Marcela Rosato

Relationships of the Woody Medicago Species (Section Dendrotelis) Assessed by Molecular Cytogenetic Analyses

BACKGROUND AND AIMS The organization of rDNA genes in the woody medic species from the agronomically important Medicago section Dendrotelis was analysed to gain insight into their taxonomic relationships, to assess the levels of infraspecific variation concerning ribosomal loci in a restricted and fragmented insular species (M. citrina) and to assess the nature of its polyploidy. METHODS Fluorescence in situ hybridization (FISH) was used for physical mapping of 5S and 45S ribosomal DNA genes in the three species of section Dendrotelis (M. arborea, M. citrina, M. strasseri) and the related M. marina from section Medicago. Genomic in situ hybridization (GISH) was used to assess the genomic relationships of the polyploid M. citrina with the putatively related species from section Dendrotelis. KEY RESULTS The diploid (2n = 16) M. marina has a single 45S and two 5S rDNA loci, a pattern usually detected in previous studies of Medicago diploid species. However, polyploid species from section Dendrotelis depart from expectations. The tetraploid species (2n = 32) M. arborea and M. strasseri have one 45S rDNA locus and two 5S rDNA loci, whereas in the hexaploid (2n = 48) M. citrina four 45S rDNA and five 5S rDNA loci have been detected. No single chromosome of M. citrina was uniformly labelled after using genomic probes from M. arborea and M. strasseri. Instead, cross-hybridization signals in M. citrina were restricted to terminal chromosome arms and NOR regions. CONCLUSIONS FISH results support the close taxonomic interrelationship between M. arborea and M. strasseri. In these tetraploid species, NOR loci have experienced a diploidization event through physical loss of sequences, a cytogenetic feature so far not reported in other species of the genus. The high number of rDNA loci and GISH results support the specific status for the hexaploid M. citrina, and it is suggested that this species is not an autopolyploid derivative of M. arborea or M. strasseri. Further, molecular cytogenetic data do not suggest the hypothesis that M. arborea and M. strasseri were involved in the origin of M. citrina. FISH mapping can be used as an efficient tool to determine the genomic contribution of M. citrina in somatic hybrids with other medic species.

Amplification, contraction and genomic spread of a satellite DNA family (E180) in Medicago (Fabaceae) and allied genera

BACKGROUND AND AIMS Satellite DNA is a genomic component present in virtually all eukaryotic organisms. The turnover of highly repetitive satellite DNA is an important element in genome organization and evolution in plants. Here we assess the presence and physical distribution of the repetitive DNA E180 family in Medicago and allied genera. Our goals were to gain insight into the karyotype evolution of Medicago using satellite DNA markers, and to evaluate the taxonomic and phylogenetic signal of a satellite DNA family in a genus hypothesized to have a complex evolutionary history. METHODS Seventy accessions from Medicago, Trigonella, Melilotus and Trifolium were analysed by PCR to assess the presence of the repetitive E180 family, and fluorescence in situ hybridization (FISH) was used for physical mapping in somatic chromosomes. KEY RESULTS The E180 repeat unit was PCR-amplified in 37 of 40 taxa in Medicago, eight of 12 species of Trigonella, six of seven species of Melilotus and in two of 11 Trifolium species. Examination of the mitotic chromosomes revealed that only 13 Medicago and two Trigonella species showed FISH signals using the E180 probe. Stronger hybridization signals were observed in subtelomeric and interstitial loci than in the pericentromeric loci, suggesting this satellite family has a preferential genomic location. Not all 13 Medicago species that showed FISH localization of the E180 repeat were phylogenetically related. However, nine of these species belong to the phylogenetically derived clade including the M. sativa and M. arborea complexes. CONCLUSIONS The use of the E180 family as a phylogenetic marker in Medicago should be viewed with caution. Its amplification appears to have been produced through recurrent and independent evolutionary episodes in both annual and perennial Medicago species as well as in basal and derived clades.

Incomplete sequence homogenization in 45S rDNA multigene families: intermixed IGS heterogeneity within the single NOR locus of the polyploid species Medicago arborea (Fabaceae)

BACKGROUND AND AIMS Ribosomal sequences have become the classical example of the genomic homogenization of nuclear multigene families. Despite theoretical advantages and modelling predictions that support concerted evolution of the 45S rDNA, several reports have found intragenomic polymorphisms. However, the origins and causes of these rDNA polymorphisms are difficult to assess because seed plants show a wide range of 45S rDNA loci number variation, especially in polyploids. Medicago arborea is a tetraploid species that has a single 45S rDNA locus. This feature makes this species a suitable case study to assess the fate of ribosomal IGS homogenization in polyploid species showing nucleolus organizer region (NOR) reduction. METHODS The intergenic spacer (IGS) region was amplified by long PCR and the fragments were cloned and sequenced by a primer-walking strategy. The physical mapping of the whole and partial IGS variants was assessed by fluorescent in situ hybridization (FISH) and fibre-FISH methods on mitotic chromosomes and extended DNA fibres, respectively. KEY RESULTS Two IGS fragments of 4·8 and 3·5 kb were obtained showing structural features of functional sequences. The shorter variant appears to be a truncated copy of the 4·8 kb fragment that lacks the duplication of the transcription initiation site region and the entire D region. The physical localization of the two IGS variants on metaphase chromosomes and extended DNA fibres using FISH corroborated their joint presence within the same locus. In addition, no spatial structure of the two variants was detected within the NOR. CONCLUSIONS The results suggest that full sequence homogenization is not operating within the NOR locus of M. arborea. The structure of the NOR locus reported here departs from the models of IGS heterogeneity present in plants and caution against assuming the widespread belief that intragenomic ribosomal heterogeneity is mainly due to sequence variation between paralogous loci.

Karyological Observations in Medicago Section Dendrotelis (Fabaceae)

Medicago section Dendrotelis comprises polyploid, woody shrubs restricted to rocky and cliff faces in coastal places of the Mediterranean basin, showing a very close morphology. This has resulted in controversial taxonomic studies, differing in the circumscription and number of recognized species (from one to three). In the present study, cytogenetic studies (meiotic analysis, C-banding, Ag-NOR banding) were made in M. arborea, M. strasseri, and M. citrina to determine their karyological and evolutionary relationships. The observation of meiocytes revealed a regular meiotic cycle in all species. At pachytene stage, chromosomes showed constitutive heterochromatic blocks (knobs) that were species-specific, and clearly discriminate M. arborea and M. strasseri from M. citrina. Conventional karyotypes of M. strasseri and M. arborea were similar concerning chromosome classes, the apparent chromosome length, and the morphology of the single NOR chromosome pair. However, C-banding technique revealed karyotypic differences between both species. In contrast, M. citrina exhibited a slightly asymmetric karyotype, the presence of two pairs of chromosomes showing secondary constrictions, a similar apparent chromosome length, and two pairs of active rDNA loci. Its C-banded karyotype strikingly differed from those observed in M. arborea and M. strasseri, and only three chromosome pairs showed heterochromatic blocks. These cytogenetic data indicate a clear evolutionary split in woody medics (tetraploid vs hexaploid species), reflecting divergent patterns of karyological evolution. Taken together, all karyological data unequivocally support the recognition of M. citrina as a distinct species.

Conserved Organisation of 45S rDNA Sites and rDNA Gene Copy Number among Major Clades of Early Land Plants.

Genes encoding ribosomal RNA (rDNA) are universal key constituents of eukaryotic genomes, and the nuclear genome harbours hundreds to several thousand copies of each species. Knowledge about the number of rDNA loci and gene copy number provides information for comparative studies of organismal and molecular evolution at various phylogenetic levels. With the exception of seed plants, the range of 45S rDNA locus (encoding 18S, 5.8S and 26S rRNA) and gene copy number variation within key evolutionary plant groups is largely unknown. This is especially true for the three earliest land plant lineages Marchantiophyta (liverworts), Bryophyta (mosses), and Anthocerotophyta (hornworts). In this work, we report the extent of rDNA variation in early land plants, assessing the number of 45S rDNA loci and gene copy number in 106 species and 25 species, respectively, of mosses, liverworts and hornworts. Unexpectedly, the results show a narrow range of ribosomal locus variation (one or two 45S rDNA loci) and gene copies not present in vascular plant lineages, where a wide spectrum is recorded. Mutation analysis of whole genomic reads showed higher (3-fold) intragenomic heterogeneity of Marchantia polymorpha (Marchantiophyta) rDNA compared to Physcomitrella patens (Bryophyta) and two angiosperms (Arabidopsis thaliana and Nicotiana tomentosifomis) suggesting the presence of rDNA pseudogenes in its genome. No association between phylogenetic position, taxonomic adscription and the number of rDNA loci and gene copy number was found. Our results suggest a likely evolutionary rDNA stasis during land colonisation and diversification across 480 myr of bryophyte evolution. We hypothesise that strong selection forces may be acting against ribosomal gene locus amplification. Despite showing a predominant haploid phase and infrequent meiosis, overall rDNA homogeneity is not severely compromised in bryophytes.

Evolutionary site-number changes of ribosomal DNA loci during speciation: complex scenarios of ancestral and more recent polyploid events.

Several genome duplications have been identified in the evolution of seed plants, providing unique systems for studying karyological processes promoting diversification and speciation. Knowledge about the number of ribosomal DNA (rDNA) loci, together with their chromosomal distribution and structure, provides clues about organismal and molecular evolution at various phylogenetic levels. In this work, we aim to elucidate the evolutionary dynamics of karyological and rDNA site-number variation in all known taxa of subtribe Vellinae, showing a complex scenario of ancestral and more recent polyploid events. Specifically, we aim to infer the ancestral chromosome numbers and patterns of chromosome number variation, assess patterns of variation of both 45S and 5S rDNA families, trends in site-number change of rDNA loci within homoploid and polyploid series, and reconstruct the evolutionary history of rDNA site number using a phylogenetic hypothesis as a framework. The best-fitting model of chromosome number evolution with a high likelihood score suggests that the Vellinae core showing x = 17 chromosomes arose by duplication events from a recent x = 8 ancestor. Our survey suggests more complex patterns of polyploid evolution than previously noted for Vellinae. High polyploidization events (6x, 8x) arose independently in the basal clade Vella castrilensis-V. lucentina, where extant diploid species are unknown. Reconstruction of ancestral rDNA states in Vellinae supports the inference that the ancestral number of loci in the subtribe was two for each multigene family, suggesting that an overall tendency towards a net loss of 5S rDNA loci occurred during the splitting of Vellinae ancestors from the remaining Brassiceae lineages. A contrasting pattern for rDNA site change in both paleopolyploid and neopolyploid species was linked to diversification of Vellinae lineages. This suggests dynamic and independent changes in rDNA site number during speciation processes and a significant lack of correlation between 45S and 5S rDNA evolutionary pathways.

Partial Sequence Homogenization in the 5S Multigene Families May Generate Sequence Chimeras and Spurious Results in Phylogenetic Reconstructions

Multigene families have provided opportunities for evolutionary biologists to assess molecular evolution processes and phylogenetic reconstructions at deep and shallow systematic levels. However, the use of these markers is not free of technical and analytical challenges. Many evolutionary studies that used the nuclear 5S rDNA gene family rarely used contiguous 5S coding sequences due to the routine use of head-to-tail polymerase chain reaction primers that are anchored to the coding region. Moreover, the 5S coding sequences have been concatenated with independent, adjacent gene units in many studies, creating simulated chimeric genes as the raw data for evolutionary analysis. This practice is based on the tacitly assumed, but rarely tested, hypothesis that strict intra-locus concerted evolution processes are operating in 5S rDNA genes, without any empirical evidence as to whether it holds for the recovered data. The potential pitfalls of analysing the patterns of molecular evolution and reconstructing phylogenies based on these chimeric genes have not been assessed to date. Here, we compared the sequence integrity and phylogenetic behavior of entire versus concatenated 5S coding regions from a real data set obtained from closely related plant species (Medicago, Fabaceae). Our results suggest that within arrays sequence homogenization is partially operating in the 5S coding region, which is traditionally assumed to be highly conserved. Consequently, concatenating 5S genes increases haplotype diversity, generating novel chimeric genotypes that most likely do not exist within the genome. In addition, the patterns of gene evolution are distorted, leading to incorrect haplotype relationships in some evolutionary reconstructions.

High and uneven levels of 45S rDNA site-number variation across wild populations of a diploid plant genus (Anacyclus, Asteraceae)

The nuclear genome harbours hundreds to several thousand copies of ribosomal DNA. Despite their essential role in cellular ribogenesis few studies have addressed intrapopulation, interpopulation and interspecific levels of rDNA variability in wild plants. Some studies have assessed the extent of rDNA variation at the sequence and copy-number level with large sampling in several species. However, comparable studies on rDNA site number variation in plants, assessed with extensive hierarchical sampling at several levels (individuals, populations, species) are lacking. In exploring the possible causes for ribosomal loci dynamism, we have used the diploid genus Anacyclus (Asteraceae) as a suitable system to examine the evolution of ribosomal loci. To this end, the number and chromosomal position of 45S rDNA sites have been determined in 196 individuals from 47 populations in all Anacyclus species using FISH. The 45S rDNA site-number has been assessed in a significant sample of seed plants, which usually exhibit rather consistent features, except for polyploid plants. In contrast, the level of rDNA site-number variation detected in Anacyclus is outstanding in the context of angiosperms particularly regarding populations of the same species. The number of 45S rDNA sites ranged from four to 11, accounting for 14 karyological ribosomal phenotypes. Our results are not even across species and geographical areas, and show that there is no clear association between the number of 45S rDNA loci and the life cycle in Anacyclus. A single rDNA phenotype was detected in several species, but a more complex pattern that included intra-specific and intra-population polymorphisms was recorded in A. homogamos, A. clavatus and A. valentinus, three weedy species showing large and overlapping distribution ranges. It is likely that part of the cytogenetic changes and inferred dynamism found in these species have been triggered by genomic rearrangements resulting from contemporary hybridisation.

Inter- and intraspecific hypervariability in interstitial telomeric-like repeats (TTTAGGG)n in Anacyclus (Asteraceae)

Background and Aims Interstitial telomeric repeat (ITR) sites, consisting of tandem repeats of telomeric motifs localized at intrachromosomal sites, have been reported in a few unrelated organisms including plants. However, the causes for the occurrence of ITRs outside of the chromosomal termini are not fully understood. One possible explanation are the chromosomal rearrangements involving telomeric sites, which could also affect the location of other structural genome elements, such as the 45S rDNA. Taking advantage of the high dynamism in 45S rDNA loci previously found in Anacyclus (Asteraceae, Anthemideae), the occurrence and patterns of variation of ITRs were explored in this genus with the aim of finding common underlying causes. Methods In total, 132 individuals from 44 populations of nine species were analysed by fluorescence in situ hybridization using an Arabidopsis-type telomeric sequence as a probe. Key results Variable presence of ITR sites was detected in six out of nine species of Anacyclus, ranging from two to 45 sites and showing contrasting chromosomal locations and a differential presence of the ITR site on homologous chromosome pairs. At the intraspecific level, the ranges were as large as 0-12 ITR sites. Although only 26 % of the total observed ITR sites were localized in chromosomes bearing 45S rDNA loci, all cases of interstitial 45S rDNA reported in a previous work co-occurred with ITRs in close proximity in the same chromosome arms. Conclusions High levels of ITR polymorphism within a single species have not been previously reported in plants and suggest that this pattern might have been overlooked due to insufficient sampling. Although ancient Robertsonian translocations or the amplification of terminal 45S rDNA sites cannot, on their own, explain all of the levels of variability in ITRs reported here, there are suggestions that they may have been involved in the evolutionary history of this genus or its ancestors in Anthemideae.