Genome size and karyotype evolution in the slipper orchids (Cypripedioideae: Orchidaceae)

Nuclear DNA contents (4C) were estimated by Feulgen microdensitometry in 27 species of slipper orchids. These data and recent information concerning the molecular systematics of Cypripedioideae allow an interesting re-evaluation of karyotype and genome size variation among slipper orchids in a phylogenetic context. DNA amounts differed 5.7-fold, from 24.4 pg in Phragmipedium longifolium to 138.1 pg in Paphiopedilum wardii. The most derived clades of the conduplicate-leaved slipper orchids have undergone a radical process of genome fragmentation that is most parsimoniously explained by Robertsonian changes involving centric fission. This process seems to have occurred independently of genome size variation. However, it may reflect environmental or selective pressures favoring higher numbers of linkage groups in the karyotype.     

Physical mapping of 35S rRNA genes and genome size variation in polyploid series of Sisyrinchium micranthum and S. rosulatum (Iridaceae: Iridoideae)

Sisyrinchium micranthum and S. rosulatum are part of a species complex in which S. micranthum displays considerable morphological variation. S. rosulatum is a tetraploid species, whereas S. micranthum plants may present three different ploidy levels (2x, 4x, and 6x), so that polyploidy might have an important role in the diversification of this group. Notwithstanding, most cytogenetic studies on these species are based on chromosome counting. Aiming to understand how polyploidy may have impacted the genomes of these species, the DNA content of 184 specimens was estimated; fluorochrome banding with chromomycin A₃ and fluorescent in situ hybridization using an 18S-5.8S-26S ribosomal DNA (rDNA) probe were also performed. The results showed a reduction in monoploid genome size (1Cx), as well as in the number of heterochromatin bands and rDNA sites per monoploid genome, from diploids to polyploids. Additionally, intraspecific and within-ploidy variations in genome size and number of rDNA sites were observed. The source of varying structure in genome organization of these plants may be the multiple independent formations of polyploids along with an ongoing diploidization process. However, the intraspecific and within-ploidy polymorphisms indicate genetic mechanisms other than genome duplication and diploidization to be important to the genome evolution of these taxa.     

Karyotype evolution and phylogenetic analyses in the genus Cardiospermum L. (Paullinieae,Sapindaceae)

Cardiospermum L. belongs to the Paullinieae tribe (Sapindaceae) and comprises 16 species. Of these, 12 species are present in South America and all occur in Brazil. Cardiospermum shows the most variable chromosome number of the tribe. Phylogenetic relationships within the genus Cardiospermum, especially with other species of the tribe, are poorly understood. This research focuses on characterisation of the karyotypic features of Cardiospermum using conventional cytogenetic methods, CMA/DAPI chromosome banding and fluorescence in situ hybridisation (FISH). To elucidate the phylogeny of the genus, the nuclear markers ITS1 and ITS2 were sequenced and analysed using maximum parsimony and Bayesian inference. Cardiospermum shows important diversity in basic numbers, with x = 7, 9, 10, 11 and 12. All species studied have metacentric and submetacentric chromosomes, some species have subtelocentric chromosomes, while telocentric chromosomes are absent. The interphase nuclei differentiate the Cardiospermum species into two groups. The CMA₃/DAPI chromosome banding revealed the presence of an AT‐rich terminal region in C. corindum, C. grandiflorum and C. urvilleoides, whereas GC‐rich regions were found in C. grandiflorum, C. halicacabum var. halicacabum, C. halicacabum var. microcarpum, C. heringeri and C. integerrimum. FISH revealed syntenic and non‐syntenic distribution of the 18‐5.8‐26S and 5S rDNA. The syntenic distribution always occurred in the short arms of the same chromosome in all of the species. The phylogenetic relationships reveal, in part, the taxonomic arrangement of the genus Cardiospermum.     

The promiscuous and the chaste: frequent allopolyploid speciation and its genomic consequences in American daisies (Melampodium sect. Melampodium; Asteraceae)

Polyploidy, an important factor in eukaryotic evolution, is especially abundant in angiosperms, where it often acts in concert with hybridization to produce allopolyploids. The application of molecular phylogenetic techniques has identified the origins of numerous allopolyploids, but little is known on genomic and chromosomal consequences of allopolyploidization, despite their important role in conferring divergence of allopolyploids from their parental species. Here, using several plastid and nuclear sequence markers, we clarify the origin of tetra- and hexaploids in a group of American daisies, allowing characterization of genome dynamics in polyploids compared to their diploid ancestors. All polyploid species are allopolyploids. Among the four diploid gene pools, the propensity for allopolyploidization is unevenly distributed phylogenetically with a few species apparently more prone to participate, but the underlying causes remain unclear. Polyploid genomes are characterized by differential loss of ribosomal DNA loci (5S and 35S rDNA), known hotspots of chromosomal evolution, but show genome size additivity, suggesting limited changes beyond those affecting rDNA loci or the presence of processes counterbalancing genome reduction. Patterns of rDNA sequence conversion and provenance of the lost loci are highly idiosyncratic and differ even between allopolyploids of identical parentage, indicating that allopolyploids deriving from the same lower-ploid parental species can follow different evolutionary trajectories.     

Cytogenetic characterization and nuclear DNA content of three North African endemic Centaurea species

Three endemic Centaurea species from North Africa are investigated for the first time by chromomycin fluorochrome banding for GC-rich DNA distribution, fluorescence in situ hybridization for physical mapping of rRNA genes, and flow cytometry for genome-size assessment. Investigated species belong to three different sections and possess three basic chromosome numbers: C. tougourensis subsp. tougourensis 2n = 4x = 36 (x = 9), C. musimonum 2n = 2x = 20 (x = 10), and C. maroccana 2n = 2x = 24 (x = 12). The number and distribution of chromomycin positive bands (CMA⁺) and 18S-5.8S-26S (35S) rDNA loci were different among investigated species and ranged from 6 to 80 chromomycin bands and from 2 to 6 35S rDNA loci. The three species have just one 5S rDNA locus at intercalary position on a separate chromosome pairs, except in the case of C. musimonum in which both rDNA loci were localized on the same chromosome. All rDNA loci were co-localized with CMA⁺ bands, except three 35S in C. musimonum. Genome size ranged from 2C = 1.66 to 2C = 2.86 pg in diploid species (C. musimonum and C. maroccana, respectively) and to 2C = 4.51 pg in tetraploid C. tougourensis subsp. tougourensis.     

Molecular cytogenetics of Xeranthemum L. and related genera (Asteraceae, Cardueae)

Abstract: Seven representatives of the genera Amphoricarpus, Chardinia, Siebera, and Xeranthemum, all of them closely related as demonstraded by molecular phylogeny, have been studied from a cytogenetic perspective. Morphometrical karyotype parameters were calculated and idiograms obtained. Fluorochrome banding was performed with chromomycin A₃ to identify GC-rich regions in the chromosomes. Fluorescence in situ hybridization allowed us to locate the sites of 18S-5.8S-26S and 5S rDNA. Silver nitrate staining was used to count the number of nucleoli and to detect the active nucleolar organizing regions. Systematic and evolutionary issues are addressed in the light of these data.     

Molecular cytogenetic characterisation and phylogenetic analysis of the seven cultivated Vigna species (Fabaceae)

The genomic organisation of the seven cultivated Vigna species, V. unguiculata, V. subterranea, V. angularis, V. umbellata, V. radiata, V. mungo and V. aconitifolia, was determined using sequential combined PI and DAPI (CPD) staining and dual‐colour fluorescence in situ hybridisation (FISH) with 5S and 45S rDNA probes. For phylogenetic analyses, comparative genomic in situ hybridisation (cGISH) onto somatic chromosomes and sequence analysis of the internal transcribed spacer (ITS) of 45S rDNA were used. Quantitative karyotypes were established using chromosome measurements, fluorochrome bands and rDNA FISH signals. All species had symmetrical karyotypes composed of only metacentric or metacentric and submetacentric chromosomes. Distinct heterochromatin differentiation was revealed by CPD staining and DAPI counterstaining after FISH. The rDNA sites among all species differed in their number, location and size. cGISH of V. umbellata genomic DNA to the chromosomes of all species produced strong signals in all centromeric regions of V. umbellata and V. angularis, weak signals in all pericentromeric regions of V. aconitifolia, and CPD‐banded proximal regions of V. mungo var. mungo. Molecular phylogenetic trees showed that V. angularis and V. umbellata were the closest relatives, and V. mungo and V. aconitifolia were relatively closely related; these species formed a group that was separated from another group comprising V. radiata, V. unguiculata ssp. sesquipedalis and V. subterranea. This result was consistent with the phylogenetic relationships inferred from the heterochromatin and cGISH patterns; thus, fluorochrome banding and cGISH are efficient tools for the phylogenetic analysis of Vigna species.     

FISH mapping of 35S and 5S rRNA genes in Artemisia subgenus Dracunculus (Asteraceae): changes in number of loci during polyploid evolution and their systematic implications

Fluorescence in situ hybridization (FISH) with 35S and 5S rDNA probes was used to characterize cytogenetically representatives of Artemisia subgenus Dracunculus and allied species and to explore their evolution following polyploidization. At the diploid level two rDNA loci were observed in most species belonging to the A. dracunculus complex, a pattern considered to be the ancestral state for diploid Artemisia. In contrast, representative species from the Eurasian grade which belong to the other major lineage of the subgenus had more heterogeneous rDNA profiles, with three to five loci at the diploid level. Divergent patterns of locus evolution were also detected in polyploids, with the number and distribution of rDNA loci broadly fitting the two main diversification lineages in the subgenus. In the polyploid complex of A. dracunculus, the number of rDNA loci was almost proportional to ploidy, although monoploid genome size was shown to decrease with increasing ploidy. However, in polyploids from the Eurasian grade we found a remarkable reduction in the number of rDNA sites, suggesting that these species might have experienced either a complete loss of loci or a significant reduction in the number of repeats following polyploid formation. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013 , 171 , 655–666.     

Comparative cytogenetic analysis of Cestrum (Solanaceae) reveals different trends in heterochromatin and rDNA sites distribution

Species of Cestrum L. (Solanaceae) exhibit large variability in the accumulation of repetitive DNA, although their species possess a stable diploid number with 2n = 16. In this study, we used chromosome banding and fluorescence in situ hybridization (FISH) to characterize the karyotypes and populations of two species, Cestrum nocturnum L. and C. mariquitense Kunth. We also performed a karyotype comparison using 16 idiograms, of which 4 were developed in this study and 12 were obtained from the literature. Cestrum nocturnum displayed more bands than C. mariquitense, but the latter exhibited greater interpopulational variation in the band patterns. There was a tendency for large bands to be located at intercalary/terminal regions and for small bands to be located at intermediate/proximal regions. The idiogram comparison revealed a large variation in the amount, distribution, and size of heterochromatic bands. FISH with rDNA probes revealed stability in the number and location of 5S sites, while 45S was more variable in size and number of sites. Although 45S rDNA always appeared in the subterminal regions, this DNA family exhibited a mobility among chromosome pairs. These data highlight the dynamic of repetitive DNA families in these genomes, as well as the contribution for intra- and interspecific karyotype differentiation in Cestrum.     

Chromosomal organization of ribosomal genes and NOR-associated heterochromatin, and NOR activity in some populations of Allium commutatum Guss. (Alliaceae)

The position and the number of 18S-5.8S-26S and 5S rDNA loci, characterization of nucleolar organizing region (NOR)-associated heterochromatin and NOR activity assessment are given for six south-eastern Adriatic populations of Allium commutatum Guss. The karyotype characteristics were identical for all the populations studied, even those of distant islands. Diploid karyotypes (2 n = 16) always possessed two NOR-bearing chromosome pairs with pericentric and median secondary constrictions (SCs) on the short arm of the chromosomes VII and VIII. Fluorescent in situ hybridization (FISH) confirmed that these were the only sites of 18S-5.8S-26S rRNA genes. NOR-associated heterochromatin was of the constitutive character as shown after C-banding. Differential fluorochrome banding with Chromomycin A3 (CMA) and 4,6-diamidino-2-phenylindole (DAPI) revealed that this heterochromatin comprises both GC- and AT-rich DNA segments. Heteromorphism of C- and CMA-bands was noticed between homologous NOR-bearing chromosomes. The maximum number of four active NORs was correlated with the maximum number of four nucleoli in interphase. Variability of NOR-activity, expressed as number and size of silver stained NORs, existed between cells and between individuals of the same population. The different size of homologous and nonhomologous silver stained NORs was correlated with the extension of SCs. The only 5S rDNA locus was in an intercalary position on short arm of the chromosome VI, at the region of AT-rich constitutive heterochromatin. Dimorphism of C-bands and DAPI/Hoechst(H)-fluorescent bands was noticed between homologous chromosomes VI. © 2002 The Linnean Society of London, Botanical Journal of the Linnean Society , 2002, 139 , 99–108.     

Karyotype constancy and genome size variation in BulgarianCrepis foetida s. l. (Asteraceae)

Ten populations ofCrepis foetida from Bulgaria belonging to the three subspeciesfoetida, rhoeadifolia, andcommutata were analyzed karyologically using haematoxylin staining, Giemsa C-banding, fluorochrome banding, Ag-NOR staining, Feulgen cytophotometry (scanning densitometry and video-based image analysis), and propidium iodide flow cytometry. The quantitatively-evaluated karyotype structure was similar among all populations, with minor variation in a few intercalary sites only and in the amount of NOR-associated heterochromatin (satellites). In contrast to the karyotypic constancy the genome size ofC. foetida subsp.commutata was about 10% lower than those of the other two subspecies, which had similar genome sizes. The genome size measurements using three different methods resulted in highly correlated data. The genome size difference adds some weight to previous taxonomic opinions treatingC. foetida subsp.commutata at species level, asC. commutata. Prof. Dr. Stefan Kozhuharov (4 January 1933–24 August 1997).     

Using chromosomal data in the phylogenetic and molecular dating framework: karyotype evolution and diversification in Nierembergia (Solanaceae) influenced by historical changes in sea level

Karyotype data within a phylogenetic framework and molecular dating were used to examine chromosome evolution in Nierembergia and to infer how geological or climatic processes have influenced in the diversification of this solanaceous genus native to South America and Mexico. Despite the numerous studies comparing karyotype features across species, including the use of molecular phylogenies, to date relatively few studies have used formal comparative methods to elucidate chromosomal evolution, especially to reconstruct the whole ancestral karyotypes. Here, we mapped on the Nierembergia phylogeny one complete set of chromosomal data obtained by conventional staining, AgNOR‐, C‐ and fluorescent chromosome banding, and fluorescent in situ hybridisation. In addition, we used a Bayesian molecular relaxed clock to estimate divergence times between species. Nierembergia showed two major divergent clades: a mountainous species group with symmetrical karyotypes, large chromosomes, only one nucleolar organising region (NOR) and without centromeric heterochromatin, and a lowland species group with asymmetrical karyotypes, small chromosomes, two chromosomes pairs with NORs and centromeric heterochromatin bands. Molecular dating on the DNA phylogeny revealed that both groups diverged during Late Miocene, when Atlantic marine ingressions, called the ‘Paranense Sea’, probably forced the ancestors of these species to find refuge in unflooded areas for about 2 Myr. This split agrees with an increased asymmetry and heterochromatin amount, and decrease in karyotype length and chromosome size. Thus, when the two Nierembergia ancestral lineages were isolated, major divergences occurred in chromosomal evolution, and then each lineage underwent speciation separately, with relatively minor changes in chromosomal characteristics.     

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.     

Physical mapping of ribosomal RNA genes in peonies (Paeonia,Paeoniaceae) by fluorescent in situ hybridization: implications for phylogeny and concerted evolution

Physical maps of the 18S–5.8S–26S ribosomal RNA genes (rDNA) were generated by fluorescent in situ hybridization for five diploid Paeonia species, P. delavayi and P. rockii of section Moutan, and P. emodi, P. tenuifolia, and P. veitchii of section Paeonia. Of five pairs of mitotic chromosomes, rDNA loci were mapped near the telomeres of chromosomes 3, 4, and 5 of P. rockii and P. tenuifolia, chromosomes 2, 3, 4, and 5 of P. delavayi, and all five pairs of chromosomes of P. emodi and P. veitchii. Combining this information with the previously obtained rDNA maps of P. brownii and P. californica of section Oneapia, we hypothesized that the most recent common ancestor of extant peony species had three rDNA loci located on chromosomes 3, 4, and 5. Increase in number of rDNA loci occurred later in each of the three sections, and the increase from three to four loci represents a parallel gain of an rDNA locus on chromosome 2 in P. delavayi of section Moutan and P. brownii of section Oneapia. The increase in number of rDNA loci likely resulted from the translocation of rDNA repeats from chromosomes bearing rDNA loci to chromosomes without them; such translocation is probably facilitated by the telomeric location of rDNA loci. For allotetraploid peony species lacking polymorphism in sequences of the internal transcribed spacers (ITS) of rDNA, the rDNAs derived from divergent diploid parents may have been homogenized through concerted evolution among at least six rDNA loci in the allotetraploids. Chromosomal location of rDNA loci has a more substantial impact on the tempo of concerted evolution than the number of loci.     

Karyotype diversification and evolution in diploid and polyploid South American Hypochaeris (Asteraceae) inferred from rDNA localization and genetic fingerprint data

Background and Aims: Changes in chromosome structure and number play an importantrole in plant evolution. A system well-suited to studying differentmodes of chromosome evolution is the genus Hypochaeris (Asteraceae)with its centre of species' diversity in South America. AllSouth American species uniformly have a chromosome base numberof x = 4 combined with variation in rDNA number and distribution,and a high frequency of polyploidy. The aim of this paper isto assess directions and mechanisms of karyotype evolution inSouth American species by interpreting both newly obtained andprevious data concerning rDNA localization in a phylogeneticcontext. Methods: Eleven Hypochaeris species from 18 populations were studiedusing fluorescence in situ hybridization (FISH) with 35S and5S rDNA probes. A phylogenetic framework was established fromneighbour-net analysis of amplified fragment length polymorphism(AFLP) fingerprint data. Key Results: A single 5S rDNA locus is invariably found on the short armof chromosome 2. Using 35S rDNA loci, based on number (one ortwo) and localization (interstitial on the long arm of chromosome2, but sometimes lacking, and terminal or interstitial on theshort arm of chromosome 3, only very rarely lacking), sevenkaryotype groups can be distinguished; five of these includepolyploids. Karyotype groups with more than one species do notform monophyletic groups. Conclusions: Early evolution of Hypochaeris in South America was characterizedby considerable karyotype differentiation resulting from independentderivations from an ancestral karyotype. There was marked diversificationwith respect to the position and evolution of the 35S rDNA locuson chromosome 3, probably involving inversions and/or transpositions,and on chromosome 2 (rarely 3) concerning inactivation and loss.Among these different karyotype assemblages, the apargioidesgroup and its derivatives constitute by far the majority ofspecies.     

Genome size variation in the genus Carthamus (Asteraceae, Cardueae): systematic implications and additive changes during allopolyploidization

BACKGROUND AND AIMS: Plant genome size is an important biological characteristic, with relationships to systematics, ecology and distribution. Currently, there is no information regarding nuclear DNA content for any Carthamus species. In addition to improving the knowledge base, this research focuses on interspecific variation and its implications for the infrageneric classification of this genus. Genome size variation in the process of allopolyploid formation is also addressed. METHODS: Nuclear DNA samples from 34 populations of 16 species of the genus Carthamus were assessed by flow cytometry using propidium iodide. KEY RESULTS: The 2C values ranged from 2.26 pg for C. leucocaulos to 7.46 pg for C. turkestanicus, and monoploid genome size (1Cx-value) ranged from 1.13 pg in C. leucocaulos to 1.53 pg in C. alexandrinus. Mean genome sizes differed significantly, based on sectional classification. Both allopolyploid species (C. creticus and C. turkestanicus) exhibited nuclear DNA contents in accordance with the sum of the putative parental C-values (in one case with a slight reduction, frequent in polyploids), supporting their hybrid origin. CONCLUSIONS: Genome size represents a useful tool in elucidating systematic relationships between closely related species. A considerable reduction in monoploid genome size, possibly due to the hybrid formation, is also reported within these taxa.     

Phylogenetic relationships in West Mediterranean Scaritina (Coleoptera: Carabidae) inferred from mitochondrial COI sequences and karyotype analysis

The phylogenetic relationships of the West Mediterranean lineages of the subtribe Scaritina have been investigated by studying the mitochondrial cytochrome oxidase gene (COI) and the mitotic and meiotic chromosomes, including the localization of rDNA genes by fluorescence in situ hybridization. These sources of data are congruent and suggest the following conclusions: (1) the subgenus Parallelomorphus (genus Scarites ) is a monophyletic group according to molecular and karyotypic data, in agreement with its geographical distribution and morphological characters; (2) in the same genus, the subgenus Scallophorites seems to be monophyletic but includes two well-separated lineages – one represented by Scarites buparius (Forster, 1771) and Scarites occidentalis (Bedel, 1895) (both with multiple sex chromosomes), and one represented by Scarites hespericus (Dejean, 1831); (3) the nominal subgenus Scarites s.s . represented by Scarites eurytus (Fischer, 1828), is cladistically more closely related to subgenus Scallophorites than to subgenus Parallelomorphus ; (4) Distichus planus (Bonelli, 1813) belongs to a distinct clade that is well separated from Scarites, and shows more plesiomorphic states of COI sequence and chromosome number. In addition, the presence of two autapomorphies, such as rDNA sites located in the X chromosome, and an A + T rich intergenic spacer of about 120 bp between the stop codon of the COI gene and the tRNAleu, agree with the ranking of Distichus as a separate genus from Scarites .     

Polyploidy: its consequences and enabling role in plant diversification and evolution

BackgroundMost, if not all, green plant (Virdiplantae) species including angiosperms and ferns are polyploids themselves or have ancient polyploid or whole genome duplication signatures in their genomes. Polyploids are not only restricted to our major crop species such as wheat, maize, potato and the brassicas, but also occur frequently in wild species and natural habitats. Polyploidy has thus been viewed as a major driver in evolution, and its influence on genome and chromosome evolution has been at the centre of many investigations. Mechanistic models of the newly structured genomes are being developed that incorporate aspects of sequence evolution or turnover (low-copy genes and regulatory sequences, as well as repetitive DNAs), modification of gene functions, the re-establishment of control of genes with multiple copies, and often meiotic chromosome pairing, recombination and restoration of fertility.ScopeWorld-wide interest in how green plants have evolved under different conditions – whether in small, isolated populations, or globally – suggests that gaining further insight into the contribution of polyploidy to plant speciation and adaptation to environmental changes is greatly needed. Forward-looking research and modelling, based on cytogenetics, expression studies, and genomics or genome sequencing analyses, discussed in this Special Issue of the Annals of Botany, consider how new polyploids behave and the pathways available for genome evolution. They address fundamental questions about the advantages and disadvantages of polyploidy, the consequences for evolution and speciation, and applied questions regarding the spread of polyploids in the environment and challenges in breeding and exploitation of wild relatives through introgression or resynthesis of polyploids.ConclusionChromosome number, genome size, repetitive DNA sequences, genes and regulatory sequences and their expression evolve following polyploidy – generating diversity and possible novel traits and enabling species diversification. There is the potential for ever more polyploids in natural, managed and disturbed environments under changing climates and new stresses.     

Physical localization of the 18S-5.8S-26S rDNA and sequence analysis of ITS regions in Thinopyrum ponticum (Poaceae: Triticeae): implications for concerted evolution

Fluorescence in situ hybridization was used in Thinopyrum ponticum, a decaploid species, and its related diploid species, to investigate the distribution of the 18S-5.8S-26S rDNA. The distribution of rDNA was similar in all three diploid species (Th. bessarabicum, Th. elongatum and Pseudoroegneria stipifolia). Two pairs of loci were observed in each somatic cell at metaphase and interphase. One pair was located near the terminal end and the other in the interstitial regions of the short arms of one pair of chromosomes. However, all of the major loci in Th. ponticum were located on the terminal end of the short arms of chromosomes, and one chromosome had only one major locus. The maximum number of major loci detected on metaphase spreads was 20, which was the sum of that of its progenitors. The interstitial loci that exist in the possible diploid genome donor species were probably 'lost' during the evolutionary process of the decaploid species. A number of minor loci were also detected on whole regions of two pairs of homologous chromosomes. These results suggested that the position of rDNA loci in the Triticeae might be changeable rather than fixed. Positional changes of 18S-5.8S-26S rDNA loci between Th. ponticum and its candidate genome donors indicate that it is almost impossible to find a genome in the polyploid species that is completely identical to that of its diploid donors. The possible evolutionary significance of the distribution of the rDNA is also discussed. Internal transcribed spacer (ITS) regions of nuclear DNA in Th. ponticum were investigated by PCR amplification and sequencing. The sequence data from five positive clones selected at random, together with restriction site analysis, indicated that the ITS repeated units are nearly homogeneous in this autoallodecapolypoid species. Combined with in situ hybridization results, the data led to the conclusion that the ITS region has experienced interlocus as well as intralocus concerted evolution. Phylogenetic analyses showed that the sequences from Th. ponticum have concerted to the E genome repeat type.