Lucie Horová

Ecological and evolutionary significance of genomic GC content diversity in monocots

Significance Our large-scale survey of genomic nucleotide composition across monocots has enabled the first rigorous testing, to our knowledge, of its biological significance in plants. We show that genomic DNA base composition (GC content) is significantly associated with genome size and holocentric chromosomal structure. GC content may also have deep ecological relevance, because changes in GC content may have played a significant role in the evolution of Earth’s biota, especially the rise of grass-dominated biomes during the mid-Tertiary. The discovery of several groups with very unusual GC contents highlights the need for in-depth analysis to uncover the full extent of genomic diversity. Furthermore, our stratified sampling method of distribution data and quantile regression-like logic of phylogenetic analyses may find wider applications in the analysis of spatially heterogeneous data. Genomic DNA base composition (GC content) is predicted to significantly affect genome functioning and species ecology. Although several hypotheses have been put forward to address the biological impact of GC content variation in microbial and vertebrate organisms, the biological significance of GC content diversity in plants remains unclear because of a lack of sufficiently robust genomic data. Using flow cytometry, we report genomic GC contents for 239 species representing 70 of 78 monocot families and compare them with genomic characters, a suite of life history traits and climatic niche data using phylogeny-based statistics. GC content of monocots varied between 33.6% and 48.9%, with several groups exceeding the GC content known for any other vascular plant group, highlighting their unusual genome architecture and organization. GC content showed a quadratic relationship with genome size, with the decreases in GC content in larger genomes possibly being a consequence of the higher biochemical costs of GC base synthesis. Dramatic decreases in GC content were observed in species with holocentric chromosomes, whereas increased GC content was documented in species able to grow in seasonally cold and/or dry climates, possibly indicating an advantage of GC-rich DNA during cell freezing and desiccation. We also show that genomic adaptations associated with changing GC content might have played a significant role in the evolution of the Earth’s contemporary biota, such as the rise of grass-dominated biomes during the mid-Tertiary. One of the major selective advantages of GC-rich DNA is hypothesized to be facilitating more complex gene regulation.

Effect of phosphorus availability on the selection of species with different ploidy levels and genome sizes in a long‐term grassland fertilization experiment

Polyploidy and increased genome size are hypothesized to increase organismal nutrient demands, namely of phosphorus (P), which is an essential and abundant component of nucleic acids. Therefore, polyploids and plants with larger genomes are expected to be selectively disadvantaged in P-limited environments. However, this hypothesis has yet to be experimentally tested. We measured the somatic DNA content and ploidy level in 74 vascular plant species in a long-term fertilization experiment. The differences between the fertilizer treatments regarding the DNA content and ploidy level of the established species were tested using phylogeny-based statistics. The percentage and biomass of polyploid species clearly increased with soil P in particular fertilizer treatments, and a similar but weaker trend was observed for the DNA content. These increases were associated with the dominance of competitive life strategy (particularly advantageous in the P-treated plots) in polyploids and the enhanced competitive ability of dominant polyploid grasses at high soil P concentrations, indicating their increased P limitation. Our results verify the hypothesized effect of P availability on the selection of polyploids and plants with increased genome sizes, although the relative contribution of increased P demands vs increased competitiveness as causes of the observed pattern requires further evaluation.

Evolution of genome size in Carex (Cyperaceae) in relation to chromosome number and genomic base composition

BACKGROUND AND AIMS The genus Carex exhibits karyological peculiarities related to holocentrism, specifically extremely broad and almost continual variation in chromosome number. However, the effect of these peculiarities on the evolution of the genome (genome size, base composition) remains unknown. While in monocentrics, determining the arithmetic relationship between the chromosome numbers of related species is usually sufficient for the detection of particular modes of karyotype evolution (i.e. polyploidy and dysploidy), in holocentrics where chromosomal fission and fusion occur such detection requires knowledge of the DNA content. METHODS The genome size and GC content were estimated in 157 taxa using flow cytometry. The exact chromosome numbers were known for 96 measured samples and were taken from the available literature for other taxa. All relationships were tested in a phylogenetic framework using the ITS tree of 105 species. KEY RESULTS The 1C genome size varied between 0·24 and 1·64 pg in Carex secalina and C. cuspidata, respectively. The genomic GC content varied from 34·8 % to 40·6 % from C. secalina to C. firma. Both genomic parameters were positively correlated. Seven polyploid and two potentially polyploid taxa were detected in the core Carex clade. A strong negative correlation between genome size and chromosome number was documented in non-polyploid taxa. Non-polyploid taxa of the core Carex clade exhibited a higher rate of genome-size evolution compared with the Vignea clade. Three dioecious taxa exhibited larger genomes, larger chromosomes, and a higher GC content than their hermaphrodite relatives. CONCLUSIONS Genomes of Carex are relatively small and very GC-poor compared with other angiosperms. We conclude that the evolution of genome and karyotype in Carex is promoted by frequent chromosomal fissions/fusions, rare polyploidy and common repetitive DNA proliferation/removal.

Intrapopulation Genome Size Dynamics in Festuca pallens

BACKGROUND AND AIMS It is well known that genome size differs among species. However, information on the variation and dynamics of genome size in wild populations and on the early phase of genome size divergence between taxa is currently lacking. Genome size dynamics, heritability and phenotype effects are analysed here in a wild population of Festuca pallens (Poaceae). METHODS Genome size was measured using flow cytometry with DAPI dye in 562 seedlings from 17 maternal plants varying in genome size. The repeatability of genome size measurements was verified at different seasons through the use of different standards and with propidium iodide dye; the range of variation observed was tested via analysis of double-peaks. Additionally, chromosome counts were made in selected seedlings. KEY RESULTS AND CONCLUSIONS Analysis of double-peaks showed that genome size varied up to 1.188-fold within all 562 seedlings, 1.119-fold within the progeny of a single maternal plant and 1.117-fold in seedlings from grains of a single inflorescence. Generally, genome sizes of seedlings and their mothers were highly correlated. However, in maternal plants with both larger and smaller genomes, genome sizes of seedlings were shifted towards the population median. This was probably due to the frequency of available paternal genomes (pollen grains) in the population. There was a stabilizing selection on genome size during the development of seedlings into adults, which may be important for stabilizing genome size within species. Furthermore, a positive correlation was found between genome size and the development rate of seedlings. A larger genome may therefore provide a competitive advantage, perhaps explaining the higher proportion of plants with larger genomes in the population studied. The reason for the observed variation may be the recent induction of genome size variation, e.g. by activity of retrotransposons, which may be preserved in the long term by the segregation of homeologous chromosomes of different sizes during gametogenesis.

Genome size and genomic GC content evolution in the miniature genome‐sized family Lentibulariaceae

• Lentibulariaceae contains species with the smallest genome size in tracheophytes, yet data are available only for 8% of its species. This prevents understanding of the history of miniaturization events and their possible reasons. Nothing is known about the variation of genomic DNA base composition. • Genome size and genomic GC content were analyzed with flow cytometry in 119 Lentibulariaceae species. The evolution of both parameters and their correspondence with several ecological traits was tested by sequence-based phylogeny. • Genome size ranged from 1C=73 to 1C=1471 Mbp, with 19 species found to be smaller than Arabidopsis. Miniaturizations have a long history in Utricularia; they also accompany the evolution of Genlisea and two species of Pinguicula. The absence of correlation between genomic parameters and ecological variables suggests that the driving forces of miniaturization are of intrinsic nature. Genome size dynamics associates with extreme variation of GC contents (34.0%–45.1%), being the highest among tracheophyte families. The extremely low GC contents, however, must clearly have evolved with contributions from processes other than sole DNA removal. • The extreme dynamics of Lentibulariaceae genomes provides a unique opportunity for studying genome miniaturization and GC content variation. Hopefully, our study will facilitate the selection of proper model species.

Measurements of genomic GC content in plant genomes with flow cytometry: a test for reliability

• Knowledge of the phylogenetic pattern and biological relevance of the base composition of large eukaryotic genomes (including those of plants) is poor. With the use of flow cytometry (FCM), the amount of available data on the guanine + cytosine (GC) content of plants has nearly doubled in the last decade. However, skepticism exists concerning the reliability of the method because of uncertainty in some input parameters. • Here, we tested the reliability of FCM for estimating GC content by comparison with the biochemical method of DNA temperature melting analysis (TMA). We conducted measurements in 14 plant species with a maximum currently known GC content range (33.6-47.5% as measured by FCM). We also compared the estimations of the GC content by FCM with genomic sequences in 11 Oryza species. • FCM and TMA data exhibited a high degree of correspondence which remained stable over the relatively wide range of binding lengths (3.39-4.09) assumed for the base-specific dye used. A high correlation was also observed between FCM results and the sequence data in Oryza, although the latter GC contents were consistently lower. • Reliable estimates of the genomic base composition in plants by FCM are comparable with estimates obtained using other methods, and so wider application of FCM in future plant genomic research, although it would pose a challenge, would be supported by these findings.

Indel patterns of the plastid DNA trnL–trnF region within the genus Poa (Poaceae)

The use of insertion/deletion (indel) patterns from sequences of the trnL intron and trnL–F intergenic spacer (IGS) in finding plastid genome types of the genus Poa L. was studied. New sequences for 23 taxa (P. alpina, P. badensis, P. bulbosa, P. crassipes, P. molinerii, P. annua, P. chaixii, P. granitica, P. pratensis, P. sibirica, P. remota, P. botryoides, P. cenisia, P. compressa, P. laxa, P. margilicola, P. media, P. nemoralis, P. palustris, P. pannonica, P. pirinica, P. riphaea, and P. sejuncta) and 18 previously published sequences, which represent 11 of the 13 sections listed for Poa in Flora Europaea, were investigated. Collections were made primarily in central Europe. Indel patterns, despite sampling less than 0.7% of the plastid genome, produced four taxa groupings that were congruent with the major divisions obtained in intensive, previously published restriction-site studies. Insertion/deletion events in the trnL intron and trnL–trnF IGS were in nearly all cases unique to a single pattern group and thus provided almost no information about relationships among these groups. Indels did, however, provide a meaningful infrageneric classification criterion for Poa. They can serve as useful tools in studying relationships within this genus.

Stabilizing selection on genome size in a population of Festuca pallens under conditions of intensive intraspecific competition

*Stabilizing selection is a key evolutionary mechanism for which there is relatively little experimental evidence. To date, stabilizing selection has never been observed at the whole-genome level. *We tested the effect of selection on genome size in a field experiment using seeds collected in a population of Festuca pallens with a highly variable genome size. Using flow cytometry, we measured the genome size in germinating seedlings and juvenile plants grown with or without high intraspecific competition (908 individuals). Above-ground biomass and leaf number were used as measurements of individual vegetative performance. The possible confounding effect of seed weight was controlled for in a separate experiment. *Growth under high competition had a significant stabilizing effect on genome size. Because no relationship was observed between genome size and vegetative performance, we assume that the elimination of plants with extreme genome sizes was the result of decreased survival as a consequence of some unrecognized stress. *Our results indicate that genome size may be under direct selection. The equal disadvantaging of either large or small genomes indicates that the selection for optimum genome size in species may be fully context dependent. This study demonstrates the power of competition experiments for the detection of weak selection processes.

Genome size microscale divergence of Cyclamen persicum in Evolution Canyon, Israel

Using DAPI flow cytometry, we examined genome size divergence of the Persian violet, Cyclamen persicum (Primulaceae) (2n=48) on close opposite slopes of Evolution Canyon (EC), Mt. Carmel, Israel. The range of genome size variation detected among measured cyclamens was 6.41% in relation to the smallest measured DNA content. Our data on C. persicum at EC showed that local variability in the 2C-value exists. Significantly less DNA was recorded in plants growing in one station of the African savannah-like south-facing slope (AS) but not in the remaining two stations of the same slope. We were not able to reject the null hypothesis that there are no significant interslope differences in the genome size between the temperate European garrigue-like north-facing slope (ES) and the drier AS. In spite of the nonsignificant interslope trend for the higher genome size in C. persicum, the data-fusion (meta-analysis) test using correlations between C-values in C. persicum, and earlier studied carob tree (Ceratonia siliqua), trifoil (Lotus peregrinus) and a beetle (Oryzaephilus surinamensis) and their distribution along the aridity gradient indicates a positive relationship between drought and genome size at the microsite.

Flow cytometry may allow microscope-independent detection of holocentric chromosomes in plants

Two chromosomal structures, known as monocentric and holocentric chromosomes, have evolved in eukaryotes. Acentric fragments of monocentric chromosomes are unequally distributed to daughter cells and/or lost, while holocentric fragments are inherited normally. In monocentric species, unequal distribution should generate chimeras of cells with different nuclear DNA content. We investigated whether such differences in monocentric species are detectable by flow cytometry (FCM) as (i) a decreased nuclear DNA content and (ii) an increased coefficient of variance (CV) of the G1 peak after gamma radiation-induced fragmentation. We compared 13 monocentric and 9 holocentric plant species. Unexpectedly, monocentrics and holocentrics did not differ with respect to parameters (i) and (ii) in their response to gamma irradiation. However, we found that the proportion of G2 nuclei was highly elevated in monocentrics after irradiation, while holocentrics were negligibly affected. Therefore, we hypothesize that DNA-damaging agents induce cell cycle arrest leading to endopolyploidy only in monocentric and not (or to much lesser extent) in holocentric plants. While current microscope-dependent methods for holocentrism detection are unreliable for small and numerous chromosomes, which are common in holocentrics, FCM can use somatic nuclei. Thus, FCM may be a rapid and reliable method of high-throughput screening for holocentric candidates across plant phylogeny.