Parental origin of the allotetraploid tobacco Nicotiana benthamiana

Nicotiana section Suaveolentes is an almost all‐Australian clade of allopolyploid tobacco species including the important plant model Nicotiana benthamiana. The homology relationships of this clade and its formation are not completely understood. To address this gap, we assessed phylogenies of all individual genes of N. benthamiana and the well studied N. tabacum (section Nicotiana) and their homologues in six diploid Nicotiana species. We generated sets of 44 424 and 65 457 phylogenetic trees of N. benthamiana and N. tabacum genes, respectively, each collectively called a phylome. Members of Nicotiana sections Noctiflorae and Sylvestres were represented as the species closest to N. benthamiana in most of the gene trees. Analyzing the gene trees of the phylome we: (i) dated the hybridization event giving rise to N. benthamiana to 4–5 MyA, and (ii) separated the subgenomes. We assigned 1.42 Gbp of the genome sequence to section Noctiflorae and 0.97 Gbp to section Sylvestres based on phylome analysis. In contrast, read mapping of the donor species did not succeed in separating the subgenomes of N. benthamiana. We show that the maternal progenitor of N. benthamiana was a member of section Noctiflorae, and confirm a member of section Sylvestres as paternal subgenome donor. We also demonstrate that the advanced stage of long‐term genome diploidization in N. benthamiana is reflected in its subgenome organization. Taken together, our results underscore the usefulness of phylome analysis for subgenome characterization in hybrid species.     

Genetic investigation of the origination of allopolyploid with virtually synthesized lines: application to the C subgenome of Brassica napus

Although there are a number of different allopolyploids in the plant kingdom, the exact ancestral parents of some allopolyploids have not been well characterized. We propose a strategy in which virtual allopolyploid lines derived from different types of parental species are used to investigate the progenitors of an allopolyploid. The genotypes of the parental lines and the natural allopolyploid were established using a set of DNA molecular markers. The genotypes of the virtual lines were then derived from those of the parental lines, and compared extensively with that of the natural allopolyploid. We applied this strategy to investigate the progenitors of the C subgenome of Brassica napus (rapeseed, AACC). A total of 39 accessions from 10 wild and 7 cultivated types of the B. oleracea cytodeme (CC), and 4 accessions of B. rapa (AA) were used to construct 156 virtual rapeseed lines. Genetic structure was compared among natural rapeseed, virtual rapeseed lines, and their parental lines by principal component analysis and analysis of ancestry. Our data showed that the C subgenome of natural rapeseed was related closely to the genome of cultivated B. oleracea and its related wild types, such as B. incana, B. bourgeaui, B. montana, B. oleracea ssp. oleracea and B. cretica. This finding indicated that these types or their progeny might be ancestral donors of the C subgenome of rapeseed. The successful application of the strategy of virtual allopolyploidy in rapeseed demonstrates that it can possibly be used to identify the progenitors of an allopolyploid species.     

A high frequency of allopolyploid speciation in the gymnospermous genus Ephedra and its possible association with some biological and ecological features

The origin and evolution of polyploids have been studied extensively in angiosperms and ferns but very rarely in gymnosperms. With the exception of three species of conifers, all natural polyploid species of gymnosperms belong to Ephedra, in which more than half of the species show polyploid cytotypes. Here, we investigated the origin and evolution of polyploids of Ephedra distributed in the Qinghai–Tibetan Plateau (QTP) and neighbouring areas. Flow cytometry (FCM) was used to measure the ploidy levels of the sampled species that are represented by multiple individuals from different populations, and then, two single‐copy nuclear genes (LFY and DDB2) and two chloroplast DNA fragments were used to unravel the possible origins and maternal donors of the polyploids. The results indicate that the studied polyploid species are allopolyploids, and suggest that allotetraploidy is a dominant mode of speciation in Ephedra. The high percentage of polyploids in the genus could be related to some of its biological attributes such as vegetative propagation, a relatively high rate of unreduced gamete formation, and a small genome size relative to most other gymnosperms. Significant ecological divergences between allotetraploids and their putative progenitors were detected by PCAs and anova and Tukey's tests, with the exception of E. saxatilis. The overlap of geographical distributions and ecological niches of some diploid species could have provided opportunities for interspecific hybridization and allopolyploid speciation.     

Diploidization and genome size change in allopolyploids is associated with differential dynamics of low‐ and high‐copy sequences

Recent advances have highlighted the ubiquity of whole‐genome duplication (polyploidy) in angiosperms, although subsequent genome size change and diploidization (returning to a diploid‐like condition) are poorly understood. An excellent system to assess these processes is provided by Nicotiana section Repandae, which arose via allopolyploidy (approximately 5 million years ago) involving relatives of Nicotiana sylvestris and Nicotiana obtusifolia. Subsequent speciation in Repandae has resulted in allotetraploids with divergent genome sizes, including Nicotiana repanda and Nicotiana nudicaulis studied here, which have an estimated 23.6% genome expansion and 19.2% genome contraction from the early polyploid, respectively. Graph‐based clustering of next‐generation sequence data enabled assessment of the global genome composition of these allotetraploids and their diploid progenitors. Unexpectedly, in both allotetraploids, over 85% of sequence clusters (repetitive DNA families) had a lower abundance than predicted from their diploid relatives; a trend seen particularly in low‐copy repeats. The loss of high‐copy sequences predominantly accounts for the genome downsizing in N. nudicaulis. In contrast, N. repanda shows expansion of clusters already inherited in high copy number (mostly chromovirus‐like Ty3/Gypsy retroelements and some low‐complexity sequences), leading to much of the genome upsizing predicted. We suggest that the differential dynamics of low‐ and high‐copy sequences reveal two genomic processes that occur subsequent to allopolyploidy. The loss of low‐copy sequences, common to both allopolyploids, may reflect genome diploidization, a process that also involves loss of duplicate copies of genes and upstream regulators. In contrast, genome size divergence between allopolyploids is manifested through differential accumulation and/or deletion of high‐copy‐number sequences.     

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.     

Polyploid evolution and plastid DNA variation in the Dactylorhiza incarnata/maculata complex (Orchidaceae) in Scandinavia

The Dactylorhiza incarnata/maculata complex (Orchidaceae) was used as a model system to understand genetic differentiation processes in a naturally occurring polyploid complex with much of ongoing diversification and wide distribution in recently glaciated areas in northern Europe. Data were obtained for 12 hypervariable regions in the plastid DNA genome. A total of 166 haplotypes were found in a sample of 1099 plants. Allopolyploid taxa have inherited their plastid genomes from D. maculata s.l. Overall haplotype diversity of the combined group of allopolyploid taxa was comparable to that of maternal D. maculata s.l., but populations of allopolyploids were also more strongly differentiated from each other and contained lower numbers of haplotypes than populations of D. maculata s.l. In addition to haplotypes found in extant D. maculata s.l., the allopolyploids also contained several distinct and widespread haplotypes that were not found in any of the parental lineages. Some of these haplotypes were shared between widespread allopolyploids. Divergent allopolyploids with small distributions did not seem to originate from local polyploidization events, but rather as segregates of already existing allopolyploids. Genetic diversification of allopolyploid Dactylorhiza is the result of repeated polyploid formation, secondary hybridization and introgression between already existing polyploids and extant representatives of parental lineages, hybridization between independently derived polyploid lineages, and phyletic diversification in the group of allopolyploids. Although some polyploid taxa must have evolved after the last glaciation, genetic material from the parental lineages has been transferred continuously for longer periods of time. This combination of processes may explain the taxonomic complexity encountered in Dactylorhiza and other polyploid complexes distributed in previously glaciated parts of Europe.     

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.     

Nuclear DNA amount and genome downsizing in natural and synthetic allopolyploids of the genera Aegilops and Triticum

Recent molecular studies in the genera Aegilops and Triticum showed that allopolyploidization (interspecific or intergeneric hybridization followed by chromosome doubling) generated rapid elimination of low-copy or high-copy, non-coding and coding DNA sequences. The aims of this work were to determine the amount of nuclear DNA in allopolyploid species of the group and to see to what extent elimination of DNA sequences affected genome size. Nuclear DNA amount was determined by the flow cytometry method in 27 natural allopolyploid species (most of which were represented by several lines and each line by several plants) as well as 14 newly synthesized allopolyploids (each represented by several plants) and their parental plants. Very small intraspecific variation in DNA amount was found between lines of allopolyploid species collected from different habitats or between wild and domesticated forms of allopolyploid wheat. In contrast to the constancy in nuclear DNA amount at the intraspecific level, there are significant differences in genome size between the various allopolyploid species, at both the tetraploid and hexaploid levels. In most allopolyploids nuclear DNA amount was significantly less than the sum of DNA amounts of the parental species. Newly synthesized allopolyploids exhibited a similar decrease in nuclear DNA amount in the first generation, indicating that genome downsizing occurs during and (or) immediately after the formation of the allopolyploids and that there are no further changes in genome size during the life of the allopolyploids. Phylogenetic considerations of the origin of the B genome of allopolyploid wheat, based on nuclear DNA amount, are discussed.     

Phylogenetic relationships in Nicotiana (Solanaceae) inferred from multiple plastid DNA regions

For Nicotiana, with 75 naturally occurring species (40 diploids and 35 allopolyploids), we produced 4656bp of plastid DNA sequence for 87 accessions and various outgroups. The loci sequenced were trnL intron and trnL-F spacer, trnS-G spacer and two genes, ndhF and matK. Parsimony and Bayesian analyses yielded identical relationships for the diploids, and these are consistent with other data, producing the best-supported phylogenetic assessment currently available for the genus. For the allopolyploids, the line of maternal inheritance is traced via the plastid tree. Nicotiana and the Australian endemic tribe Anthocercideae form a sister pair. Symonanthus is sister to the rest of Anthocercideae. Nicotiana sect. Tomentosae is sister to the rest of the genus. The maternal parent of the allopolyploid species of N. sect. Polydicliae were ancestors of the same species, but the allopolyploids were produced at different times, thus making such sections paraphyletic to their extant diploid relatives. Nicotiana is likely to have evolved in southern South America east of the Andes and later dispersed to Africa, Australia, and southwestern North America.     

RECONSTRUCTING THE COMPLEX EVOLUTIONARY ORIGIN OF WILD ALLOPOLYPLOID TOBACCOS (NICOTIANA SECTION SUAVEOLENTES)

Nicotiana (Solanaceae) provides an ideal system for understanding polyploidization, a pervasive and powerful evolutionary force in plants, as this genus contains several groups of allotetraploids that formed at different times from different diploid progenitors. However, the parental lineages of the largest group of allotetraploids, Nicotiana section Suaveolentes, have been problematic to identify. Using data from four regions of three low‐copy nuclear genes, nuclear ribosomal DNA, and regions of the plastid genome, we have reconstructed the evolutionary origin of sect. Suaveolentes and identified the most likely diploid progenitors by using a combination of gene trees and network approaches to uncover the most strongly supported evidence of species relationships. Our analyses best support a scenario where a member of the sect. Sylvestres lineage acted as the paternal progenitor and a member of either sect. Petunioides or sect. Noctiflorae that also contained introgressed DNA from the other, or a hypothetical hybrid species between these two sections, was the maternal progenitor. Nicotiana exemplifies many of the factors that can complicate the reconstruction of polyploid evolutionary history and highlights how reticulate evolution at the diploid level can add even greater complexity to allopolyploid genomes.     

Nonadditive changes in genome size during allopolyploidization in the wheat (aegilops-triticum) group

Interspecific or intergeneric hybridization, followed by chromosome doubling, can lead to the formation of new allopolyploid species. Recent studies indicate that allopolyploid formation is associated with genetic and epigenetic changes. Despite these studies, it is not yet clear whether the C value of an allopolyploid is the sum of its diploid parents. To address this question, six newly synthesized wheat allopolyploids and their parental plants were investigated. It was found that allopolyploids have a genome size significantly smaller than the expected value. The reduction of the nuclear genome size in the synthetic allotetraploids and allohexaploids was 2 pg DNA at 2C. It was also found that changes in the genome size already existed in the first generation amphiploids, indicating that the change was a rapid event. There was no difference in the reduction of nuclear genome size between the allotetraploid and the allohexaploid. These data clearly show that genome differentiation in allopolyploids was not related to the ploidy level. The data obtained clearly suggested that the nonadditive change in genome size that occurred during allopolyploidization may represent a preprogrammed adaptive response to genomic stress caused by hybridization and allopolyploidy, which serves to stabilize polyploid genomes.     

Chloroplast nucleoids are highly dynamic in ploidy,number, and structure during angiosperm leaf development

Chloroplast nucleoids are large, compact nucleoprotein structures containing multiple copies of the plastid genome. Studies on structural and quantitative changes of plastid DNA (ptDNA) during leaf development are scarce and have produced controversial data. We have systematically investigated nucleoid dynamics and ptDNA quantities in the mesophyll of Arabidopsis, tobacco, sugar beet, and maize from the early post‐meristematic stage until necrosis. DNA of individual nucleoids was quantified by DAPI‐based supersensitive epifluorescence microscopy. Nucleoids occurred in scattered, stacked, or ring‐shaped arrangements and in recurring patterns during leaf development that was remarkably similar between the species studied. Nucleoids per organelle varied from a few in meristematic plastids to >30 in mature chloroplasts (corresponding to about 20–750 nucleoids per cell). Nucleoid ploidies ranged from haploid to >20‐fold even within individual organelles, with average values between 2.6‐fold and 6.7‐fold and little changes during leaf development. DNA quantities per organelle increased gradually from about a dozen plastome copies in tiny plastids of apex cells to 70–130 copies in chloroplasts of about 7 μm diameter in mature mesophyll tissue, and from about 80 plastome copies in meristematic cells to 2600–3300 copies in mature diploid mesophyll cells without conspicuous decline during leaf development. Pulsed‐field electrophoresis, restriction of high‐molecular‐weight DNA from chloroplasts and gerontoplasts, and CsCl equilibrium centrifugation of single‐stranded and double‐stranded ptDNA revealed no noticeable fragmentation of the organelle DNA during leaf development, implying that plastid genomes in mesophyll tissues are remarkably stable until senescence.     

Allopolyploidy-Induced Rapid Genome Evolution in the Wheat (Aegilops–Triticum) Group

To better understand genetic events that accompany allopolyploid formation, we studied the rate and time of elimination of eight DNA sequences in F1 hybrids and newly formed allopolyploids of Aegilops and Triticum. In total, 35 interspecific and intergeneric F1 hybrids and 22 derived allopolyploids were analyzed and compared with their direct parental plants. The studied sequences exist in all the diploid species of the Triticeae but occur in only one genome, either in one homologous pair (chromosome-specific sequences [CSSs]) or in several pairs of the same genome (genome-specific sequences [GSSs]), in the polyploid wheats. It was found that rapid elimination of CSSs and GSSs is a general phenomenon in newly synthesized allopolyploids. Elimination of GSSs was already initiated in F1 plants and was completed in the second or third allopolyploid generation, whereas elimination of CSSs started in the first allopolyploid generation and was completed in the second or third generation. Sequence elimination started earlier in allopolyploids whose genome constitution was analogous to natural polyploids compared with allopolyploids that do not occur in nature. Elimination is a nonrandom and reproducible event whose direction was determined by the genomic combination of the hybrid or the allopolyploid. It was not affected by the genotype of the parental plants, by their cytoplasm, or by the ploidy level, and it did not result from intergenomic recombination. Allopolyploidy-induced sequence elimination occurred in a sizable fraction of the genome and in sequences that were apparently noncoding. This finding suggests a role in augmenting the differentiation of homoeologous chromosomes at the polyploid level, thereby providing the physical basis for the diploid-like meiotic behavior of newly formed allopolyploids. In our view, this rapid genome adjustment may have contributed to the successful establishment of newly formed allopolyploids as new species.     

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.     

The plastid genome of the red alga Laurencia

We present the 174,935 nt long plastid genome of the red alga Laurencia sp. JFC0032. It is the third plastid genome characterized for the largest order of red algae (Ceramiales). The circular‐mapping plastid genome is small compared to most florideophyte red algae, and our comparisons show a trend toward smaller plastid genome sizes in the family Rhodomelaceae, independent from a similar trend in Cyanidiophyceae. The Laurencia genome is densely packed with 200 annotated protein‐coding genes (188 widely conserved, 3 open reading frames shared with other red algae and 9 hypothetical coding regions). It has 29 tRNAs, a single‐copy ribosomal RNA cistron, a tmRNA, and the RNase P RNA.