Repeat Dynamics across Timescales: A Perspective from Sibling Allotetraploid Marsh Orchids (Dactylorhiza majalis s.l.).

To provide insights into the fate of transposable elements (TEs) across timescales in a post-polyploidization context, we comparatively investigate five sibling Dactylorhiza allotetraploids (Orchidaceae) formed independently and sequentially between 500 and 100K generations ago by unidirectional hybridization between diploids D. fuchsii and D. incarnata. Our results first reveal that the paternal D. incarnata genome shows a marked increased content of LTR retrotransposons compared to the maternal species, reflected in its larger genome size and consistent with a previously hypothesized bottleneck. With regard to the allopolyploids, in the youngest D. purpurella both genome size and TE composition appear to be largely additive with respect to parents, whereas for polyploids of intermediate ages we uncover rampant genome expansion on a magnitude of multiple entire genomes of some plants such as Arabidopsis. The oldest allopolyploids in the series are not larger than the intermediate ones. A putative tandem repeat, potentially derived from a non-autonomous miniature inverted-repeat TE (MITE) drives much of the genome dynamics in the allopolyploids. The highly dynamic MITE-like element is found in higher proportions in the maternal diploid, D. fuchsii, but is observed to increase in copy number in both subgenomes of the allopolyploids. Altogether, the fate of repeats appears strongly regulated and therefore predictable across multiple independent allopolyploidization events in this system. Apart from the MITE-like element, we consistently document a mild genomic shock following the allopolyploidizations investigated here, which may be linked to their relatively large genome sizes, possibly associated with strong selection against further genome expansions.

Differential Genome Size and Repetitive DNA Evolution in Diploid Species of Melampodium sect. Melampodium (Asteraceae).

Plant genomes vary greatly in composition and size mainly due to the diversity of repetitive DNAs and the inherent propensity for their amplification and removal from the host genome. Most studies addressing repeatome dynamics focus on model organisms, whereas few provide comprehensive investigations across the genomes of related taxa. Herein, we analyze the evolution of repeats of the 13 species in Melampodium sect. Melampodium, representing all but two of its diploid taxa, in a phylogenetic context. The investigated genomes range in size from 0.49 to 2.27 pg/1C (ca. 4.5-fold variation), despite having the same base chromosome number (x = 10) and very strong phylogenetic affinities. Phylogenetic analysis performed in BEAST and ancestral genome size reconstruction revealed mixed patterns of genome size increases and decreases across the group. High-throughput genome skimming and the RepeatExplorer pipeline were utilized to determine the repeat families responsible for the differences in observed genome sizes. Patterns of repeat evolution were found to be highly correlated with phylogenetic position, namely taxonomic series circumscription. Major differences found were in the abundances of the SIRE (Ty1-copia), Athila (Ty3-gypsy), and CACTA (DNA transposon) lineages. Additionally, several satellite DNA families were found to be highly group-specific, although their overall contribution to genome size variation was relatively small. Evolutionary changes in repetitive DNA composition and genome size were complex, with independent patterns of genome up- and downsizing throughout the evolution of the analyzed diploids. A model-based analysis of genome size and repetitive DNA composition revealed evidence for strong phylogenetic signal and differential evolutionary rates of major lineages of repeats in the diploid genomes.

The Repetitive DNA Composition in the Natural Pesticide Producer Tanacetum cinerariifolium: Interindividual Variation of Subtelomeric Tandem Repeats.

Dalmatian pyrethrum (Tanacetum cinerariifolium (Trevir.) Sch. Bip.), a plant species endemic to the east Adriatic coast, is used worldwide for production of the organic insecticide, pyrethrin. Most studies concerning Dalmatian pyrethrum have focused on its morphological and biochemical traits relevant for breeding. However, little is known about the chromosomal evolution and genome organization of this species. Our study aims are to identify, classify, and characterize repetitive DNA in the T. cinerariifolium genome using clustering analysis of a low coverage genomic dataset. Repetitive DNA represents about 71.63% of the genome. T. cinerariifolium exhibits linked 5S and 35S rDNA configuration (L-type). FISH reveals amplification of interstitial telomeric repeats (ITRs) in T. cinerariifolium. Of the three newly identified satellite DNA families, TcSAT1 and TcSAT2 are located subterminally on most of T. cinerariifolium chromosomes, while TcSAT3 family is located intercalary within the longer arm of two chromosome pairs. FISH reveals high levels of polymorphism of the TcSAT1 and TcSAT2 sites by comparative screening of 28 individuals. TcSAT2 is more variable than TcSAT1 regarding the number and position of FISH signals. Altogether, our data highlights the dynamic nature of DNA sequences associated with subtelomeres in T. cinerariifolium and suggests that subtelomeres represent one of the most dynamic and rapidly evolving regions in eukaryotic genomes.

Dating the Species Network: Allopolyploidy and Repetitive DNA Evolution in American Daisies (Melampodium sect. Melampodium, Asteraceae).

Allopolyploidy has played an important role in the evolution of the flowering plants. Genome mergers are often accompanied by significant and rapid alterations of genome size and structure via chromosomal rearrangements and altered dynamics of tandem and dispersed repetitive DNA families. Recent developments in sequencing technologies and bioinformatic methods allow for a comprehensive investigation of the repetitive component of plant genomes. Interpretation of evolutionary dynamics following allopolyploidization requires both the knowledge of parentage and the age of origin of an allopolyploid. Whereas parentage is typically inferred from cytogenetic and phylogenetic data, age inference is hampered by the reticulate nature of the phylogenetic relationships. Treating subgenomes of allopolyploids as if they belonged to different species (i.e., no recombination among subgenomes) and applying cross-bracing (i.e., putting a constraint on the age difference of nodes pertaining to the same event), we can infer the age of allopolyploids within the framework of the multispecies coalescent within BEAST2. Together with a comprehensive characterization of the repetitive DNA fraction using the RepeatExplorer pipeline, we apply the dating approach in a group of closely related allopolyploids and their progenitor species in the plant genus Melampodium (Asteraceae). We dated the origin of both the allotetraploid, Melampodium strigosum, and its two allohexaploid derivatives, Melampodium pringlei and Melampodium sericeum, which share both parentage and the direction of the cross, to the Pleistocene ($<$1.4 Ma). Thus, Pleistocene climatic fluctuations may have triggered formation of allopolyploids possibly in short intervals, contributing to difficulties in inferring the precise temporal order of allopolyploid species divergence of M. sericeum and M. pringlei. The relatively recent origin of the allopolyploids likely played a role in the near-absence of major changes in the repetitive fraction of the polyploids' genomes. The repetitive elements most affected by the postpolyploidization changes represented retrotransposons of the Ty1-copia lineage Maximus and, to a lesser extent, also Athila elements of Ty3-gypsy family.

rDNA Loci Evolution in the Genus Glechoma (Lamiaceae).

Glechoma L. (Lamiaceae) is distributed in eastern Asia and Europe. Understanding chromosome evolution in Glechoma has been strongly hampered by its small chromosomes, constant karyotype and polyploidy. Here phylogenetic patterns and chromosomal variation in Glechoma species are considered, using genome sizes, chromosome mapping of 5S and 35S rDNAs by fluorescence in situ hybridisation (FISH), and phylogenetic analyses of internal transcribed spacers (nrITS) of 35S rDNA and 5S rDNA NTS sequences. Species and populations of Glechoma are tetraploid (2n = 36) with base chromosome number of x = 9. Four chromosomes carry pericentric 5S rDNA sites in their short arms in all the species. Two to four of these chromosomes also carry 35S rDNA in subterminal regions of the same arms. Two to four other chromosomes have 35S rDNA sites, all located subterminally within short arms; one individual possessed additional weak pericentric 35S rDNA signals on three other chromosomes. Five types of rDNA locus distribution have been defined on the basis of 35S rDNA variation, but none is species-specific, and most species have more than one type. Glechoma hederacea has four types. Genome size in Glechoma ranges from 0.80 to 0.94 pg (1C), with low levels of intrapopulational variation in all species. Phylogenetic analyses of ITS and NTS sequences distinguish three main clades coinciding with geographical distribution: European (G. hederacea-G. hirsuta), Chinese and Korean (G. longituba), and Japanese (G. grandis). The paper presents the first comparative cytogenetic analyses of Glechoma species including karyotype structure, rDNA location and number, and genome size interpreted in a phylogenetic context. The observed variation suggests that the genus is still in genomic flux. Genome size, but not rDNA loci number and distribution, provides a character for species delimitation which allows better inferences of interspecific relationships to be made, in the absence of well-defined morphological differentiation.

The Impact of Reconstruction Methods, Phylogenetic Uncertainty and Branch Lengths on Inference of Chromosome Number Evolution in American Daisies (Melampodium, Asteraceae).

Chromosome number change (polyploidy and dysploidy) plays an important role in plant diversification and speciation. Investigating chromosome number evolution commonly entails ancestral state reconstruction performed within a phylogenetic framework, which is, however, prone to uncertainty, whose effects on evolutionary inferences are insufficiently understood. Using the chromosomally diverse plant genus Melampodium (Asteraceae) as model group, we assess the impact of reconstruction method (maximum parsimony, maximum likelihood, Bayesian methods), branch length model (phylograms versus chronograms) and phylogenetic uncertainty (topological and branch length uncertainty) on the inference of chromosome number evolution. We also address the suitability of the maximum clade credibility (MCC) tree as single representative topology for chromosome number reconstruction. Each of the listed factors causes considerable incongruence among chromosome number reconstructions. Discrepancies between inferences on the MCC tree from those made by integrating over a set of trees are moderate for ancestral chromosome numbers, but severe for the difference of chromosome gains and losses, a measure of the directionality of dysploidy. Therefore, reliance on single trees, such as the MCC tree, is strongly discouraged and model averaging, taking both phylogenetic and model uncertainty into account, is recommended. For studying chromosome number evolution, dedicated models implemented in the program ChromEvol and ordered maximum parsimony may be most appropriate. Chromosome number evolution in Melampodium follows a pattern of bidirectional dysploidy (starting from x = 11 to x = 9 and x = 14, respectively) with no prevailing direction.

Molecular and cytogenetic evidence for an allotetraploid origin of Chenopodium quinoa and C. berlandieri (Amaranthaceae).

Most of the cultivated chenopods are polyploids, but their origin and evolutionary history are still poorly understood. Phylogenetic analyses of DNA sequences of four plastid regions, nrITS and nuclear 5S rDNA spacer region (NTS) of two tetraploid chenopods (2n=4x=36), Andean C. quinoa and North American C. berlandieri, and their diploid relatives allowed inferences of their origin. The phylogenetic analyses confirmed allotetraploid origin of both tetraploids involving diploids of two different genomic groups (genomes A and B) and suggested that these two might share very similar parentage. The hypotheses on the origin of the two allopolyploid species were further tested using genomic in situ hybridization (GISH). Several diploid Chenopodium species belonging to the two lineages, genome A and B, suggested by phylogenetic analyses, were tested as putative parental taxa. GISH differentiated two sets of parental chromosomes in both tetraploids and further corroborated their allotetraploid origin. Putative diploid parental taxa have been suggested by GISH for C. quinoa and C. berlandieri. Genome sizes of the analyzed allotetraploids fit nearly perfectly the expected additive values of the putative parental taxa. Directional and uniparental loss of rDNA loci of the maternal A-subgenome was revealed for both C. berlandieri and C. quinoa.