Nuclear DNA contents, rDNAs, and karyotype evolution in Vicia subgenus Vicia: II. Section Peregrinae

Summary. Nuclear DNA contents, automated karyotype analyses, and sequences of rDNA spacers have been determined for the species of Vicia belonging to sect. Peregrinae, as well as for V. mollis. The phylogenetic data generated from the comparison of rDNA sequences and karyomorphological results would both indicate that Vicia mollis is a sister group to sect. Peregrinae. The relationships among the species belonging to the Peregrinae section and species enclosed in sections Faba, Narbonensis, and Bithynicae have been also investigated: a clade including V. mollis and sect. Peregrinae is a sister group to a clade including V. bithynica and sect. Narbonensis. With our choice of outgroup, Vicia faba (including subsp. paucijuga) is external to the above mentioned inclusive group. Correspondence and reprints: Dipartimento di Agrobiologia ed Agrochimica, Università della Tuscia, via San Camillo de Lellis, 01100 Viterbo, Italy.     

Nuclear DNA contents, rDNAs, and karyotype evolution in subgenus Vicia: III. The heterogeneous section Hypechusa

Summary. Nuclear DNA contents, automated karyotype analyses, and sequences of internal transcribed spacers from ribosomal genes have been determined in the species belonging to section Hypechusa of the subgenus Vicia. Karyomorphological results and phylogenetic data generated from the comparison of rDNA (genes coding for rRNA) sequences showed that sect. Hypechusa is not monophyletic; however, some monophyletic units are apparent (one including Vicia galeata, V. hyrcanica, V. noeana, and V. tigridis, another including V. assyriaca, V. hybrida, V. melanops, V. mollis, and V. sericocarpa), which partly correspond to morphology-based infrasectional groups. The relationships among these species and the species in sections Faba, Narbonensis, Bithynicae, and Peregrinae have been also investigated. Correspondence and reprints: Dipartimento di Agrobiologia ed Agrochimica, Università della Tuscia, via San Camillo de Lellis, 01100 Viterbo, Italy.     

Phylogenetic relationships betweenVicia faba (Fabaceae) and related species inferred from chloroplasttrnL sequences

The chloroplasttrnL intron from 46 differentVicia accessions, representing five of the nine sections of the genusVicia subg.Vicia sensuMaxted (1991a) were amplified by the Polymerase Chain Reaction (PCR) using oligonucleotide primers homologous to conserved regions intrnL. The products fell into two distinct groups; those of approximately 250 nt and those of around 450 nt in length. Of these, products from 17 differentVicia species were cloned and their nucleotide sequences determined. Multiple alignments were assembled and phylogenetic trees constructed by the weighted least-squares distance method. ALathyrus latifolius trnL intron sequence was used as an outgroup. The resulting trees clearly group and separate the sectt.Narbonensis, Bithynica andFaba species but were less able to distinguish species from sectt.Hypechusa andPeregrinae. Based on these sequence data,V. faba appears to be more distant from sect.Narbonensis than sectt.Hypechusa andPeregrinae. The results are in general agreement with a recent treatment ofVicia subg.Vicia (Maxted 1993) and lend further support to placingV. faba in the monospecific sect.Faba.     

Microarray-based survey of repetitive genomic sequences in Vicia spp.

A modified DNA microarray-based technique was devised for preliminary screening of short fragment genomic DNA libraries from three Vicia species (V. melanops, V. narbonensis, and V. sativa) to isolate representative highly abundant DNA sequences that show different distribution patterns among related legume species. The microarrays were sequentially hybridized with labeled genomic DNAs of thirteen Vicia and seven other Fabaceae species and scored for hybridization signals of individual clones. The clones were then assigned to one of the following groups characterized by hybridization to: (1) all tested species, (2) most of the Vicia and Pisum species, (3) only a few Vicia species, and (4) preferentially a single Vicia species. Several clones from each group, 65 in total, were sequenced. All Group I clones were identified as rDNA genes or fragments of chloroplast genome, whereas the majority of Group II clones showed significant homologies to retroelement sequences. Clones in Groups III and IV contained novel dispersed repeats with copy numbers 10²–10⁶/1C and two genus-specific tandem repeats. One of these belongs to the VicTR-B repeat family, and the other clone (S12) contains an amplified portion of the rDNA intergenic spacer. In situ hybridization using V. sativa metaphase chromosomes revealed the presence of the S12 sequences not only within rDNA genes, but also at several additional loci. The newly identified repeats, as well as the retroelement-like sequences, were characterized with respect to their abundance within individual genomes. Correlations between the repeat distributions and the current taxonomic classification of these species are discussed.     

Electrophoretic seed albumin patterns and species relationships inVicia sect.Faba (Fabaceae)

Electrophoretic analysis of seed albumins (PAGE) covered 173 accessions representing nine species ofVicia sect.Faba. The number of albumin bands recorded in particular species varied from three inV. eristaloides to 23 inV. faba; in total, 38 bands were distinguished in the investigated material. The examined species, exceptV. eristalioides, showed intraspecific variation with respect to the number and relative staining intensity of albumin bands; individual variation was especially marked inV. faba and inV. narbonensis. Hierarchical clustering of the investigated taxa was based onBhattacharyya distances calculated from the electrophoretic data. The taxa grouped in three main clusters.Vicia faba and the rather remotely relatedV. kalakhensis formed one cluster. The second cluster was composed ofV. narbonensis distantly related toV. hyaeniscyamus. The third cluster comprised three subgroups: 1.V. johannis, V. galilaea andV. serratifolia, 2.V. eristalioides, and 3.V. bithynica. The obtained results are discussed with reference to taxonomic relationships inVicia sect.Faba.     

Ribosomal DNA repeat unit polymorphism in 49 Vicia species

DNA restriction endonuclease fragment analysis was used to obtain new information on the genomic organization of Vicia ribosomal DNA (rDNA), more particularly among V. faba and its close relatives and the taxa within three (Narbonensis, Villosa, Sativa) species' complexes. Total genomic DNA of 90 accessions representing 49 Vicia species was restricted with 11 enzymes, and the restriction fragments were probed with three ribosomal clones. Twenty-eight repeat unit length classes were identified. The number of length classes (1–2) per accession did not correspond to the number of nucleolar organizing regions (NORs). The number of rRNA genes was independent of the 2C nuclear DNA amount present in the taxon. Each of the 90 accessions had 2 (rarely 1)-4 DraI sites. Those taxa with the same number of DraI sites generally could be distinguished from each other by different configurations. Probing of the DNA samples digested with tetranucleotide recognition restriction endonucleases emphasized differences between divergent spacer regions and enabled relative homologies between the coding regions to be established. Overall, rDNA restriction site variation among the species showed a good correlation with taxonomic classification. The rDNA analysis indicated evolutionary relatedness of the various taxa within the Narbonensis species complex. rDNA diversity within two other species complexes (Villosa, Sativa), on the other hand, was more extensive than expected. With few exceptions, data on the two complexes give evidence of taxon-specific divergences not seen with other approaches. The restriction site variability and repeat length heterogeneity in the rDNA repeat exhibited startling differences between V.faba and its close wild relatives included in the Narbonensis species complex. This analysis provides new evidence that none of the species within the complex can be considered to be putative allies of broad bean.     

Cytology of Vicia species. X. Karyotype evolution and phylogenetic implication in Vicia species of the sections Atossa, Microcarinae, Wiggersia and Vicia

Automated karyotype analyses and sequence of rDNA spacers have been analysed for the species belonging to sections Atossa, Microcarinae, Wiggersia and Vicia. Karyomorphological parameters, based on Rec, Syi and TF% indices, have been determined and evidenced that, in term of symmetry, the karyotype of Vicia lathyroides was the most asymmetric one. A multivariate analysis using 34 karyological parameters, in addition to the symmetry indices, has been carried out and the corresponding dendrogram of linkage distances showed six different groups. Molecular investigations on the inclusive group in study by employing ITS DNA sequences indicated a different pattern of relationships. The cladistic analysis combining the molecular data set with karyological parameters evidenced that the species of sections Vicia and Atossa join closely to each other in a paraphyletic group, which includes the monophyletic section Wiggersia. Therefore, our karyological and molecular data provide information about the phylogenetic position of the analysed species inside the subgenus Vicia and are discussed in relation to previous results obtained by morphology, isozymes and ribosomal genes analyses.     

Retrotransposon populations of <Emphasis Type="Italic">Vicia</Emphasis> species with varying genome size

The (non-LTR) LINE and Ty3-gypsy-type LTR retrotransposon populations of three Vicia species that differ in genome size (Vicia faba, Vicia melanops and Vicia sativa) have been characterised. In each species the LINE retrotransposons comprise a complex, very heterogeneous set of sequences, while the Ty3-gypsy elements are much more homogeneous. Copy numbers of all three retrotransposon groups (Ty1-copia, Ty3-gypsy and LINE) in these species have been estimated by random genomic sequencing and Southern hybridisation analysis. The Ty3-gypsy elements are extremely numerous in all species, accounting for 18–35% of their genomes. The Ty1-copia group elements are somewhat less abundant and LINE elements are present in still lower amounts. Collectively, 20–45% of the genomes of these three Vicia species are comprised of retrotransposons. These data show that the three retrotransposon groups have proliferated to different extents in members of the Vicia genus and high proliferation has been associated with homogenisation of the retrotransposon population.Electronic Supplementary Material Supplementary material is available for this article at .     

TheTy1-copia group retrotransposons inVicia species: copy number, sequence heterogeneity and chromosomal localisation

We present an in-depth study of theTy1-copia group of retrotransposons within the plant genusVicia, which contains species with widely differing genome sizes. We have compared the numbers and sequence heterogeneities of these genetic elements in three diploidVicia species chosen to represent large (V. faba, 1C=13.3 pg), medium (V. melanops, 1C=11.5 pg) and small (V. sativa, 1C=2.3 pg) genomes within the genus. The copy numbers of the retrotransposons are all high but vary greatly, withV. faba containing approximately 10⁶ copies,V. melanops about 1000 copies andV. sativa 5000 copies. The degree of sequence heterogeneity ofTy1-copia group elements correlates with their copy number within each genome, but neither heterogeneity nor copy number are related to the genome size of the host. In situ hybridization to metaphase chromosomes shows that the retrotransposons inV. faba are distributed throughout all chromosomes but are much less abundant in certain heterochromatic regions. These results are discussed in the context of plant retrotransposon evolution.     

The karyotype and 5S rRNA genes from Spanish individuals of the bat species <Emphasis Type="Italic">Rhinolophus</Emphasis><Emphasis Type="Italic">hipposideros</Emphasis> (Rhinolophidae; Chiroptera)

The karyotype of individuals of the species Rhinolophus hipposideros from Spain present a chromosome number of 2n = 54 (NFa = 62). The described karyotype for these specimens is very similar to another previously described in individual from Bulgaria. However, the presence of one additional pair of autosomal acrocentric chromosomes in the Bulgarian karyotype and the differences in X chromosome morphology indicated that we have described a new karyotype variant in this species. In addition, we have analyzed several clones of 1.4 and 1 kb of a PstI repeated DNA sequence from the genome of R. hipposideros. The repeated sequence included a region with high identity with the 5S rDNA genes and flanking regions, with no homology with GenBank sequences. Search for polymerase III regulatory elements demonstrated the presence of type I promoter elements (A-box, Intermediate Element and C-box) in the 5S rDNA region. In addition, upstream regulatory elements, as a D-box and Sp1 binding sequences, were present in flanking regions. All data indicated that the cloned repeated sequences are the functional rDNA genes from this species. Finally, FISH demonstrated the presence of rDNA in nine chromosome pairs, which is surprising as most mammals have only one carrier chromosome pair.     

The analysis of core and symbiotic genes of rhizobia nodulating Vicia from different continents reveals their common phylogenetic origin and suggests the distribution of Rhizobium leguminosarum strains together with Vicia seeds

In this work, we analysed the core and symbiotic genes of rhizobial strains isolated from Vicia sativa in three soils from the Northwest of Spain, and compared them with other Vicia endosymbionts isolated in other geographical locations. The analysis of rrs, recA and atpD genes and 16S–23S rRNA intergenic spacer showed that the Spanish strains nodulating V. sativa are phylogenetically close to those isolated from V. sativa and V. faba in different European, American and Asian countries forming a group related to Rhizobium leguminosarum. The analysis of the nodC gene of strains nodulating V. sativa and V. faba in different continents showed they belong to a phylogenetically compact group indicating that these legumes are restrictive hosts. The results of the nodC gene analysis allow the delineation of the biovar viciae showing a common phylogenetic origin of V. sativa and V. faba endosymbionts in several continents. Since these two legume species are indigenous from Europe, our results suggest a world distribution of strains from R. leguminosarum together with the V. sativa and V. faba seeds and a close coevolution among chromosome, symbiotic genes and legume host in this Rhizobium–Vicia symbiosis.     

Changes in DNA composition in the evolution of Vicia species

Summary The composition of nuclear DNA in 3 Vicia species are compared. The species V. eriocarpa, V. johannis and V. melanops are from three separate subgeneric sections of Vicia and show a fourfold variation in their amounts of nuclear DNA. DNA melting experiments, buoyant density gradient analysis and Cot reassociation experiments show that the quantitiative change in nuclear DNA between the three species is achieved by changes in the amounts of both repetitive and nonrepetitive DNA sequences. It is suggested that while the increase in the repetitive fraction is achieved by the proliferation of repetitive base sequences the increase in the nonrepetitive fraction is due to the steady accretion of highly diverged base sequences resulting from mutations, deletions, insertions and base sequence rearrangements among families of repetitive sequences.     

Electrophoretic seed albumin patterns inVicia species of sectt.Hypechusa andPeregrinae (Fabaceae)

This work is a continuation of electrophoretic investigations aimed at revealing a wild relative ofVicia faba. Electrophoretic analysis (PAGE) of seed albumins covered 52 accessions representing eightVicia species of sect.Hypechusa and two species of sect.Peregrinae. Most of the examined species showed an intraspecific variation due to differences between accessions and/or individual variation within accessions. In spite of the intraspecific variation, marked interspecific differences were recorded. However, none of the investigated species displayed electrophoretic seed albumin patterns similar to those reported earlier forV. faba. Contribution of the obtained results to characterization of the examined taxa is discussed.     

Phylogenetic analyses within three sections of the genus Vicia

The averaged genomic similarities based on multilocus randomly amplified polymorphic DNA (RAPD) were calculated for eight species representing three sections of the genus Vicia: faba, bithynica and narbonensis. The frequency of appearance of the sequences corresponding to 25 decamers selected at random from genomes of different Fabace species was checked, and a high correlation with the frequency observed for Vicia allowed us to assume their similar weight in typing Vicia species. The RAPD-based similarity coefficients compared with those related to whole genome hybridization with barley rDNA and those based on restriction fragment length polymorphism (RFLP) revealed similar interspecies relationships. The averaged RAPD-based similarity coefficient (Pearson’s) was 0.68 for all the species, and was sectionspecific: 0.43 (bithynica), 0.50 (faba) and 0.73 (narbonensis). The averaged similarity coefficient for V. serratifolia (0.63) placed it apart from the rest (0.75) of its section. The results correspond to the interspecies relationships built upon non-genetic data. The averaged similarity coefficient for particular RAPD was related to the presence and type of tandemly repeated motif in a primer: 0.7–0.8 for heterodimers (GC, AG, CA, GT, CT), 0.5–0.6 for homodimers (CC, GG) and 0.6 for no repeat, indicating the sensitivity of diversity range to the type of target sequences.     

Molecular phylogenetic relationships of nonpathogenic grass mycosymbionts and clavicipitaceous plant pathogens

Acremonium sect.Albo-lanosa (Fungi Imperfecti) includes beneficial, endophytic mycosymbionts of various grasses of the subfamilyPooideae, and also the anamorph of the grass choke pathogen,Epichloë typhina (Clavicipitaceae, Ascomycotina). These fungi are seed-disseminated, thus stably maintained for many host generations. To investigate the possibility of long-term coevolution, isolates ofE. typhina and anamorphs were obtained from eight grass species, sequences of their rRNA gene internal transcribed spacers were aligned with those from otherClavicipitaceae, and cladograms were generated by maximum parsimony. The results indicated that the nonpathogenic endophytes have not necessarily coevolved with their host species and that they arose fromE. typhina on multiple occasions.     

The identification of gibberellins in immature seeds of Vicia faba,and some chemotaxonomic considerations

GA₁₇, GA₁₉, GA₂₀, GA₂₉, GA₄₄ and 13-hydroxy-GA₁₂, now named GA₅₃, were identified by GC-MS in immature seeds of Vicia faba (broad bean). Also identified were a GA catabolite, two polyhydroxykauranoic acids, and abscisic, phaseic and dihydrophaseic acids. The GAs of Vicia are hydroxylated at C-13, in common with those of other legumes. However the GAs of Vicia are not hydroxylated at C-3, nor do they appear to be readily conjugated. In these respects Vicia resembles Pisum, another member of the tribe Viciae. Vicia differs from Phaseolus and Vigna, of the tribe Phaseoleae, in both these respects.Abbreviations ABA abscisic acid - DPA dihydrophaseic acid - GA_n gibberellin A_n - GC gas chromatography - GC-MS gas chromatography mass spectrometry - KA kauranoic acid - PA phaseic acid - TLC thin layer chromatography     

Isoenzyme variation in the wild relatives ofVicia faba (Fabaceae)

Electrophoretic analysis of five enzyme systems, LAP, PGI, SKDH, SOD and 6-PGDH, among 102Vicia accessions representingV. bithynica and seven species of theV. narbonensis complex, namelyV. eristalioides, V. kalakhensis, V. johannis, V. galilaea, V. serratifolia, V. narbonensis andV. hyaeniscyamus, has been performed. The recorded variation was tentatively assigned to 41 allelic genes at eight loci; intraspecific variation was observed in all species except forV. eristalioides. The results obtained were compared with the corresponding data reported earlier forV. faba. Hierarchical grouping of the investigated taxa, includingV. faba, was based onNei's genetic identities calculated from the allozyme frequency data.Vicia faba andV. bithynica were shown to be most distantly related to one another and to the remaining species investigated.Vicia serratifolia appeared to be a peripheral member of theV. narbonensis complex. The results are discussed with reference to genetic diversity and taxonomic relationships of the species under study.     

Molecular phylogeny of Russulaceae (Basidiomycetes; Russulales) inferred from the nucleotide sequences of nuclear large subunit rDNA

Phylogenetic relationships of the genera Russula and Lactarius were investigated using sequence data from the nuclear-encoded large subunit ribosomal DNA (LSU rDNA). Ninety-five sequences belonging to the genera Russula and Lactarius, including 31 sequences from the databases, were used in this study. Analysis of the LSU rDNA region indicated that Russulaceae was divided into six groups (group A–F) in the neighbor-joining (NJ) tree. Lactarius consisted of one large clade (group A). Therefore, this genus was found to be monophyletic. However, the monophyly of genus Russula remained unclear. The genus Russula consisted of five groups in the NJ tree. Group B includes sects. Plorantes and Archaeinae (Heim), and group C includes sects. Delicoarchaeae and Russula in the NJ tree. Neither of the two groups formed a single clade in the most parsimonius (MP) tree. Group D includes many taxa having colored spore prints and amyloid in suprahilar plage of spores in sect. Russula and sect. Rigidae. Group E consists of only sect. Compactae and is further divided into three subclades, represented by R. densifolia, R. nigricans, and R. subnigricans, respectively. Group F contains sects. Rigidae, Ingratae, and Pelliculariae. Sect. Compactae and sect. Plorantes should not be as closely related as previously supposed. Russula earlei may be placed in sect. Archaeinae Heim. Russula flavida (subsect. Amoeninae) is placed in sect. Russula with R. aurea with a high bootstrap value (99%). The nuclear LSU rDNA region is a useful tool in recognization of species of Russulaceae and may provide information concerning phylogenetic relationships between the genera Russula and Lactarius.     

Molecular systematics of the Old World Astragalus (Fabaceae) as inferred from nrDNA ITS sequence data

This study represents a nuclear rDNA ITS-based phylogenetic analyses of a greater sampling of the Old WorldAstragalus compared to our previous work (212 vs. 134 taxa). Phylogenetic relationships among 212 species (213 accessions) of the Old WorldAstragalus, including newly segregated monotypic genusPodlechiella, the two aneuploid New WorldAstragalus, and five related genera, were inferred from analyses of nuclear rDNA ITS sequences using maximum parsimony. A total of 658 nucleotide sites and four binary characters for indels were analyzed. The results of phylogenetic analyses suggest sect.Phyllolobium, comprising mostly the Chinese species, is placed outside of the so-calledAstragalus s. str. and is a well-supported monophyletic group. The monotypic annual segregate genusThlaspidium (≡Astragalus sect.Thlaspidium, A. thlaspi), is clearly nested withinAstragalus s. str. Among the many sections analyzed here, only sects.Cenanthrum, Caraganella, Eremophysa, Incani, Laxiflori, andLotidium are strongly supported as monophyletic. Our analysis, in agreement with previous studies, shows that the North American euploidAstragalus species are scattered throughout the Old World groups of the genus.     

Ribosomal DNA non-transcribed spacer polymorphism in subterranean mole rats: genetic differentiation,environmental correlates and phylogenetic relationships

Summary An analysis is presented of genetic differentiation in the non-transcribed spacers of ribosomal DNA (NTS rDNA). Diversity, environmental correlates and the phylogenetic relationships are examined within and between species of the actively speciating subterranean mole rat, superspeciesSpalax ehrenbergi (2n=52, 54, 58, 60) in Israel. This analysis is based on a previous study of the geographic distribution of restriction fragment length polymorphisms of NTS rDNA. Here we present results indicating that NTS rDNA diversity exists mostly (66%) within populations, while 20% is between populations within species, and 14% between species. Multivariate discriminant analysis succeeded in separating 10 of the 13 populations (77%) into their correct chromosomal species, on the basis of the combination of three NTS rDNA repetypes. The phylogenetic relationships suggest that the complex involves two pairs of closely related species (2n=52–54 and 2n=58–60). NTS rDNA diversity, as well as the decrease southward in frequency of repetype C, are correlated with climatic factors of humidity and temperature. These data are discussed in terms of the evolutionary forces of migration and selection which may cause NTS rDNA differentiation. Climatic selection appears to be the major differentiating factor of NTS rDNA.