What does the nr5S DNA multigene family tell us about the genomic relationship between Dasypyrum breviaristatum and D. villosum (Triticeae: Poaceae)?

The genus Dasypyrum contains two species: the annual and widespread D. villosum (2x = 2n = 14) and the perennial and generally rare D. breviaristatum (2x = 2n = 14 and 4x = 2n = 28). The origin of the latter and its genome constitution have been subject of several studies. There is agreement that the genome of the diploid D. villosum (VV) is different from the diploid cytotype of D. breviaristatum (VbVb), but there is no agreement of the constitution of the tetraploid cytotype, specifically whether is it an autotetraploid or an allotetraploid. This is a long-standing disagreement that this study aims to resolve using the 5S nrDNA as a genomic marker. Our studies suggest that the 4x D. breviaristatum is an allotetraploid (VVVbVb).     

Identification of the chromosome complement and the spontaneous 1R/1V translocations in allotetraploid <Emphasis Type="Italic">Secale cereale</Emphasis> × <Emphasis Type="Italic">Dasypyrum villosum</Emphasis> hybrids through cytogenetic approaches

Genome modifications that occur at the initial interspecific hybridization event are dynamic and can be consolidated during the process of stabilization in successive generations of allopolyploids. This study identifies the number and chromosomal location of ribosomal DNA (rDNA) sites between Secale cereale, Dasypyrum villosum, and their allotetraploid S. cereale × D. villosum hybrids. For the first time, we show the advantages of FISH to reveal chromosome rearrangements in the tetraploid Secale × Dasypyrum hybrids. Based on the specific hybridization patterns of ribosomal 5S, 35S DNA and rye species-specific pSc200 DNA probes, a set of genotypes with numerous Secale/Dasypyrum translocations of 1R/1V chromosomes were identified in successive generations of allotetraploid S. cereale × D. villosum hybrids. In addition we analyse rye chromosome pairs using FISH with chromosome-specific DNA sequences on S. cereale × D. villosum hybrids.     

Genomic organization and phylogenetic relationships in the genus Dasypyrum analysed by Southern and in situ hybridization of total genomic and cloned DNA probes

Molecular cytogenetic methods have been used to study the controversial phylogenetic relationships between the species Dasypyrum villosum (L.) Candargy (2n=2x=14) and D. breviaristatum (Lindb. f.) Frederiksen (2n=4x=28). Using total genomic DNA from the two species as probes for in situ hybridization to chromosomes, we found that the pericentromeric regions of the chromosome arms of both species are similar, while distal regions show substantial differences. Two dispersed repetitive DNA sequences were isolated: pDbKB45 is distributed along the chromosomes but amplified in the subtelomeric regions of D. breviaristatum chromosomes, while pDbKB49, in both species, is less amplified in terminal regions. Size-separated restriction enzyme digests of DNA showed many repetitive fragments, but few in common between the two species. After probing Southern transfers with D. breviaristatum genomic DNA, all lanes showed similar hybridization patterns although one extra small band was evident in the D. breviaristatum lanes. In contrast, probing with D. villosum DNA showed very substantial differences between the two species. Genomic in situ hybridization to meiotic metaphases from an interspecific hybrid showed seven bivalents of D. breviaristatum origin and seven univalents from D. villosum. We also analysed the physical organization of 5S rDNA, 18S-25S rDNA and a tandemly repeated sequence from rye. Our data support an autotetraploid origin for D. breviaristatum, but its genome and that of D. villosum show extensive differences, so the tetraploid is unlikely to be directly derived from D. villosum. Received: 29 March 1996; in revised form: 28 December 1996 / Accepted: 2 February 1997     

The comparative reproductive biology of a tetraploid species,Hedychium villosum,and its diploid progenitor H. tenuiflorum (Zingiberaceae)

The evolutionary advantages of polyploidy may result from a number of changes in floral traits and breeding system, which may enable polyploids to exploit new habitats and become widespread. In this study, we comparatively investigated the floral biology of the tetraploid species Hedychium villosum and its diploid progenitor H. tenuiflorum, to assess reproductive divergence between the two species. The results showed that flowers of the tetraploid species last longer and produce more nectar than did diploid species. The flowering times of the two species did not overlap at all. Observations of floral visitors in natural populations demonstrated that butterflies and hawkmoths were effective pollinators of both species, but there was a significant difference in butterfly and hawkmoth assemblages between the two species. The hand‐pollination experiments and pollen tube growth experiments suggested that diploid H. tenuiflorum was self‐incompatible, while tetraploid H. villosum was completely self‐compatible. H. villosum has a much wider distribution range and occupies more diverse habitats than H. tenuiflorum. Polyploidisation may enable tetraploid H. villosum to exploit new habitats previously unavailable to diploid H. tenuiflorum.     

Genome sizes of Eucomis L’Hér. (Hyacinthaceae) and a description of the new species Eucomis grimshawii G.D.Duncan & Zonneveld

Nuclear genome size, as measured by flow cytometry with propidium iodide, was used to investigate the relationships within the genus Eucomis L’Hér. (Hyacinthaceae). Most species of Eucomis have the same basic chromosome number, x = 15. However, the somatic DNA 2C-value (2C) is shown to range from 21 to 31 pg for the diploids. The largest genome contains roughly 10¹⁰ more base pairs than the smallest. Genome sizes are evaluated here in combination with available morphological and geographical data. Therefore, the taxonomy proposed here is not based on genome size alone. The genus Eucomis, as here determined, has 12 species. These can be divided into two groups: mainly dwarf diploid species and large-sized, tetraploid species. A small diploid plant, Eucomis (autumnalis subsp.) amaryllidifolia, is restored to species status, as a diploid subspecies seems incongruent with an allotetraploid Eucomis autumnalis. Moreover, as a diploid it is separated reproductively from the allotetraploid E. autumnalis. A new diploid species that has the lowest C value, E. grimshawii, is described here. On the basis of DNA content and other morphological characters, possible parents are suggested for all tetraploid species. Nuclear DNA content as measured by using flow cytometry may conveniently be used to produce systematic data. It is applicable even in dormant bulbs or sterile plants for the monitoring of the trade in bulbous species.     

Independent origins of tetraploid cryptic species in the fern <Emphasis Type="Italic">Ceratopteris thalictroides</Emphasis>

Ceratopteris thalictroides (L.) Brongn is a tetraploid fern species that contains at least three cryptic species, the south, the north and the third type. In this study we combined data from both chloroplast DNA (cpDNA) and nuclear DNA sequences of three diploid species and three cryptic species of C. thalictroides to unravel the origin of the cryptic species, particularly of the reticulate relationships among the diploid and tetraploid taxa in the genus Ceratopteris. Of the three diploid species examined, C. cornuta had cpDNA identical to that of the tetraploid third type plants, and this diploid species is a possible maternal ancestor of the tetraploid third type. Analysis of the homologue of the Arabidopsis thaliana LEAFY gene (CLFY1) identified ten alleles in the genus Ceratopteris, with six alleles found in C. thalictroides. The unrooted tree of the CLFY1 gene revealed four clusters. Each cryptic species showed fixed heterozygosity at the CLFY1 locus and had two alleles from different clusters of the CLFY1 tree. Consideration of the cpDNA sequences, CLFY1 genotypes of the cryptic species and CLFY1 gene tree in concert suggested that the cryptic species of C. thalictroides had originated through independent allopolyploidization events involving C. cornuta and two unknown hypothetical diploid species.     

Restriction fragment patterns of chloroplast and mitochondrial DNA of Dasypyvum villosum (L.) candargy and wheats

To elucidate the phylogenetic relationships and cytoplasmic types, restriction endonuclease fragment patterns of chloroplast (cp) and mitochondrial (mt) DNAs isolated from two different accessions of Dasypyrum villosum (L.) candargy were compared with those of tetraploid wheat (Triticum durum Desf., PI265007), hexaploid wheat (Triticum aestivum L., cv Chinese Spring), Aegilops longissimum (S. and M., in Muschli) Bowden and Hordeum vulgare L. T. aestivum and T. durum had identical restriction patterns for their cp and mtDNAs in digestions with four different enzymes. Likewise, no differences were found between the restriction fragment patterns of two accessions of D. villosum. But, there were distinct differences in chloroplast and mitochondrial DNA restriction fragment patterns between D. villosum and tetraploid and hexaploid wheats. A. longissimum (G609) showed a similar pattern to those wheats for PstI digestion of cpDNA. Organellar DNA from Hordeum vulgare (cv Himalaya) showed a distinctly different restriction pattern from those of wheat and D. villosum. These results suggest that D. villosum is unlikely to be the donor of cytoplasm to wheats, and its cytoplasmic organelles were also different from those of A. longissimum.Contribution No. 92-522-J from the Kansas Agricultural Experiment Station; Kansas State University, Manhattan, Kansas, USA     

Nuclear and cytoplasmic genome relationships in the genus Avena: Analysis by isoelectric focusing of ribulose biphosphate carboxylase subunits

Comparisons of the isoelectric points of small and large subunits of ribulose biphosphate carboxylase extracted from a number of diploid, tetraploid, and hexaploid Avena species have been used to obtain information on the nuclear and cytoplasmic genome relationships within the genus. All species tested had small subunits with similar isoelectric points, so their analysis provided no information of taxonomic value. Three types of large subunits could be distinguished by this method, and the distribution of each among the available species provides strong evidence against the involvement of a C genome diploid (such as A. ventricosa) as the maternal parent in the formation of either tetraploid or hexaploid species. One type of large subunit was confined to the perennial tetraploid, A. macrostachya, and its position in the genus and possible origin are discussed. The value of this approach in studying genome relationships within the genus Avena and related genera is assessed.     

Chromosome numbers in callirhoe (Malvaceae)

Forty-four chromosome counts from the North American genusCallirhoe are reported, including first-recorded counts ofC. alcaeoides, C. digitata, C. bushii, C. triangulata, andC. involucrata var.tenuissima. Information concerning chromosome numbers from present and previous reports is summarized and evaluated. Observations are presented on meiosis for the genus in general, for male sterile individuals of gynodioecious species, and for selected F₁ hybrids. Pollen fertility data are recorded for the latter. Chromosomally three species groups are recognized: an exinvolucellate, diploid species group withn=14; an involucellate, tetraploid and octoploid species pair,n=28 and 56; and an involucellate, diploid and tetraploid species group withn=15 and 30. Intrageneric relationships and those within the tribe Malveae subtribe Malvinae are discussed.     

HYBRIDIZATION AND CYTOTAXONOMY OF DICENTRA

Biosystematic relationships among species assigned to three subgenera of the genus Dicentra were investigated with respect to hybridization and chromosomal constitution and fertility of the hybrids. Four species of subgenus Dicentra, D. formosa, D. eximia, D. nevadensis, and D. peregrina, were intercrossed in various ways to form diploid, triploid, and tetraploid hybrids. Hybrids at the tetraploid level in this subgenus invariably were highly fertile. Triploid hybrids, as expected, were mostly very sterile. Diploid hybrids varied in this respect, but none was highly fertile. Crosses with two of the remaining four species of subgenus Dicentra produced no hybrids, but abundant seed was obtained in one instance. The two species of the subgenus Chrysocapnos, D. chrysaniha and D. ochroleuca, cross to produce a partially fertile tetraploid hybrid, but cross-pollinations involving these species with those of other subgenera failed. The single species of subgenus Hedycapnos, D. spectablis (diploid) produced no hybrids when cross-pollinated with members of the other two sections. These results are fully concordant with presumed affinities based on morphological similarity In addition, preliminary results of hybridization between the monocotyledonous D. peregrina and a number of dicotyledonous species of Dicentra are reported.     

Polyploidy in the Australian leptodactylid frog genus Neobatrachus

Karyotypic analysis of six species of the Australian leptodactylid frog genus Neobatrachus showed that N. pictus, N. centralis, N. pelobatoides and N. wilsmorei are diploid (2n=24) while N. sudelli and N. sutor are tetraploid (4n=48). Polyploidy has not been reported previously among Australian anurans. Idiograms of the six species indicate that they are similar to the other Australian leptodactylids so far discribed. DNA values of the tetraploids are approximately double the values for diploids. Tetraploid nuclear and cell sizes are greater compared with diploids but total body size shows no increase. At diakinesis in primary spermatocytes of tetraploids, mainly tetravalents together with a few bivalents are present. Silver staining of metaphase spreads clearly demonstrates the location of NORs at the secondary constrictions and their frequent association in the tetraploid N. sutor. Nucleolar number in interphase nuclei provides a reliable guide for distinguishing tetraploid from diploid frogs in the absence of chromosome analysis and can be determined for both living and preserved specimens. The possible origins and relationships of the tetraploid species are discussed.     

Integrated taxonomy of the <Emphasis Type="Italic">Asplenium normale</Emphasis> complex (Aspleniaceae) in China and adjacent areas

The Asplenium normale D. Don complex comprises several taxa that are either diploid or tetraploid. The tetraploids are assumed to have originated from diploid ancestors by relatively recent autopolyploidization or allopolyploidization. Some of the diploids are readily recognized morphologically but most of the taxa have until now been placed into a single species. However, phylogenetic studies have challenged this treatment and emphasized the notion that the taxonomic treatment of this complex needs to be revised. An integrative taxonomic approach was employed to delimit species in the complex using cytological, morphological, and DNA sequence data. Initially, we employed a diploid first approach to establish a robust taxonomic framework. Special efforts were made to collect and identify the diploid progenitors of each polyploid lineage identified in the plastid DNA based phylogenetic hypothesis. A total of six distinct diploid species were identified. The distinctive nature of the six diploids is strongly supported by sequence differences in plastid DNA and nuclear loci, as well as by the results of morphometric analysis. Diagnostic morphological characters were identified to distinguish the six diploid species, resulting in their revised taxonomy, which includes two novel species, namely, Asplenium normaloides and A. guangdongense. Further studies to strengthen the taxonomic classification of all of the tetraploid taxa are warranted.     

Phylogenetic analysis of tetraploid wheat based on nuclear DMC1 gene

Tetraploid wheat (AABB or AAGG, 2n = 4x = 28) holds an important place in Triticum. It includes two allopolyploid species, Triticum turgidum and Triticum timopheevii. Many problems concerning the phylogenetic relationships among tetraploid wheat species remain unresolved. In this study, sequences data for the nuclear DMC1 gene from 61 accessions of Triticum and Aegilops species, representing diploid and tetraploid species, were used to examine the phylogenetic relationships among tetraploid wheat. Phylogenetic trees were constructed using maximum-likelihood and neighbor-joining approaches, and gene flow and genetic differentiation values were computed. The results indicated that the A genome of tetraploid wheat originated from T. urartu rather than T. monococcum, and Aegilops speltoides was the donor of the B and G genomes. Hulled tetraploid wheat accessions formed a subclade, and naked tetraploid wheat got other subclade, indicating that at least two intermediary subspecies were involved in the evolution of T. turgidum. Triticum turgidum and T. timopheevii might have simultaneously originated from a hybridization events. These results indicated that the DMC1 gene sequences are useful for resolution of the molecular phylogenetic relationships of tetraploid wheat.     

Flow Sorting and Molecular Cytogenetic Identification of Individual Chromosomes of Dasypyrum villosum L. (H. villosa) by a Single DNA Probe

Dasypyrum villosum (L.) Candargy (sin. Haynaldia villosa) is an annual wild diploid grass species (2n = 2x = 14; genome VV) belonging to the Poaceae family, which is considered to be an important source of biotic and abiotic stress resistance genes for wheat breeding. Enhanced characterization of D. villosum chromosomes can facilitate exploitation of its gene pool and its use in wheat breeding programs. Here we present the cytogenetic identification of D. villosum chromosomes on slide by fluorescent in situ hybridization (FISH), with the GAA simple sequence repeat (SSR) as a probe. We also describe the isolation and the flow cytometric analysis of D. villosum chromosomes in suspension, resulting in a distinguished flow karyotype. Chromosomes were flow sorted into three fractions, according their DNA content, one of which was composed of a single type of chromosome, namely 6 V, sorted with over 85% purity. Chromosome 6 V is known to carry genes to code for important resistance and seed storage characteristics, and its isolation represents a new source of genetic traits and specific markers useful for wheat improvement.     

Patterns of chloroplast diversity among western European Dactylorhiza species (Orchidaceae)

In Europe, the genus Dactylorhiza comprises a bewildering variety of forms that are difficult to sort into discrete species. Most Dactylorhiza species are diploid or tetraploid and contrasting hypotheses have been proposed to explain the complex variation within this group. Using PCR-RFLP analysis in eight putative species, we could identify four highly differentiated chloroplast DNA lineages. The first lineage (clade A) included the unique haplotype found in D. sambucina. Clade B grouped four haplotypes belonging mostly to D. incarnata. Clades C and D included 27 haplotypes detected in diploid D. fuchsii and in all tetraploid species investigated. Eighty percent of the chloroplast variation were consistent with currently accepted species boundaries. The imperfect agreement between chloroplast variation and species boundaries may be ascribed to incomplete lineage sorting and/or reticulation. Our cpDNA results provide strong evidence that the allotetrapolyploids have been formed through asymmetric hybridization with a member of the D. fuchsii / maculata group as the maternal parent.     

Phylogeny and chromosomal variations in East Asian Carex, Siderostictae group (Cyperaceae), based on DNA sequences and cytological data

Carex (Cyperaceae) is one of the largest genera of the flowering plants, and comprises more than 2,000 species. In Carex, section Siderostictae with broader leaves distributed in East Asia is thought to be an ancestral group. We aimed to clarify the phylogenetic relationships and chromosomal variations within the section Siderostictae, and to examine the relationship of broad-leaved species of the sections Hemiscaposae and Surculosae from East Asia, inferred from DNA sequences and cytological data. Our results indicate that a monophyletic Siderostictae clade, including the sections Hemiscaposae, Siderostictae and Surculosae, as the earliest diverging group in the tribe Cariceae. Low chromosome numbers, 2n = 12 or 24, with large sizes were observed in these three sections. Our results suggest that the genus Carex might have originated or relictly restricted in the East Asia. Geographical distributions of diploid species are restricted in narrower areas, while those of tetraploid species are wider in East Asia. It is concluded that chromosomal variations in Siderostictae clade may have been caused by polyploidization and that tetraploid species may have been able to exploit their habitats by polyploidization.     

Species relationships in the genus Tulipa

The relationships of diploid and polyploid species belonging to the sub-section Eriostemones of the genus Tulipa have been investigated from the point of view of chromosome morphology, relative DNA values, and meiotic pairing properties. Karyotype morphology is basically the same for all species and eight principal chromosome classes can be distinguished. The range of DNA values obtained by Feulgen cytophotometry for the diploid species is relatively small and provides little assistance in ascertaining the pattern of ploidy involved in the evolution of the group. Partial asynapsis and reverse loop pairing in several of the diploids at pachytene reveals a degree of structural differentiation which may be the outcome of hybridity between species. Significantly, these anomalies are rarely encountered at the equivalent tetraploid levels and a scheme is proposed to account for this behaviour.     

SPECIATION BY POLYPLOIDY IN TREEFROGS: MULTIPLE ORIGINS OF THE TETRAPLOID,HYLA VERSICOLOR

Speciation by polyploidy is rare in animals, yet, in vertebrates, there is a disproportionate concentration of polyploid species in anuran amphibians. Sequences from the cytochrome b gene of the mitochondrial DNA (mtDNA) were used to determine phylogenetic relationships among 37 populations of the diploid-tetraploid species pair of gray treefrogs, Hyla chrysoscelis and Hyla versicolor. The diploid species, H. chrysoscelis, consists of an eastern and a western lineage that have 2.3% sequence divergence between them. The tetraploid species, H. versicolor, had at least three separate, independent origins. Two of the tetraploid lineages are more closely related to one or the other of the diploid lineages (0.18%–1.4% sequence divergence) than they are to each other (1.9%–3.4% sequence divergence). The maternal ancestor of the third tetraploid lineage is unknown. The phylogenetic relationships between the two species and among lineages within each species support the hypothesis of multiple origins of the tetraploid lineages.     

Chromosomal Locations of 5S and 45S rDNA in Gossypium Genus and Its Phylogenetic Implications Revealed by FISH

We investigated the locations of 5S and 45S rDNA in Gossypium diploid A, B, D, E, F, G genomes and tetraploid genome (AD) using multi-probe fluorescent in situ hybridization (FISH) for evolution analysis in Gossypium genus. The rDNA numbers and sizes, and synteny relationships between 5S and 45S were revealed using 5S and 45S as double-probe for all species, and the rDNA-bearing chromosomes were identified for A, D and AD genomes with one more probe that is single-chromosome-specific BAC clone from G. hirsutum (A₁D₁). Two to four 45S and one 5S loci were found in diploid-species except two 5S loci in G . incanum (E₄), the same as that in tetraploid species. The 45S on the 7th and 9th chromosomes and the 5S on the 9th chromosomes seemed to be conserved in A, D and AD genomes. In the species of B, E, F and G genomes, the rDNA numbers, sizes, and synteny relationships were first reported in this paper. The rDNA pattern agrees with previously reported phylogenetic history with some disagreements. Combined with the whole-genome sequencing data from G . raimondii (D₅) and the conserved cotton karyotype, it is suggested that the expansion, decrease and transposition of rDNA other than chromosome rearrangements might occur during the Gossypium evolution.