Hybridization is a key mechanism involved in lineage diversification and speciation, especially in ecosystems that experienced repeated environmental oscillations. Recently radiated plant groups, which have evolved in mountain ecosystems impacted by historical climate change provide an excellent model system for studying the impact of gene flow on speciation. We combined organellar (whole-plastome) and nuclear genomic data (RAD-seq) with a cytogenetic approach (rDNA FISH) to investigate the effects of hybridization and introgression on evolution and speciation in the genus Soldanella (snowbells, Primulaceae). Pervasive introgression has already occurred among ancestral lineages of snowbells and has persisted throughout the entire evolutionary history of the genus, regardless of the ecology, cytotype, or distribution range size of the affected species. The highest extent of introgression has been detected in the Carpathian species, which is also reflected in their extensive karyotype variation. Introgression occurred even between species with dysploid and euploid cytotypes, which were considered to be reproductively isolated. The magnitude of introgression detected in snowbells is unprecedented in other mountain genera of the European Alpine System investigated hitherto. Our study stresses the prominent evolutionary role of hybridization in facilitating speciation and diversification on the one hand, but also enriching previously isolated genetic pools. [chloroplast capture; diversification; dysploidy; European Alpine system; introgression; nuclear-cytoplasmic discordance; ribosomal DNA.].

• MeSH
• Ecology MeSH
• Ecosystem * MeSH
• Phylogeny MeSH
• Genome MeSH
• Primulaceae * genetics   MeSH
• DNA, Ribosomal MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA, Ribosomal MeSH

Native American hawkweeds are mainly mountainous species that are distributed all over the New World. They are severely understudied with respect to their origin, colonization of the vast distribution area, and species relationships. Here, we attempt to reconstruct the evolutionary history of the group by applying seven molecular markers (plastid, nuclear ribosomal and low-copy genes). Phylogenetic analyses revealed that Chionoracium is a subgenus of the mainly Eurasian genus Hieracium, which originated from eastern European hawkweeds about 1.58-2.24 million years ago. Plastid DNA suggested a single origin of all Chionoracium species. They colonized the New World via Beringia and formed several distinct lineages in North America. Via one Central American lineage, the group colonized South America and radiated into more than a hundred species within about 0.8 million years, long after the closure of the Isthmus of Panama and the most recent uplift of the Andes. Despite some incongruences shown by different markers, most of them revealed the same crown groups of closely related taxa, which were, however, largely in conflict with traditional sectional classifications. We provide a basic framework for further elucidation of speciation patterns. A thorough taxonomic revision of Hieracium subgen. Chionoracium is recommended.

• Keywords
• Chionoracium, Hieracium, Stenotheca, molecular dating, molecular markers, phylogenetic analysis,
• Publication type
• Journal Article MeSH

Molecular evolution of ribosomal DNA can be highly dynamic. Hundreds to thousands of copies in the genome are subject to concerted evolution, which homogenizes sequence variants to different degrees. If well homogenized, sequences are suitable for phylogeny reconstruction; if not, sequence polymorphism has to be handled appropriately. Here we investigate non-coding rDNA sequences (ITS/ETS, 5S-NTS) along with the chromosomal organization of their respective loci (45S and 5S rDNA) in diploids of the Hieraciinae. The subtribe consists of genera Hieracium, Pilosella, Andryala, and Hispidella and has a complex evolutionary history characterized by ancient intergeneric hybridization, allele sharing among species, and incomplete lineage sorting. Direct or cloned Sanger sequences and phased alleles derived from Illumina genome sequencing were subjected to phylogenetic analyses. Patterns of homogenization and tree topologies based on the three regions were compared. In contrast to most other plant groups, 5S-NTS sequences were generally better homogenized than ITS and ETS sequences. A novel case of ancient intergeneric hybridization between Hispidella and Hieracium was inferred, and some further incongruences between the trees were found, suggesting independent evolution of these regions. In some species, homogenization of ITS/ETS and 5S-NTS sequences proceeded in different directions although the 5S rDNA locus always occurred on the same chromosome with one 45S rDNA locus. The ancestral rDNA organization in the Hieraciinae comprised 4 loci of 45S rDNA in terminal positions and 2 loci of 5S rDNA in interstitial positions per diploid genome. In Hieracium, some deviations from this general pattern were found (3, 6, or 7 loci of 45S rDNA; three loci of 5S rDNA). Some of these deviations concerned intraspecific variation, and most of them occurred at the tips of the tree or independently in different lineages. This indicates that the organization of rDNA loci is more dynamic than the evolution of sequences contained in them and that locus number is therefore largely unsuitable to inform about species relationships in Hieracium. No consistent differences in the degree of sequence homogenization and the number of 45S rDNA loci were found, suggesting interlocus concerted evolution.

• Keywords
• 45S rDNA, 5S rDNA, Andryala, Hieracium, Pilosella, concerted evolution, in situ hybridization, molecular phylogeny,
• Publication type
• Journal Article MeSH

The repetitive content of the plant genome (repeatome) often represents its largest fraction and is frequently correlated with its size. Transposable elements (TEs), the main component of the repeatome, are an important driver in the genome diversification due to their fast-evolving nature. Hybridization and polyploidization events are hypothesized to induce massive bursts of TEs resulting, among other effects, in an increase of copy number and genome size. Little is known about the repeatome dynamics following hybridization and polyploidization in plants that reproduce by apomixis (asexual reproduction via seeds). To address this, we analyzed the repeatomes of two diploid parental species, Hieracium intybaceum and H. prenanthoides (sexual), their diploid F1 synthetic and their natural triploid hybrids (H. pallidiflorum and H. picroides, apomictic). Using low-coverage next-generation sequencing (NGS) and a graph-based clustering approach, we detected high overall similarity across all major repeatome categories between the parental species, despite their large phylogenetic distance. Medium and highly abundant repetitive elements comprise ∼70% of Hieracium genomes; most prevalent were Ty3/Gypsy chromovirus Tekay and Ty1/Copia Maximus-SIRE elements. No TE bursts were detected, neither in synthetic nor in natural hybrids, as TE abundance generally followed theoretical expectations based on parental genome dosage. Slight over- and under-representation of TE cluster abundances reflected individual differences in genome size. However, in comparative analyses, apomicts displayed an overabundance of pararetrovirus clusters not observed in synthetic hybrids. Substantial deviations were detected in rDNAs and satellite repeats, but these patterns were sample specific. rDNA and satellite repeats (three of them were newly developed as cytogenetic markers) were localized on chromosomes by fluorescence in situ hybridization (FISH). In a few cases, low-abundant repeats (5S rDNA and certain satellites) showed some discrepancy between NGS data and FISH results, which is due partly to the bias of low-coverage sequencing and partly to low amounts of the satellite repeats or their sequence divergence. Overall, satellite DNA (including rDNA) was markedly affected by hybridization, but independent of the ploidy or reproductive mode of the progeny, whereas bursts of TEs did not play an important role in the evolutionary history of Hieracium.

• Keywords
• RepeatExplorer, apomixis, hawkweed, hybridization, next-generation sequencing, polyploidization, repeatome,
• Publication type
• Journal Article MeSH

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.

• Keywords
• chloroplast DNA, germination, introgression, microsatellites, polyploidy, tension zone,
• MeSH
• Genetic Variation MeSH
• Genome, Chloroplast * MeSH
• Genetic Introgression * MeSH
• Microsatellite Repeats * MeSH
• Alnus genetics   MeSH
• Polyploidy MeSH
• Selection, Genetic MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Thorough understanding of biodiversity is a fundamental prerequisite for biological research. A lack of taxonomic knowledge and species misidentifications are particularly critical for conservation. Here we present an example of Potamogeton floridanus, the Florida Pondweed, an endangered taxon endemic to a small area in the Florida panhandle, whose taxonomic status remained controversial for more than a century, and all previous attempts to elucidate its identity have failed. We applied molecular approaches to tackle the origin of the mysterious taxon and supplemented them with morphological and anatomical investigations of both historical herbarium collections and plants recently collected in the type area for a comprehensive taxonomic reassessment. Sequencing of two nuclear ribosomal markers and one chloroplast non-coding spacer resulted in the surprising discovery that P. floridanus is a hybrid of P. pulcher and P. oakesianus, with the former being the maternal parent. The hybrid colony is currently geographically isolated from the distribution range of P. oakesianus. We show that previous molecular analyses have failed to reveal its hybrid identity due to inadequate nuclear DNA sequence editing. This is an example how the uncritical use of automized sequence reads can hamper molecular species identifications and also affect phylogenetic tree construction and interpretation. This unique hybrid taxon, P. ×floridanus, adds another case study to the debate on hybrid protection; consequences for its conservation are discussed.

• MeSH
• Biodiversity MeSH
• Phylogeny MeSH
• Endangered Species MeSH
• Potamogetonaceae anatomy & histology   genetics   MeSH
• RNA, Ribosomal, 5S genetics   MeSH
• Sequence Analysis MeSH
• Sequence Homology, Amino Acid MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• United States MeSH
• Names of Substances
• RNA, Ribosomal, 5S MeSH

PREMISE OF THE STUDY: Microsatellite markers were developed for the Pilosella alpicola group (Asteraceae), comprising four closely related species distributed in subalpine areas of Europe. These species are believed to have diverged recently, but display contrasting cytogeographic patterns and variation in breeding systems, representing a promising model system for studying plant speciation, adaptation, and recent polyploidization. METHODS AND RESULTS: We developed 17 microsatellite markers for the P. alpicola group using 454 sequencing. Sixteen markers were polymorphic, with the number of alleles per locus ranging from seven to 16 and observed and expected heterozygosity ranging from 0.45 to 0.84 and 0.72 to 0.92, respectively. Ten and five loci amplified in the related species, P. echioides and P. officinarum, respectively, but only two in Andryala and one in Hieracium s. str. CONCLUSIONS: The developed microsatellite markers have high potential to become useful tools to study microevolutionary processes in the P. alpicola group and related Pilosella species.

• Keywords
• Pilosella alpicola, hybridization, polyploidy, population genetics, simple sequence repeat (SSR) markers,
• Publication type
• Journal Article MeSH

The Hieracium and Pilosella (Lactuceae, Asteraceae) genera of closely related hawkweeds contain species with two different modes of gametophytic apomixis (asexual seed formation). Both genera contain polyploid species, and in wild populations, sexual and apomictic species co-exist. Apomixis is known to co-exist with sexuality in apomictic Pilosella individuals, however, apomictic Hieracium have been regarded as obligate apomicts. Here, a developmental analysis of apomixis within 16 Hieracium species revealed meiosis and megaspore tetrad formation in 1 to 7% of ovules, for the first time indicating residual sexuality in this genus. Molecular markers linked to the two independent, dominant loci LOSS OF APOMEIOSIS (LOA) and LOSS OF PARTHENOGENESIS (LOP) controlling apomixis in Pilosella piloselloides subsp. praealta were screened across 20 phenotyped Hieracium individuals from natural populations, and 65 phenotyped Pilosella individuals from natural and experimental cross populations, to examine their conservation, inheritance and association with reproductive modes. All of the tested LOA and LOP-linked markers were absent in the 20 Hieracium samples irrespective of their reproductive mode. Within Pilosella, LOA and LOP-linked markers were essentially absent within the sexual plants, although they were not conserved in all apomictic individuals. Both loci appeared to be inherited independently, and evidence for additional genetic factors influencing quantitative expression of LOA and LOP was obtained. Collectively, these data suggest independent evolution of apomixis in Hieracium and Pilosella and are discussed with respect to current knowledge of the evolution of apomixis.

• MeSH
• Apomixis genetics   MeSH
• Asteraceae genetics   MeSH
• Biological Evolution * MeSH
• DNA, Chloroplast genetics   MeSH
• DNA, Plant genetics   MeSH
• Genetic Loci * MeSH
• Genetic Markers MeSH
• Haplotypes MeSH
• Conserved Sequence MeSH
• Molecular Sequence Data MeSH
• Genetics, Population MeSH
• Gene Expression Regulation, Plant MeSH
• Genes, Plant * MeSH
• Seeds genetics   MeSH
• Inheritance Patterns MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Chloroplast MeSH
• DNA, Plant MeSH
• Genetic Markers MeSH

Phylogeny reconstruction based on multiple unlinked markers is often hampered by incongruent gene trees, especially in closely related species complexes with high degrees of hybridization and polyploidy. To investigate the particular strengths and limitations of chloroplast DNA (cpDNA), low-copy nuclear and multicopy nuclear markers for elucidating the evolutionary history of such groups, we focus on Hieracium s.str., a predominantly apomictic genus combining the above-mentioned features. Sequences of the trnV-ndhC and trnT-trnL intergenic spacers were combined for phylogenetic analyses of cpDNA. Part of the highly variable gene for squalene synthase (sqs) was applied as a low-copy nuclear marker. Both gene trees were compared with previous results based on the multicopy external transcribed spacer (ETS) of the nuclear ribosomal DNA. The power of the different markers to detect hybridization varied, but they largely agreed on particular hybrid and allopolyploid origins. The same crown groups of species were recognizable in each dataset, but basal relationships were strongly incongruent among cpDNA, sqs and ETS trees. The ETS tree was considered as the best approximation of the species tree. Both cpDNA and sqs trees showed basal polytomies as well as merging or splitting of species groups of non-hybrid taxa. These patterns can be best explained by a rapid diversification of the genus with ancestral polymorphism and incomplete lineage sorting. A hypothetical scenario of Hieracium speciation based on all available (including non-molecular) evidence is depicted. Incorporation of seemingly contradictory information helped to better understand species origins and evolutionary patterns in this notoriously difficult agamic complex.

• MeSH
• Asteraceae genetics   MeSH
• DNA, Chloroplast genetics   MeSH
• Farnesyl-Diphosphate Farnesyltransferase genetics   MeSH
• Phylogeny * MeSH
• Genetic Markers MeSH
• Crosses, Genetic MeSH
• DNA, Ribosomal Spacer genetics   MeSH
• Models, Genetic MeSH
• Molecular Sequence Data MeSH
• Plant Proteins genetics   MeSH
• Sequence Analysis, DNA MeSH
• Genetic Speciation * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Chloroplast MeSH
• Farnesyl-Diphosphate Farnesyltransferase MeSH
• Genetic Markers MeSH
• DNA, Ribosomal Spacer MeSH
• Plant Proteins MeSH

BACKGROUND: Hieracium 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 Hieracium 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 (ETS) and chloroplast (trnT-trnL) sequences in order to disentangle the phylogenetic relationships and to infer the origins of the polyploids. RESULTS: 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 ETS variants. Surprisingly, many diploid species were of hybrid origin whilst several polyploid taxa showed no evidence of reticulation. Intra-individual ETS sequence polymorphism generally exceeded interspecific variation and was either independent of, or additional to, additive patterns accounted for by hybrid origin. Several ETS ribotypes occurred in different hybrid taxa, but never as the only variant in any species analyzed. CONCLUSION: The high level of intra-individual ETS 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.

• MeSH
• Asteraceae classification   genetics   MeSH
• Bayes Theorem MeSH
• Diploidy MeSH
• DNA, Chloroplast genetics   MeSH
• DNA, Plant genetics   MeSH
• Species Specificity MeSH
• Phylogeny * MeSH
• Hybridization, Genetic * MeSH
• DNA, Ribosomal Spacer genetics   MeSH
• Evolution, Molecular * MeSH
• Polymorphism, Genetic MeSH
• Polyploidy MeSH
• Likelihood Functions MeSH
• Sequence Analysis, DNA MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Chloroplast MeSH
• DNA, Plant MeSH
• DNA, Ribosomal Spacer MeSH