A Multigene Phylogeny of Native American Hawkweeds (Hieracium Subgen. Chionoracium, Cichorieae, Asteraceae): Origin, Speciation Patterns, and Migration Routes.

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.

Horizontally Acquired nrDNAs Persist in Low Amounts in Host Hordeum Genomes and Evolve Independently of Native nrDNA.

Nuclear ribosomal DNA (nrDNA) has displayed extraordinary dynamics during the evolution of plant species. However, the patterns and evolutionary significance of nrDNA array expansion or contraction are still relatively unknown. Moreover, only little is known of the fate of minority nrDNA copies acquired between species via horizontal transfer. The barley genus Hordeum (Poaceae) represents a good model for such a study, as species of section Stenostachys acquired nrDNA via horizontal transfer from at least five different panicoid genera, causing long-term co-existence of native (Hordeum-like) and non-native (panicoid) nrDNAs. Using quantitative PCR, we investigated copy number variation (CNV) of nrDNA in the diploid representatives of the genus Hordeum. We estimated the copy number of the foreign, as well as of the native ITS types (ribotypes), and followed the pattern of their CNV in relation to the genus' phylogeny, species' genomes size and the number of nrDNA loci. For the native ribotype, we encountered an almost 19-fold variation in the mean copy number among the taxa analysed, ranging from 1689 copies (per 2C content) in H. patagonicum subsp. mustersii to 31342 copies in H. murinum subsp. glaucum. The copy numbers did not correlate with any of the genus' phylogeny, the species' genome size or the number of nrDNA loci. The CNV was high within the recognised groups (up to 13.2 × in the American I-genome species) as well as between accessions of the same species (up to 4×). Foreign ribotypes represent only a small fraction of the total number of nrDNA copies. Their copy numbers ranged from single units to tens and rarely hundreds of copies. They amounted, on average, to between 0.1% (Setaria ribotype) and 1.9% (Euclasta ribotype) of total nrDNA. None of the foreign ribotypes showed significant differences with respect to phylogenetic groups recognised within the sect. Stenostachys. Overall, no correlation was found between copy numbers of native and foreign nrDNAs suggesting the sequestration and independent evolution of native and non-native nrDNA arrays. Therefore, foreign nrDNA in Hordeum likely poses a dead-end by-product of horizontal gene transfer events.

A Panicum-derived chromosomal segment captured by Hordeum a few million years ago preserves a set of stress-related genes.

Intra-specific variability is a cornerstone of evolutionary success of species. Acquiring genetic material from distant sources is an important adaptive mechanism in bacteria, but it can also play a role in eukaryotes. In this paper, we investigate the nature and evolution of a chromosomal segment of panicoid (Poaceae, Panicoideae) origin occurring in the nuclear genomes of species of the barley genus Hordeum (Pooideae). The segment, spanning over 440 kb in the Asian Hordeum bogdanii and 219 kb in the South American Hordeum pubiflorum, resides on a pair of nucleolar organizer region (NOR)-bearing chromosomes. Conserved synteny and micro-collinearity of the segment in both species indicate a common origin of the segment, which was acquired before the split of the respective barley lineages 5-1.7 million years ago. A major part of the foreign DNA consists of several approximately 68 kb long repeated blocks containing five stress-related protein-coding genes and transposable elements (TEs). Whereas outside these repeats, the locus was invaded by multiple TEs from the host genome, the repeated blocks are rather intact and appear to be preserved. The protein-coding genes remained partly functional, as indicated by conserved reading frames, a low amount of non-synonymous mutations, and expression of mRNA. A screen across Hordeum species targeting the panicoid protein-coding genes revealed the presence of the genes in all species of the section Stenostachys. In summary, our study shows that grass genomes can contain large genomic segments obtained from distantly related species. These segments usually remain undetected, but they may play an important role in the evolution and adaptation of species.

Molecular Quantification of Arbuscular Mycorrhizal Fungal Root Colonization.

Specific quantification of root-colonizing arbuscular mycorrhizal fungi (AMF) by quantitative real-time PCR is a high-throughput technique, most suitable for determining abundances of AMF species or isolates in previously characterized experimental systems. The principal steps are the choice and validation of an appropriate assay to specifically amplify a gene fragment of the target AMF, preparation of templates from root samples, and quantification of the fungal gene copy numbers in these templates. The use of a suitable assay is crucial for a correct data collection but also highly specific for each experimental system and is therefore covered by general recommendations. Subsequently, specific steps are described for the validation of the assay using a standard dilution series, the determination of appropriate dilutions of DNA extracts from roots, and the quantification of the gene copy numbers in samples including calculations.

Evolutionary history and genetic diversity of apomictic allopolyploids in Hieracium s.str.: morphological versus genomic features.

PREMISE: The origin of allopolyploids is believed to shape their evolutionary potential, ecology, and geographical ranges. Morphologically distinct apomictic types sharing the same parental species belong to the most challenging groups of polyploids. We evaluated the origins and variation of two triploid taxa (Hieracium pallidiflorum, H. picroides) presumably derived from the same diploid parental pair (H. intybaceum, H. prenanthoides). METHODS: We used a suite of approaches ranging from morphological, phylogenetic (three unlinked molecular markers), and cytogenetic analyses (in situ hybridization) to genome size screening and genome skimming. RESULTS: Genotyping proved the expected parentage of all analyzed accessions of H. pallidiflorum and H. picroides and revealed that nearly all of them originated independently. Genome sizes and genome dosage largely corresponded to morphology, whereas the maternal origin of the allopolyploids had no discernable effect. Polyploid accessions of both parental species usually contained genetic material from other species. Given the phylogenetic distance of the parents, their chromosomes appeared only weakly differentiated in genomic in situ hybridization (GISH), as well as in overall comparisons of the repetitive fraction of their genomes. Furthermore, the repeatome of a phylogenetically more closely related species (H. umbellatum) differed significantly more. CONCLUSIONS: We proved (1) multiple origins of hybridogeneous apomicts from the same diploid parental taxa, and (2) allopolyploid origins of polyploid accessions of the parental species. We also showed that the evolutionary dynamics of very fast evolving markers such as satellite DNA or transposable elements does not necessarily follow patterns of speciation.

Effects of inoculum additions in the presence of a preestablished arbuscular mycorrhizal fungal community.

Communities of arbuscular mycorrhizal fungi (AMF) are crucial for promoting plant productivity in most terrestrial systems, including anthropogenically managed ecosystems. Application of AMF inocula has therefore become a widespread practice. It is, however, pertinent to understand the mechanisms that govern AMF community composition and their performance in order to design successful manipulations. Here we assess whether the composition and plant growth-promotional effects of a synthetic AMF community can be altered by inoculum additions of the isolates forming the community. This was determined by following the effects of three AMF isolates, each inoculated in two propagule densities into a preestablished AMF community. Fungal abundance in roots and plant growth were evaluated in three sequential harvests. We found a transient positive response in AMF abundance to the intraspecific inoculation only in the competitively weakest isolate. The other two isolates responded negatively to intra- and interspecific inoculations, and in some cases plant growth was also reduced. Our results suggest that increasing the AMF density may lead to increased competition among fungi and a trade-off with their ability to promote plant productivity. This is a key ecological aspect to consider when introducing AMF into soils.

Intraradical dynamics of two coexisting isolates of the arbuscular mycorrhizal fungus Glomus intraradices sensu lato as estimated by real-time PCR of mitochondrial DNA.

Real-time PCR in nuclear ribosomal DNA (nrDNA) is becoming a well-established tool for the quantification of arbuscular mycorrhizal (AM) fungi, but this genomic region does not allow the specific amplification of closely related genotypes. The large subunit of mitochondrial DNA (mtDNA) has a higher-resolution power, but mtDNA-based quantification has not been previously explored in AM fungi. We applied real-time PCR assays targeting the large subunit of mtDNA to monitor the DNA dynamics of two isolates of Glomus intraradices sensu lato coexisting in the roots of medic (Medicago sativa). The mtDNA-based quantification was compared to quantification in nrDNA. The ratio of copy numbers determined by the nrDNA- and mtDNA-based assays consistently differed between the two isolates. Within an isolate, copy numbers of the nuclear and the mitochondrial genes were closely correlated. The two quantification approaches revealed similar trends in the dynamics of both isolates, depending on whether they were inoculated alone or together. After 12 weeks of cultivation, competition between the two isolates was observed as a decrease in the mtDNA copy numbers of one of them. The coexistence of two closely related isolates, which cannot be discriminated by nrDNA-based assays, was thus identified as a factor influencing the dynamics of AM fungal DNA in roots. Taken together, the results of this study show that real-time PCR assays targeted to the large subunit of mtDNA may become useful tools for the study of coexisting AM fungi.

Development of novel low-copy nuclear markers for Hieraciinae (Asteraceae) and their perspective for other tribes.

PREMISE OF THE STUDY: The development of three low-copy nuclear markers for low taxonomic level phylogenies in Asteraceae with emphasis on the subtribe Hieraciinae is reported. METHODS AND RESULTS: Marker candidates were selected by comparing a Lactuca complementary DNA (cDNA) library with public DNA sequence databases. Interspecific variation and phylogenetic signal of the selected genes were investigated for diploid taxa from the subtribe Hieraciinae and compared to a reference phylogeny. Their ability to cross-amplify was assessed for other Asteraceae tribes. All three markers had higher variation (2.1-4.5 times) than the internal transcribed spacer (ITS) in Hieraciinae. Cross-amplification was successful in at least seven other tribes of the Asteraceae. Only three cases indicating the presence of paralogs or pseudogenes were detected. CONCLUSIONS: The results demonstrate the potential of these markers for phylogeny reconstruction in the Hieraciinae as well as in other Asteraceae tribes, especially for very closely related species.