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.     

Chromosomal stasis in diploids contrasts with genome restructuring in auto- and allopolyploid taxa of Hepatica (Ranunculaceae)

Polyploidization and chromosomal rearrangements are recognized as major forces in plant evolution. Their role is investigated in the disjunctly distributed northern hemisphere Hepatica (Ranunculaceae). Chromosome numbers, karyotype morphology, banding patterns, 5S and 35S rDNA localization in all known species were investigated and interpreted in a phylogenetic context established from nuclear internal transcribed spacer (ITS) and plastid matK sequences. All species had a chromosome base number of x = 7. The karyotype was symmetric and showed little variation among diploids with one locus each of 5S and 35S rDNA, except for interpopulational variation concerning 35S rDNA loci number and localization in H. asiatica. Tetraploids exhibited chromosomal changes, including asymmetry and/or loss of rDNA loci. Nuclear and plastid sequences resulted in incongruent topologies because of the positions of some tetraploid taxa. The diversification of Hepatica occurred not earlier than the Pliocene. Genome restructuring, especially involving 35S rDNA, within a few million yr or less characterizes evolution of both auto- and allopolyploids and of the diploid species H. asiatica, which is the presumptive ancestor of two other diploid species.     

Long-term genome diploidization in allopolyploid Nicotiana section Repandae (Solanaceae)

Here, we analyze long-term evolution in Nicotiana allopolyploid section Repandae (the closest living diploids are N. sylvestris, the maternal parent, and N. obtusifolia, the paternal parent). We compare data with other more recently formed Nicotiana allopolyploids. We investigated 35S and 5S nuclear ribosomal DNA (rDNA) chromosomal location and unit divergence. A molecular clock was applied to the Nicotiana phylogenetic tree to determine allopolyploid ages. N. tabacum and species of Repandae were c. 0.2 and 4.5 Myr old, respectively. In all Repandae species, the numbers of both 35S and 5S rDNA loci were less than the sum of those of the diploid progenitors. Trees based on 5S rDNA spacer sequences indicated units of only the paternal parent. In recent Nicotiana allopolyploids, the numbers of rDNA loci equal the sum of those of their progenitors. In the Repandae genomes, diploidization is associated with locus loss. Sequence analysis indicates that 35S and 5S units most closely resemble maternal and paternal progenitors, respectively. In Nicotiana, 4.5 Myr of allopolyploid evolution renders genomic in situ hybridization (GISH) unsuitable for the complete resolution of parental genomes.     

Interpopulation hybridization generates meiotically stable rDNA epigenetic variants in allotetraploid Tragopogon mirus

Uniparental silencing of 35S rRNA genes (rDNA), known as nucleolar dominance (ND), is common in interspecific hybrids. Allotetraploid Tragopogon mirus composed of Tragopogon dubius (d) and Tragopogon porrifolius (p) genomes shows highly variable ND. To examine the molecular basis of such variation, we studied the genetic and epigenetic features of rDNA homeologs in several lines derived from recently and independently formed natural populations. Inbred lines derived from T. mirus with a dominant d‐rDNA homeolog transmitted this expression pattern over generations, which may explain why it is prevalent among natural populations. In contrast, lines derived from the p‐rDNA dominant progenitor were meiotically unstable, frequently switching to co‐dominance. Interpopulation crosses between progenitors displaying reciprocal ND resulted in d‐rDNA dominance, indicating immediate suppression of p‐homeologs in F₁ hybrids. Original p‐rDNA dominance was not restored in later generations, even in those segregants that inherited the corresponding parental rDNA genotype, thus indicating the generation of additional p‐rDNA and d‐rDNA epigenetic variants. Despite preserved intergenic spacer (IGS) structure, they showed altered cytosine methylation and chromatin condensation patterns, and a correlation between expression, hypomethylation of RNA Pol I promoters and chromatin decondensation was apparent. Reversion of such epigenetic variants occurred rarely, resulting in co‐dominance maintained in individuals with distinct genotypes. Generally, interpopulation crosses may generate epialleles that are not present in natural populations, underlying epigenetic dynamics in young allopolyploids. We hypothesize that highly expressed variants with distinct IGS features may induce heritable epigenetic reprogramming of the partner rDNA arrays, harmonizing the expression of thousands of genes in allopolyploids.     

Switch them off or not: selective rRNA gene repression in grasses

Nucleolar dominance (ND) is selective epigenetic silencing of 35-48S rDNA loci. In allopolyploids, it is frequently manifested at the cytogenetic level by the inactivation of nucleolar organiser region(s) (NORs) inherited from one or several evolutionary ancestors. Grasses are ecologically and economically one of the most important land plant groups, which have frequently evolved through hybridisation and polyploidisation events. Here we review common and unique features of ND phenomena in this monocot family from cytogenetic, molecular, and genomic perspectives. We highlight recent advances achieved by using an allotetraploid model grass, Brachypodium hybridum, where ND commonly occurs at a population level, and we cover modern genomic approaches that decipher structural features of core arrays of NORs.     

Molecular cytogenetic analysis of recently evolved Tragopogon (Asteraceae) allopolyploids reveal a karyotype that is additive of the diploid progenitors

Tragopogon mirus and T. miscellus (both 2n = 4x = 24) are recent allotetraploids derived from T. dubius × T. porrifolius and T. dubius × T. pratensis (each 2n = 2x = 12), respectively. The genome sizes of T. mirus are additive of those of its diploid parents, but at least some populations of T. miscellus have undergone genome downsizing. To survey for genomic rearrangements in the allopolyploids, four repetitive sequences were physically mapped. TPRMBO (unit size 160 base pairs [bp]) and TGP7 (532 bp) are tandemly organized satellite sequences isolated from T. pratensis and T. porrifolius, respectively. Fluorescent in situ hybridization to the diploids showed that TPRMBO is a predominantly centromeric repeat on all 12 chromosomes, while TGP7 is a subtelomeric sequence on most chromosome arms. The distribution of tandem repetitive DNA loci (TPRMBO, TGP7, 18S-5.8S-26S rDNA, and 5S rDNA) gave unique molecular karyotypes for the three diploid species, permitting the identification of the parental chromosomes in the polyploids. The location and number of these loci were inherited without apparent changes in the allotetraploids. There was no evidence for major genomic rearrangements in Tragopogon allopolyploids that have arisen multiple times in North America within the last 80 yr.     

Analysis of 5S rDNA changes in synthetic allopolyploids Triticum x Aegilops

By the example of three synthetic allopolyploids: Aegilops sharonensis x Ae. umbellulata (2n =28), Triticum urartu x Ae. tauschii (2n =28), T. dicoccoides x Ae. tauschii (2n =42) the 5S rDNA changes at the early stage of allopolyploidization were investigated. Using fluorescent in situ hybridization (FISH), the quantitative changes affecting the separate loci of one of the parental genomes were revealed in plants of S3 generation of each hybrid combination. Souther hybridization with genomic DNA of allopolyploid T. urartu x Ae. tauschii (TMU38 x TQ27) revealed lower intensity of the fragments from Ae. tauschii compared with the T. urartu fragments. It may be confirmation of the reduction of signal on 1D chromosome that was revealed in this hybrid using FISH. Both appearance of a new 5S rDNA fragments and full disappearance of fragments from parental species were not showed by Southern hybridization, as well as PCR-analysis of 5-15 plants of S2-S3 generations. The changes were not found under comparison of primary structure of nine 5S rDNA sequences of allopolyploid TMU38 x TQ27 with analogous sequences from parental species genomes. The observable similarity by FISH results of one of the studied synthetic allopolyploids with natural allopolyploid of similar genome composition indicates the early formation of unique for each allopolyploid 5S rDNA organization.     

Recent and recurrent polyploidy in Tragopogon (Asteraceae): cytogenetic, genomic and genetic comparisons

Tragopogon mirus Ownbey and T. miscellus Ownbey are allopolyploids that formed repeatedly during the past 80 years following the introduction of three diploids (T. dubius Scop., T. pratensis L. and T. porrifolius L.) from Europe to western North America. These polyploid species of known parentage are useful for studying the consequences of recent and recurrent polyploidization. We summarize recent analyses of the cytogenetic, genomic and genetic consequences of polyploidy in Tragopogon. Analyses of rDNA ITS (internal transcribed spacer) + ETS (external transcribed spacer) sequence data indicate that the parental diploids are phylogenetically well separated within Tragopogon (a genus of perhaps 150 species), in agreement with isozymic and cpDNA data. Using Southern blot and cloning experiments on tissue from early herbarium collections of T. mirus and T. miscellus (from 1949) to represent the rDNA repeat condition closer to the time of polyploidization than samples collected today, we have demonstrated concerted evolution of rDNA. Concerted evolution is ongoing, but has not proceeded to completion in any polyploid population examined; rDNA repeats of the diploid T. dubius are typically lost or converted in both allopolyploids, including populations of independent origin. Molecular cytogenetic studies employing rDNA probes, as well as centromeric and subtelomeric repeats isolated from Tragopogon, distinguished all chromosomes among the diploid progenitors (2n = 12). The diploid chromosome complements are additive in both allopolyploids (2n = 24); there is no evidence of major chromosomal rearrangements in populations of either T. mirus or T. miscellus. cDNA‐AFLP display revealed differences in gene expression between T. miscellus and its diploid parents, as well as between populations of T. miscellus of reciprocal origin. Approximately 5% of the genes examined in the allopolyploid populations have been silenced, and an additional 4% exhibit novel gene expression relative to their diploid parents. Some of the differences in gene expression represent maternal or paternal effects. Multiple origins of a polyploid species not only affect patterns of genetic variation in natural populations, but also contribute to differential patterns of gene expression and may therefore play a major role in the long‐term evolution of polyploids. © 2004 The Linnean Society of London, Biological Journal of the Linnean Society, 2004, 82 , 485–501.     

Concerted evolution of 18–5.8–26S rDNA repeats in Nicotiana allotetraploids

We review and extend data showing concerted evolution of parental 18–5.8–26S nuclear ribosomal DNA (18–26S rDNA) gene families in three natural Nicotiana allotetraploids ( N. tabacum , N. rustica and N. arentsii , each 2 n  = 4 x  = 48) and one synthetic N. tabacum line (Th37, ♀ N. sylvestris (2 n  = 24) × ♂ N. tomentosiformis (2 n  = 24)). The origin of the gene families was analysed by sequence polymorphisms in the intergenic spacer (IGS) region and the number of chromosomal loci by fluorescence in situ hybridization (FISH). FISH revealed that the number and locations of 18–26S rDNA in the natural allopolyploids was the sum of those found in the diploid progenitors. However, the rDNA restriction patterns showed polymorphisms in the IGS that were not additive, suggesting that parental rDNA clusters were partially ( N. tabacum, N. rustica ) or completely ( N. arentsii ) overwritten by hybrid-specific units. Thus the Nicotiana allotetraploids show evidence of concerted evolution, including both intralocus and interlocus gene conversion. A feral N. tabacum collected in Bolivia had a higher proportion of unconverted parental rDNA units than cultivated tobacco varieties, suggesting either that rDNA homogenization is accelerated by inbreeding or multiple origins of tobacco. There is no evidence for the elimination of N. sylvestris- derived rDNA units in the synthetic Th37 tobacco line as occurred in natural tobacco, although several novel rDNA unit variants were found in most but not all the hybrid plants. Factors that may control the occurrence and extent of rDNA homogenization are discussed for allopolyploids in Nicotiana and other taxa.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 82 , 615–625.     

Diverse evolutionary pathways shaped 5S rDNA of species of tribe Anemoneae (Ranunculaceae) and reveal phylogenetic signal

Anemone sensu lato (including Pulsatilla and Hepatica), tribe Anemoneae (Ranunculaceae), is arranged into two subgenera, Anemone and Anemonidium, with basic chromosome numbers x = 8 and x = 7, respectively. We elucidated the level of divergence of 5S rDNA unit arrays between the subgenera, determined intra‐individual and interspecific sequence variation and tested 5S rDNA phylogenetic signal in revealing the origin of polyploid species. High intra‐individual nucleotide diversity and the presence of 5S rDNA unit array length variants and pseudogenes indicate that weak homogenization forces have shaped 5S rDNA in the investigated species. Our results show that 5S rDNA evolved through two major changes: diversification of 5S rDNA into two lineages, one with long (subgenus Anemone) and one with short 5S rDNA unit arrays (subgenus Anemonidium); and subsequent contraction and expansion of 5S rDNA unit arrays. Phylogenetic analysis based on 5S rDNA supports the hypothesis that A. parviflora could be a parental species and donor of the subgenome D to the allopolyploids A. multifida (BBDD) and A. baldensis (AABBDD). In A. baldensis interlocus exchange possibly occurred, followed by subsequent replacement of the 5S rDNA from subgenome D with those from subgenome B. Here we present evidence that both models, concerted and birth‐and‐death evolution, were probably involved in the evolution of the 5S rDNA multigene family in subgenera Anemone and Anemonidium.     

Evolution of parental ITS regions of nuclear rDNA in allopolyploid Aegilops (Poaceae) species

The genus Aegilops comprises approximately 25 diploid, tetraploid and hexaploid species, in which the genome types of all allopolyploids involve either U or D genome, or both of them. The internal transcribed spacer (ITS) region of 18S-26S nuclear ribosomal DNA (rDNA) from 11 allopolyploid species and 7 related diploid species in the genus were directly sequenced by pooled PCR products. Phylogenetic analyses for tracing evolutionary patterns of parental rDNA in allopolyploid species were performed using the neighbor-joining method. The D genome involved tree included three clades (CC-DDCC, DDMM-DDMMSS-DDMMUU, and MM-MhMh-DDNN), but did not include Ae. squarrosa (DD). It indicated that the rDNA of ancestral D genome had been somewhat differentiated in allopolyploids. The U genome involved tree showed that the allopolyploids and their common ancestor, Ae. umbellulata, formed a clade, suggesting that rDNA in UUMM and UUSS genomes has been homogenizing toward that of ancestral U genome. The phylogenetic pattern of U genome based on ITS sequences also supported the "pivotal-differential" hypothesis.     

Physical mapping of rDNA loci in Brassica species.

The number of major rDNA loci (the genes coding for 18S-5.8S-26S rRNA) was investigated in the economically important Brassica species and their wild relatives by in situ hybridization of an rDNA probe to metaphase chromosomes and interphase nuclei. The diploid species B. nigra (B genome) has two major pairs of rDNA loci, B. oleracea (C genome) has two major pairs and one minor pair of loci, while B. campestris (A genome) has five pairs of loci. Among the three tetraploid species arising from these three diploid ancestors, B. carinata (BBCC genomes) has four loci, B. juncea (AABB genomes) has five major pairs and one minor pair of loci, and B. napus (AACC genomes) has six pairs of loci, indicating that the number of loci has been reduced during evolution. The complexity of the known rDNA restriction fragment length polymorphism patterns gave little indication of number of rDNA loci. It is probable that chromosome rearrangements have occurred during evolution of the amphidiploid species. The data will be useful for physical mapping of genes relative to rDNA loci, micro- and macro-evolutionary studies and analysis of aneuploids including addition and substitution lines used in Brassica breeding programs.     

Uniparental loss of ribosomal DNA in the allotetraploid grass Zingeria trichopoda (2n = 8).

Analysis of the grass Zingeria trichopoda (2n = 8, 2C = 5.3 pg) revealed a dynamic evolution with the following characteristics. (i) Genomic in situ hybridization (GISH) demonstrates that Z. trichopoda evolved from an interspecific hybrid involving a species like contemporary Zingeria biebersteiniana (2n = 4) and a second species with a similar low number of chromosomes. The nucleus of Z. trichopoda is spatially organized at the genome level and the two parental genomes occupy distinct and separate domains of lateral arrangements. (ii) The copy number of the Z. biebersteiniana specific pericentromeric tandem repeat family Zbcen1 is drastically reduced in Z. trichopoda. (iii) GISH in combination with labeled rDNA sequences simultaneously discriminated the two parental genomes and the corresponding 5S and 45S rDNA sites. Hence, following allopolyploidization of Z. trichopoda the Z. biebersteiniana like parental chromosomes probably underwent drastic loss of 45S rDNA. This could have arisen either through the loss of Z. biebersteiniana derived 45S rDNA or through Z. trichopoda genome-wide homogenization of Z. biebersteiniana type 45S rDNA and subsequent elimination of 45S rDNA loci from Z. biebersteiniana derived chromosomes. Finally, 5S rDNA loci are present in both subgenomes of Z. trichopoda and the chromosomal position of these loci is similar for both Z. biebersteiniana and the Z. biebersteiniana like parental genome of Z. trichopoda.     

Analysis of 5S rDNA changes in synthetic allopolyploids <Emphasis Type="Italic">Triticum</Emphasis> × <Emphasis Type="Italic">Aegilops</Emphasis>

Changes of 5S rDNA at the early stage of allopolyploidization were investigated in three synthetic allopolyploids: Aegilops sharonensis × Ae. umbellulata (2n = 28), Triticum urartu × Ae. tauschii (2n = 28), and T. dicoccoides × Ae. tauschii (2n = 42). Fluorescent in situ hybridization (FISH) revealed quantitative changes affecting separate loci of one of the parental genomes in S₃ plants of each hybrid combination. Southern hybridization with genomic DNA of the allopolyploid T. urartu × Ae. tauschii (TMU38 × TQ27) revealed a lower intensity of signals from Ae. tauschii fragments compared with those derived from T. urartu. This confirmed the signal reduction revealed for chromosome 1D of this hybrid by FISH. Neither Southern hybridization nor PCR testing of 5–15 plants of the S₂-S₃ generations revealed an appearance of new 5S rDNA fragments or a complete disappearance of parental fragments from the allopolyploids under study. No changes were found by aligning nine 5S rDNA sequences of the allopolyploid TMU38 × TQ27 with corresponding sequences of the parental species. The similarity between one of the synthetic allopolyploids examined and a natural allopolyploid with the same genome composition points to an early formation of the 5S rDNA organization unique for each allopolyploid.     

Heterochromatin patterns and ribosomal DNA loci distribution in diploid and polyploid Crotalaria species (Leguminosae, Papilionoideae), and inferences on karyotype evolution

Most Crotalaria species display a symmetric karyotype with 2n = 16, but 2n = 14 is found in Chrysocalycinae subsection Incanae and 2n = 32 in American species of the section Calycinae. Seven species of the sections Chrysocalycinae, Calycinae, and Crotalaria were analyzed for the identification of heterochromatin types with GC- and AT-specific fluorochromes and chromosomal location of ribosomal DNA loci using fluorescent in situ hybridization (FISH). A major 45S rDNA locus was observed on chromosome 1 in all the species, and a variable number of minor ones were revealed. Only one 5S rDNA locus was observed in the species investigated. Chromomycin A(3) (CMA) revealed CMA(+) bands colocalized with most rDNA loci, small bands unrelated to ribosomal DNA on two chromosome pairs in Crotalaria incana, and CMA(+) centromeric bands that were quenched by distamycin A were detected in species of Calycinae and Crotalaria sections. DAPI(+) bands were detected in C. incana. The results support the species relationships based on flower specialization and were useful for providing insight into mechanisms of karyotype evolution. The heterochromatin types revealed by fluorochromes suggest the occurrence of rearrangements in repetitive DNA families in these heterochromatic blocks during species diversification. This DNA sequence turnover and the variability in number/position of rDNA sites could be interpreted as resulting from unequal crossing over and (or) transposition events. The occurrence of only one 5S rDNA locus and the smaller chromosome size in the polyploids suggest that DNA sequence losses took place following polyploidization events.