work was supported by a grant (GM-14577-01) from the National Institute of General Medical Sciences U. S. Public Health Service.     

Further studies on polyploid amphibians (Ceratophrydidae)

Meiosis and sex-ratio of artificial interspecific triploid hybrids (3n=33) produced by mating of Odontophrynus cultripes males (2n=22) with O.americanus females (4n=44) are described. — Metaphases I from the triploids show pairing of homologues in configurations of trivalents, bivalents and univalents. Metaphases II revealed a variation of chromosome numbers in the range of 11 to 22. Buploid gametes with 11, 22 and 33 dyads were also found. The latter (3n) arose by pre-meiotic duplication of the chromosomes and/or by absence of meiotic reduction. — Sex-ratio of 11 males to females in one sample of the triploid interspecific hybrids was obtained.This work was supported by U. S. Public Health Service grant GM-14577-03 from the National Institute of General Medical Sciences, by the Fundo de Pesquisas de Instituto Butantan and by the Conselho Nacional de Pesquisas.     

Evidence of interpopulation variation in the germination of Eryngium maritimum L. (Apiaceae)

Plant Ecology - Germination is considered as one of the most crucial steps in a plant’s lifetime. The germination of Eryngium maritimum, a dune species located across all European coasts, has...     

Phylogeography of the endangered Eryngium alpinum L. (Apiaceae) in the European Alps

We studied the phylogeography of Eryngium alpinum by sequencing two intergenic chloroplast spacers, trnH-psbA and trnS-trnG (1322 bp). The sampling design included 36 populations and 397 individuals spanning the entire distribution range of the species, from France to Bosnia. Twenty-one haplotypes were characterized and polymorphism was observed both within and among populations. Population differentiation was strong (F(ST) = 0.92) and largely explained by the distinction of five geographic regions: Southwestern, Western, Middle, Eastern Alps and Balkans (F(CT) = 0.62). Moreover, N(ST) was significantly higher than G(ST) (P < 0.05), showing the existence of a phylogeographic pattern. Six major lineages were recognized using samova and median-joining networks. One lineage, highly divergent from the other ones, was only found in the Balkans and probably persisted in situ during last glaciations. All other lineages might have survived in a Southwestern refugium (Mercantour) and colonized the entire Alpine arc (Southwestern, Western, Middle and Eastern Alps) through repeated colonization events at different time periods. This is the first empirical study suggesting Southern refugia for calcareous Alpine plants, although the existence of a secondary refugium in northern Italy/Austria is also suspected. We also observed recent haplotype diversification, especially in the Southwestern Alps.     

Karyotype analysis for diploid and polyploid species of the Solanum L.

Solanum species were cytogenetically analysed to localize species-specific markers. Solanum atropurpureum Schrank, S. dulcamara L., S. gilo Raddi., S. melongena L., S. nitidibaccatum Bitter and S. paniculatum L. showed 2n = 24, whereas S. luteum Mill., S. nigrum L. and S. laciniatum Ait. showed 2n = 48, 2n = 72 and 2n = 92, respectively. All species demonstrated a symmetrical karyotype. The average chromosome size varied between diploid (1.95 μm) and polyploid (1.31 μm) species. Application of the CMA₃/DAPI technique showed two CMA₃ ⁺/DAPI^? terminal blocks in S. dulcamara, S. atropurpureum and S. luteum, indicating one homologous pair. In S. nitidibaccatum two large CMA₃ ⁺/DAPI^? segments were observed apart from several terminal blocks in almost all chromosomes. The same CMA₃ ⁺ telomeric standard was also found in prometaphases of S. luteum. Similar results were obtained with FISH with 45S rDNA probes: fluorochrome CMA₃ ⁺ telomeric blocks associated with satellites, with two blocks in S. atropurpureum, S. dulcamara, S. nitidibaccatum and S. luteum and four blocks in S. nigrum and S. lacinatum. Localization of species-specific markers was successful, allowing recognition of particular cytological features in most of the species analysed.     

Isolation and characterization of microsatellites in a perennial Apiaceae,Eryngium alpinum L.

In order to investigate the genetic structure in an endangered Alpine plant (Eryngium alpinum L.), we developed microsatellites. Two different approaches were used: an enrichment protocol and the classical technique of hybridization on nylon membranes. We identified 25 loci, 13 of which revealed to be polymorphic. The polymorphism was rather low (2–6 alleles; H_E = 0.49 ± 0.16), probably due to the short size of microsatellites (6–10 dinucleotide repeats) and to the fine spatial scale investigated. However, these markers are expected to provide a new insight about the genetic processes at work within and among E. alpinum populations.     

Genetic diversity and differentiation in Eryngium alpinum L. (Apiaceae): comparison of AFLP and microsatellite markers

Genetic diversity and structure of 12 populations of Eryngium alpinum L. were investigated using 63 dominant amplified fragment length polymorphism (AFLP) and seven codominant microsatellite (48 alleles) markers. Within-population diversity estimates obtained with both markers were not correlated, but the microsatellite-based fixation index Fis was correlated with both AFLP diversity indices (number of polymorphic bands and Nei's expected heterozygosity). Only AFLP diversity indices increased with the size of populations, although they did not significantly differ among them (Kruskall-Wallis test). The discrepancy between AFLPs and microsatellites may be explained by a better coverage of the genome with numerous AFLPs, the higher mutation rates of microsatellites or the absence of significant difference among within-population diversity estimates. Genetic differentiation was higher with AFLPs (theta=0.40) than with microsatellites (theta=0.23), probably due to the higher polymorphism of microsatellites. Thus, we considered global qualitative patterns rather than absolute estimates to compare the performance of both types of markers. On a large geographic scale, the Mantel test and multivariate analysis showed that genetic patterns were more congruent with the spatial arrangement of populations when inferred from microsatellites than from AFLPs, suggesting higher homoplasy of AFLP markers. On a small spatial scale, AFLPs managed to discriminate individuals from neighboring populations whereas microsatellites did not (multivariate analysis), and the percentage of individuals correctly assigned to their population of origin was higher with AFLPs than with microsatellites. However, dominant AFLPs cannot be used to study heterozygosity-related topics. Thus, distinct molecular markers should be used depending on the biological question and the geographical scale investigated.     

Cytophotometric studies on the DNA content in diploid and polyploid enchytraeidae (Oligochaeta)

The amount of DNA in nerve nuclei was determined by Feulgen cytophotometry in 45 species and cytotypes. The DNA value of spermatids, spermatogonial nuclei and nerve nuclei in newly hatched worms was determined in some species. The total variation in DNA content ranges from a relative value of 0.40 to 4.85. The DNA content in diploid species ranges from 0.40 to 1.12. In two genera (Lumbricillus and Enchytraeus) wide diversification in chromosome numbers at the diploid level is accompanied by wide variation in DNA values, whereas other genera and species groups (Mesenchytraeus, Henlea and Marionina), which are conservative in this respect, are closer in DNA values. In the genus Fridericia constancy in chromosome number is associated with wide variation in DNA values, this may be due to an increase in DNA taking place in old individuals. In 9 out of 10 cases nearly identical ratios are found between chromosome numbers and DNA values in polyploids and related diploids or lower polyploids. Differential polyteny is recorded in two cases.Dedicated to Professor Dr. J. Seiler on the occasion of his eightieth birthday.     

Lignans,phenylpropanoids and polyacetylenes from Chaerophyllum aureum L. (Apiaceae)

Sub-aerial parts of Chaerophyllum aureum L. yielded two polyacetylenes, falcarinol (1), falcarindiol (2), three lignans, namely nemerosin (3), deoxypodorhizone (4), deoxypodo-phyllotoxin (5), two phenylpropanoids, 1'-hydroxymyristicin (6) and its angeloyl ester (7). Compounds 6 and 7 were isolated for the first time from plant material and their structures were elucidated by means of extensive 1- and 2-dimensional NMR spectroscopy and high resolution mass spectrometry. In bioautographic tests on TLC plates the dichloromethane extract showed a significant antimicrobial activity. Falcarindiol was identified as the main active principle whereas the phenylpropanoids and lignans showed no activity.     

A newFerulago (Apiaceae) species from Turkey

Ferulago antiochia Saya & Miski is newly described: it is known only from the type locality in Hatay province and has affinities toF. trachycarpa Boiss. of sect.Anisotaenia.     

Genetic diversity in an endangered alpine plant, Eryngium alpinum L. (Apiaceae), inferred from amplified fragment length polymorphism markers

Eryngium alpinum L. is an endangered species found across the European Alps. In order to obtain base-line data for the conservation of this species, we investigated levels of genetic diversity within and among 14 populations from the French Alps. We used the amplified fragment length polymorphism (AFLP) technique with three primer pairs and scored a total of 62 unambiguous, polymorphic markers in 327 individuals. Because AFLP markers are dominant, within-population genetic structure (e.g. FIS) could not be assessed. Analyses based either on the assumption of random-mating or on complete selfing lead to very similar results. Diversity levels within populations were relatively high (mean Nei's expected heterozygosity = 0.198; mean Shannon index = 0.283), and a positive correlation was detected between both genetic diversity measurements and population size (Spearman rank correlation: P = 0. 005 and P = 0.002, respectively). Moreover, FST values and exact tests of differentiation revealed high differentiation among populations (mean pairwise FST = 0.40), which appeared to be independent of geographical distance (nonsignificant Mantel test). Founder events during postglacial colonizations and/or bottlenecks are proposed to explain this high but random genetic differentiation. By contrast, we detected a pattern of isolation by distance within populations and valleys. Predominant local gene flow by pollen or seed is probably responsible for this pattern. Concerning the management of E. alpinum, the high genetic differentiation leads us to recommend the conservation of a maximum number of populations. This study demonstrates that AFLP markers enable a quick and reliable assessment of intraspecific genetic variability in conservation genetics.     

Cytogenetic and phylogenetic studies of diploid and polyploid members of tribe Anemoninae (Ranunculaceae)

The ancestry, phylogenetic differentiation and systematic classification of the worldwide-distributed genus Anemone have been debated for many years. In this paper 11 Anemone, three Pulsatilla species and Hepatica nobilis were subjected to detailed karyotype analysis with the aim of obtaining new cytogenetic data that will contribute to karyotype evolutionary studies of the tribe Anemoninae. The results are interpreted in a phylogenetic context, established from the intergenic nontranscribed spacer (NTS) of 5S rDNA and internal transcribed spacer (ITS) of 35S rDNA. One to three 35S and one to three 5S rDNA loci are present in diploid and polyploid taxa. The 35S rDNA loci are located terminally on the short arm of acrocentric chromosomes, while for 5S rDNA there is no preferential chromosomal position as it exhibits terminal, subterminal, interstitial or pericentromeric positions, and is located either on acrocentric or metacentric chromosomes. The karyotype of hexaploid A. baldensis (2n = 6x = 48) is presented for the first time, and A. sylvestris is proposed as one of its putative parental species. Chromosome fusion/translocation is proposed as the key mechanism involved in reduction of the basic chromosome number from 8 in the Anemone subgenus to 7 in the Anemonidium subgenus. The cytogenetic data obtained are mainly supported by ITS and NTS phylogeny. Diversification of the genus Anemone was accompanied by a large reduction of heterochromatin, from the Mediterranean anemones that have large amounts of heterochromatin to the New World anemones without any detectable heterochromatic blocks.     

Four new species of Eryngium (Umbelliferae) from Mexico

Four new monocotyloid Eryngia,E. ferrisiae, E. venustum, E. mexiae, andE. tzeltal from Mexico, are described, illustrated, and contrasted with related taxa.     

A new species of Lomatium (Apiaceae) from Utah

A new species ofLomatium belonging to theCynomarathrum group is described and its relationships to other species in the group are indicated.Lomatium junceum appears to be restricted to Emery Co., Utah, on barren clay and shale slopes in the San Rafael Swell and adjacent eastern base of the Wasatch Plateau.     

Cytological studies on diploid and polyploid taxa of the genus Pennisetum Rich

The cytology of thirteen taxa and two hybrids in the genus Pennisetum indicated the distribution of the taxa among the four basic chromosome numbers 5, 7, 8 and 9. The diploid nature of P. ramosum and P. typhoides and the genomic allotetraploid status of P. purpureum was further confirmed. P. massaicum (2n=32), P. orientale (2n=36) and P. subangustum (2n=36) suggested probable autotetraploid nature and the three hexaploids (2n=54) investigated (P. polystachyon, P. longistylis and P. squamulatum) revealed allohexaploid constitution. The natural triploid, P. ruppellii (2n=27) and pentaploid P. villosum (2n=45) were found to be apomicts and they were allotriploid and allopentaploid respectively. The tetraploid-hexaploid complex of P. pedicellatum showed them to be cytotypes only. The cytogenetical behaviour of the hybrid, P. typhoides x P. purpureum with 2n=21 and the trispecies hybrid (P. typhoides x P. purpureum) x P. squamulatum with 2n=48 brought out the homology within the genomes of x=7 and x=9 and also between the genomes with x=7 and x=9. The significance of the inter- and intragenomic chromosome pairing had been brought out from the interspecific hybrids and the natural allotriploid and allopentaploid species having one genome in the haploid condition.The morphological sections of this genus did not correspond with the cytological groups. A high degree of evolutionary specialization was evident in species of the section Gymnothrix. A complete series of polyploids and high degree of heterogeneity from the morphological point of view was brought out in species with x=9.