Molekulární fylogeneze a evoluční trendy v rodě Hieracium (Asteraceae, Lactuceae)

The hawkweed subgenus Hieracium s. str. is notoriously known for its extreme morphological variability and variation in ploidy levels that is associated with differences in modes of reproduction. Extensive past hybridization is supposed for the subgenus, but recent hybridization was evidenced only in few cases. The subgenus attracts the attention of botanists already for more than a century. Therefore the species diversity is largely examined and the taxonomy of the subgenus is well elaborated, although several contradictory taxonomic concepts exist. However the relationships among the species are unknown and haven't been studied yet. The investigation of these relationships from a phylogenetic perspective using molecular approaches was the main aim of the presented thesis. Basic species (both diploid and polyploid), representing morphologically unique taxa, that are supposed to be the basic evolutionary units of the subgenus were studied. The sequences of two intergenic spacers of the cpDNA (trnT-trnL and trnV-ndhC) and the external transcribed spacer of the nuclear ribosomal DNA (nrDNA ETS) were analyzed. Moreover, three new low-copy nuclear markers with higher variability than nrDNA and cpDNA markers were developed and their suitability for phylogenetic studies in Hieracium s. str. was...Pravé jestřábníky (Hieracium podrod Hieracium s. str.) se vyznačuje extrémní morfologickou variabilitou a různorodostí ploidních úrovní, která je spojená s různýma způsoby reprodukce. Předpokládá se, že v minulosti probíhala v rámci podrodu rozsáhlá hybridizace. V současné době je však hybridizace velice vzácná. Taxonomie podrodu je dost dobře propracována a diverzita na úrovni druhů je podrobně popsaná. Na druhé straně evoluční vztahy mezi jednotlivými druhy zatím nebyli studovány. Tato studie představuje první a zatím jedinou práci zaměřenou na objasnění těchto vztahů pomocí molekulárně biologických metod. Do analýz byly zahrnuty výlučně základní druhy (diploidní i polyploidní), které jsou morfologicky dobře odlišitelné a jsou chápány jako základní evoluční linie v rámci podrodu. Byli analyzovány sekvence dvou úseků chloroplastové DNA (trnT-trnL and trnV-ndhC) a mezerníkového úseku ETS, jádrové ribosomální DNA (nrDNA). V rámci studie byli vyvinuty tři nové low-copy jaderné markery, vhodné pro fylogenetické analýzy u Hieracium s. str., a každý z nich vykazoval vyšší variabilitu než nrDNA ETS. Jeden z nich, konkrétně část genu kódujícího enzym Squalen syntetázu (sqs), byl využit v rámci studie jako další jaderný úsek. Na základě analýzy sekvencí nrDNA ETS byly v podrodu Hieracium s. str....Department of BotanyKatedra botanikyFaculty of SciencePřírodovědecká fakult

Intra-individual polymorphism in diploid and apomictic polyploid hawkweeds (Hieracium, Lactuceae, Asteraceae): disentangling phylogenetic signal, reticulation, and noise

<p>Abstract</p> <p>Background</p> <p><it>Hieracium </it>s.str. is a complex species-rich group of perennial herbs composed of few sexual diploids and numerous apomictic polyploids. The existence of reticulation and the near-continuity of morphological characters across taxa seriously affect species determination, making <it>Hieracium </it>one of the best examples of a 'botanist's nightmare'. Consequently, its species relationships have not previously been addressed by molecular methods. Concentrating on the supposed major evolutionary units, we used nuclear ribosomal (<it>ETS</it>) and chloroplast (<it>trnT</it>-<it>trnL</it>) sequences in order to disentangle the phylogenetic relationships and to infer the origins of the polyploids.</p> <p>Results</p> <p>Despite relatively low interspecific variation, the nuclear data revealed the existence of two major groups roughly corresponding to species with a Western or Eastern European origin. Extensive reticulation was mainly inferred from the character additivity of parental <it>ETS </it>variants. Surprisingly, many diploid species were of hybrid origin whilst several polyploid taxa showed no evidence of reticulation. Intra-individual <it>ETS </it>sequence polymorphism generally exceeded interspecific variation and was either independent of, or additional to, additive patterns accounted for by hybrid origin. Several <it>ETS </it>ribotypes occurred in different hybrid taxa, but never as the only variant in any species analyzed.</p> <p>Conclusion</p> <p>The high level of intra-individual <it>ETS </it>polymorphism prevented straightforward phylogenetic analysis. Characterization of this variation as additive, shared informative, homoplasious, or unique made it possible to uncover the phylogenetic signal and to reveal the hybrid origin of 29 out of 60 accessions. Contrary to expectation, diploid sexuals and polyploid apomicts did not differ in their molecular patterns. The basic division of the genus into two major clades had not previously been intimated on morphological grounds. Both major groups are thought to have survived in different glacial refugia and to have hybridized as a result of secondary contact. Several lines of evidence suggest the data is best explained by the presence of an extinct range of variation and a larger diversity of ancestral diploids in former times rather than by unsampled variation. Extinct diversity and extensive reticulation are thought to have largely obscured the species relationships. Our study illustrates how multigene sequences can be used to disentangle the evolutionary history of agamic complexes or similarly difficult datasets.</p

Uniparental expression of ribosomal RNA in ×Festulolium grasses: a link between the genome and nucleolar dominance

Genome or genomic dominance (GD) is a phenomenon observed in hybrids when one parental genome becomes dominant over the other. It is manifested by the replacement of chromatin of the submissive genome by that of the dominant genome and by biased gene expression. Nucleolar dominance (ND) – the functional expression of only one parental set of ribosomal genes in hybrids – is another example of an intragenomic competitive process which, however, concerns ribosomal DNA only. Although GD and ND are relatively well understood, the nature and extent of their potential interdependence is mostly unknown. Here, we ask whether hybrids showing GD also exhibit ND and, if so, whether the dominant genome is the same. To test this, we used hybrids between Festuca and Lolium grasses (Festulolium), and between two Festuca species in which GD has been observed (with Lolium as the dominant genome in Festulolium and F. pratensis in interspecific Festuca hybrids). Using amplicon sequencing of ITS1 and ITS2 of the 45S ribosomal DNA (rDNA) cluster and molecular cytogenetics, we studied the organization and expression of rDNA in leaf tissue in five hybrid combinations, four generations and 31 genotypes [F. pratensis × L. multiflorum (F1, F2, F3, BC1), L. multiflorum × F. pratensis (F1), L. multiflorum × F. glaucescens (F2), L. perenne × F. pratensis (F1), F. glaucescens × F. pratensis (F1)]. We have found that instant ND occurs in Festulolium, where expression of Lolium-type rDNA reached nearly 100% in all F1 hybrids and was maintained through subsequent generations. Therefore, ND and GD in Festulolium are manifested by the same dominant genome (Lolium). We also confirmed the concordance between GD and ND in an interspecific cross between two Festuca species

Natural History of a Satellite DNA Family: From the Ancestral Genome Component to Species-Specific Sequences, Concerted and Non-Concerted Evolution

Satellite DNA (satDNA) is the most variable fraction of the eukaryotic genome. Related species share a common ancestral satDNA library and changing of any library component in a particular lineage results in interspecific differences. Although the general developmental trend is clear, our knowledge of the origin and dynamics of satDNAs is still fragmentary. Here, we explore whole genome shotgun Illumina reads using the RepeatExplorer (RE) pipeline to infer satDNA family life stories in the genomes of Chenopodium species. The seven diploids studied represent separate lineages and provide an example of a species complex typical for angiosperms. Application of the RE pipeline allowed by similarity searches a determination of the satDNA family with a basic monomer of ~40 bp and to trace its transformation from the reconstructed ancestral to the species-specific sequences. As a result, three types of satDNA family evolutionary development were distinguished: (i) concerted evolution with mutation and recombination events; (ii) concerted evolution with a trend toward increased complexity and length of the satellite monomer; and (iii) non-concerted evolution, with low levels of homogenization and multidirectional trends. The third type is an example of entire repeatome transformation, thus producing a novel set of satDNA families, and genomes showing non-concerted evolution are proposed as a significant source for genomic diversity

The major satellite DNA families of the diploid Chenopodium album aggregate species: Arguments for and against the “library hypothesis”

Satellite DNA (satDNA) is one of the major fractions of the eukaryotic nuclear genome. Highly variable satDNA is involved in various genome functions, and a clear link between satellites and phenotypes exists in a wide range of organisms. However, little is known about the origin and temporal dynamics of satDNA. The "library hypothesis" indicates that the rapid evolutionary changes experienced by satDNAs are mostly quantitative. Although this hypothesis has received some confirmation, a number of its aspects are still controversial. A recently developed next-generation sequencing (NGS) method allows the determination of the satDNA landscape and could shed light on unresolved issues. Here, we explore low-coverage NGS data to infer satDNA evolution in the phylogenetic context of the diploid species of the Chenopodium album aggregate. The application of the Illumina read assembly algorithm in combination with Oxford Nanopore sequencing and fluorescent in situ hybridization allowed the estimation of eight satDNA families within the studied group, six of which were newly described. The obtained set of satDNA families of different origins can be divided into several categories, namely group-specific, lineage-specific and species-specific. In the process of evolution, satDNA families can be transmitted vertically and can be eliminated over time. Moreover, transposable element-derived satDNA families may appear repeatedly in the satellitome, creating an illusion of family conservation. Thus, the obtained data refute the "library hypothesis", rather than confirming it, and in our opinion, it is more appropriate to speak about "the library of the mechanisms of origin".Peer reviewe

The structural diversity of CACTA transposons in genomes of Chenopodium (Amaranthaceae, Caryophyllales) species: specific traits and comparison with the similar elements of Angiosperms

Background: CACTA transposable elements (TEs) comprise one of the most abundant superfamilies of Class 2 (cut-and-paste) transposons. Over recent decades, CACTA elements were widely identified in species from the plant, fungi, and animal kingdoms, but sufficiently studied in the genomes of only a few model species although non-model genomes can bring additional and valuable information. It primarily concerned the genomes of species belonging to clades in the base of large taxonomic groups whose genomes, to a certain extent, can preserve relict and/or possesses specific traits. Thus, we sought to investigate the genomes of Chenopodium (Amaranthaceae, Caryophyllales) species to unravel the structural variability of CACTA elements. Caryophyllales is a separate branch of Angiosperms and until recently the diversity of CACTA elements in this clade was unknown. Results: Application of the short-read genome assembly algorithm followed by analysis of detected complete CACTA elements allowed for the determination of their structural diversity in the genomes of 22 Chenopodium album aggregate species. As a result: (i) the presence of two CACTA transposons subtypes coexisting in single genome; (ii) gaining of additional protein conserved domains within the coding sequence; (iii) the presence of captured gene fragments, including key genes for flower development; and (iv) presence of captured satDNA arrays were identified. Wide comparative database analysis revealed that identified events are scattered through Angiosperms in different proportions. Conclusions: Our study demonstrated that while preserving the basic element structure a wide range of coding and non-coding additions to CACTA transposons occur in genomes of C. album aggregate species. Ability to relocate additions inside genome in combination with proposed novel functional features of structurally-different CACTA elements can impact evolutionary trajectory of the host genome.Peer reviewe

Multiple hybridization events in Cardamine (Brassicaceae) during the last 150 years: revisiting a textbook example of neoallopolyploidy

Background and Aims Recently formed allopolyploid species represent excellent subjects for exploring early stages of polyploid evolution. The hexaploid Cardamine schulzii was regarded as one of the few nascent allopolyploid species formed within the past ∼150 years that presumably arose by autopolyploidization of a triploid hybrid, C. × insueta; however, the most recent investigations have shown that it is a trigenomic hybrid. The aims of this study were to explore the efficiency of progenitor-specific microsatellite markers in detecting the hybrid origins and genome composition of these two allopolyploids, to estimate the frequency of polyploid formation events, and to outline their evolutionary potential for long-term persistence and speciation. Methods Flow-cytometric ploidy-level screening and genotyping by progenitor-specific microsatellite markers (20 microsatellite loci) were carried out on samples focused on hybridizing populations at Urnerboden, Switzerland, but also including comparative material of the parental species from other sites in the Alps and more distant areas. Key Results It was confirmed that hybridization between the diploids C. amara and C. rivularis auct. gave rise to triploid C. × insueta, and it is inferred that this has occurred repeatedly. Evidence is provided that C. schulzii comprises three parental genomes and supports its origin from hybridization events between C. × insueta and the locally co-occurring hypotetraploid C. pratensis, leading to two cytotypes of C. schulzii: hypopentaploid and hypohexaploid. Each cytotype of C. schulzii is genetically uniform, suggesting their single origins. Conclusions Persistence of C. schulzii has presumably been achieved only by perennial growth and clonal reproduction. This contrasts with C. × insueta, in which multiple origins and occasional sexual reproduction have generated sufficient genetic variation for long-term survival and evolutionary success. This study illustrates a complex case of recurrent hybridization and polyploidization events, and highlights the role of triploids that promoted the origin of trigenomic hybrid

Molecular phylogeny and evolutionary trends in Hieracium (Asteraceae, Lactuceae)

The hawkweed subgenus Hieracium s. str. is notoriously known for its extreme morphological variability and variation in ploidy levels that is associated with differences in modes of reproduction. Extensive past hybridization is supposed for the subgenus, but recent hybridization was evidenced only in few cases. The subgenus attracts the attention of botanists already for more than a century. Therefore the species diversity is largely examined and the taxonomy of the subgenus is well elaborated, although several contradictory taxonomic concepts exist. However the relationships among the species are unknown and haven't been studied yet. The investigation of these relationships from a phylogenetic perspective using molecular approaches was the main aim of the presented thesis. Basic species (both diploid and polyploid), representing morphologically unique taxa, that are supposed to be the basic evolutionary units of the subgenus were studied. The sequences of two intergenic spacers of the cpDNA (trnT-trnL and trnV-ndhC) and the external transcribed spacer of the nuclear ribosomal DNA (nrDNA ETS) were analyzed. Moreover, three new low-copy nuclear markers with higher variability than nrDNA and cpDNA markers were developed and their suitability for phylogenetic studies in Hieracium s. str. was..

Analyses of Hybrid Viability across a Hybrid Zone between Two Alnus Species Using Microsatellites and cpDNA Markers

Diploid Alnus glutinosa s. str. and autotetraploid A. rohlenae form a narrow hybrid zone in a study area in southern Serbia, which results in triploid hybrid formation. The vast majority of previous studies have been focused on studies of maternal plants, but the offspring resulting from their crossing have not been much studied. Here, we use the variability of microsatellites and chloroplast DNA between these species and their putative hybrids to create an overall picture of the development of the hybrid zone and its predicted type. To elucidate the gene transfer within both species, the origins of individual ploidies and especially the role of triploid hybrids, a germination experiment was carried out linked with a flow cytometry study of the resulting seedlings. The tension zone model seems to offer the most adequate explanation of our observations, with selection against triploid hybrids and the spatial positioning of the hybrid zone. Despite selection against them, the triploid hybrids play an important role in the exchange of genes between the two species and therefore serve as a bridge for introgression. The presence of fertile triploids is essential for enriching the haplotype diversity between these species and for the development of new genetic lineages

Atriplex_alignment_samples

Aligned of all cpDNA sequences of A. tatarica and A. sagittata. The alignment is based on the concatenated sequences of the four analyzed cpDNA regions: rpl32-trnL, psbD-trnT, atpI-atpH, trnD-trnT