The global distribution of angiosperm genome size is shaped by climate.

Angiosperms, which inhabit diverse environments across all continents, exhibit significant variation in genome sizes, making them an excellent model system for examining hypotheses about the global distribution of genome size. These include the previously proposed large genome constraint, mutational hazard, polyploidy-mediated, and climate-mediated hypotheses. We compiled the largest genome size dataset to date, encompassing 16 017 (> 5% of known) angiosperm species, and analyzed genome size distribution using a comprehensive geographic distribution dataset for all angiosperms. We observed that angiosperms with large range sizes generally had small genomes, supporting the large genome constraint hypothesis. Climate was shown to exert a strong influence on genome size distribution along the global latitudinal gradient, while the frequency of polyploidy and the type of growth form had negligible effects. In contrast to the unimodal patterns along the global latitudinal gradient shown by plant size traits and polyploid proportions, the increase in angiosperm genome size from the equator to 40-50°N/S is probably mediated by different (mostly climatic) mechanisms than the decrease in genome sizes observed from 40 to 50°N northward. Our analysis suggests that the global distribution of genome sizes in angiosperms is mainly shaped by climatically mediated purifying selection, genetic drift, relaxed selection, and environmental filtering.

Chromosome size matters: genome evolution in the cyperid clade.

BACKGROUND AND AIMS: While variation in genome size and chromosome numbers and their consequences are often investigated in plants, the biological relevance of variation in chromosome size remains poorly known. Here, we examine genome and mean chromosome size in the cyperid clade (families Cyperaceae, Juncaceae and Thurniaceae), which is the largest vascular plant lineage with predominantly holocentric chromosomes. METHODS: We measured genome size in 436 species of cyperids using flow cytometry, and augment these data with previously published datasets. We then separately compared genome and mean chromosome sizes (2C/2n) amongst the major lineages of cyperids and analysed how these two genomic traits are associated with various environmental factors using phylogenetically informed methods. KEY RESULTS: We show that cyperids have the smallest mean chromosome sizes recorded in seed plants, with a large divergence between the smallest and largest values. We found that cyperid species with smaller chromosomes have larger geographical distributions and that there is a strong inverse association between mean chromosome size and number across this lineage. CONCLUSIONS: The distinct patterns in genome size and mean chromosome size across the cyperids might be explained by holokinetic drive. The numerous small chromosomes might function to increase genetic diversity in this lineage where crossovers are limited during meiosis.

Kinetochore size scales with chromosome size in bimodal karyotypes of Agavoideae.

BACKGROUND AND AIMS: In eukaryotes, the total kinetochore size (defined as a chromosomal region containing CENH3-positive nucleosomes) per nucleus strongly correlates with genome size, a relationship that has been hypothesized to stem from general intracellular scaling principles. However, if larger chromosomes within a karyotype required larger kinetochores to move properly, it could also be derived from the mechanics of cell division. METHODS: We selected seven species of the plant subfamily Agavoideae whose karyotypes are characterized by the presence of small and very large chromosomes. We visualized the kinetochore regions and chromosomes by immunolabelling with an anti-CENH3 antibody and DAPI (6'-diamidino-2-phenylindole) staining. We then employed 2D widefield and 3D super-resolution microscopy to measure chromosome and kinetochore areas and volumes, respectively. To assess the scaling relationship of kinetochore size to chromosome size inside a karyotype, we log-transformed the data and analysed them with linear mixed models which allowed us to control for the inherent hierarchical structure of the dataset (metaphases within slides and species). KEY RESULTS: We found a positive intra-karyotype relationship between kinetochore and chromosome size. The slope of the regression line of the observed relationship (0.277 for areas, 0.247 for volumes) was very close to the theoretical slope of 0.25 for chromosome width based on the expected physics of chromosome passage through the cytoplasm during cell division. We obtained similar results by reanalysing available data from human and maize. CONCLUSIONS: Our findings suggest that the total kinetochore size to genome size scaling observed across eukaryotes may also originate from the mechanics of cell division. Moreover, the potential causal link between kinetochore and chromosome size indicates that evolutionary mechanisms capable of leading kinetochore size changes to fixation, such as centromere drive, could promote the size evolution of entire chromosomes and genomes.

Centromere size scales with genome size across Eukaryotes.

Previous studies on grass species suggested that the total centromere size (sum of all centromere sizes in a cell) may be determined by the genome size, possibly because stable scaling is important for proper cell division. However, it is unclear whether this relationship is universal. Here we analyze the total centromere size using the CenH3-immunofluorescence area as a proxy in 130 taxa including plants, animals, fungi, and protists. We verified the reliability of our methodological approach by comparing our measurements with available ChIP-seq-based measurements of the size of CenH3-binding domains. Data based on these two independent methods showed the same positive relationship between the total centromere size and genome size. Our results demonstrate that the genome size is a strong predictor (R-squared = 0.964) of the total centromere size universally across Eukaryotes. We also show that this relationship is independent of phylogenetic relatedness and centromere type (monocentric, metapolycentric, and holocentric), implying a common mechanism maintaining stable total centromere size in Eukaryotes.

Holocentric Chromosomes Probably Do Not Prevent Centromere Drive in Cyperaceae.

Centromere drive model describes an evolutionary process initiated by centromeric repeats expansion, which leads to the recruitment of excess kinetochore proteins and consequent preferential segregation of an expanded centromere to the egg during female asymmetric meiosis. In response to these selfish centromeres, the histone protein CenH3, which recruits kinetochore components, adaptively evolves to restore chromosomal parity and counter the detrimental effects of centromere drive. Holocentric chromosomes, whose kinetochores are assembled along entire chromosomes, have been hypothesized to prevent expanded centromeres from acquiring a selective advantage and initiating centromere drive. In such a case, CenH3 would be subjected to less frequent or no adaptive evolution. Using codon substitution models, we analyzed 36 CenH3 sequences from 35 species of the holocentric family Cyperaceae. We found 10 positively selected codons in the CenH3 gene [six codons in the N-terminus and four in the histone fold domain (HFD)] and six branches of its phylogeny along which the positive selection occurred. One of the positively selected codons was found in the centromere targeting domain (CATD) that directly interacts with DNA and its mutations may be important in centromere drive suppression. The frequency of these positive selection events was comparable to the frequency of positive selection in monocentric clades with asymmetric female meiosis. Taken together, these results suggest that preventing centromere drive is not the primary adaptive role of holocentric chromosomes, and their ability to suppress it likely depends on their kinetochore structure in meiosis.

Is the evolution of carnivory connected with genome size reduction?

PREMISE: As repeatedly shown, the remarkable variation in the genome size of angiosperms can be shaped by extrinsic selective pressures, including nutrient availability. Carnivory has evolved independently in 10 angiosperm clades, but all carnivorous plants share a common affinity to nutrient-poor habitats. As such, carnivory and genome reduction could be responses to the same environmental pressure. Indeed, the smallest genomes among flowering plants are found in the carnivorous family Lentibulariaceae, where a unique mutation in cytochrome c oxidase (COX) is suspected to promote genome miniaturization. Despite these hypotheses, a phylogenetically informed test of genome size and nutrient availability across carnivorous clades has so far been missing. METHODS: Using linear mixed models, we compared genome sizes of 127 carnivorous plants from 7 diverse angiosperm clades with 1072 of their noncarnivorous relatives. We also tested whether genome size in Lentibulariaceae reflects the presence of the COX mutation. RESULTS: The genome sizes of carnivorous plants do not differ significantly from those of their noncarnivorous relatives. Based on available data, no significant association between the COX mutation and genome miniaturization could be confirmed, not even when considering polyploidy. CONCLUSIONS: Carnivory alone does not seem to significantly affect genome size decrease. Plausibly, it might actually counterbalance the effect of nutrient limitation on genome size evolution. The role of the COX mutation in genome miniaturization needs to be evaluated by analysis of a broader data set because current knowledge of its presence across Lentibulariaceae covers less than 10% of the species diversity in this family.

Endopolyploidy is a common response to UV-B stress in natural plant populations, but its magnitude may be affected by chromosome type.

BACKGROUND AND AIMS: Ultraviolet-B radiation (UV-B) radiation damages the DNA, cells and photosynthetic apparatus of plants. Plants commonly prevent this damage by synthetizing UV-B-protective compounds. Recent laboratory experiments in Arabidopsis and cucumber have indicated that plants can also respond to UV-B stress with endopolyploidy. Here we test the generality of this response in natural plant populations, considering their monocentric or holocentric chromosomal structure. METHODS: We measured the endopolyploidy index (flow cytometry) and the concentration of UV-B-protective compounds in leaves of 12 herbaceous species (1007 individuals) from forest interiors and neighbouring clearings where they were exposed to increased UV-B radiation (103 forest + clearing populations). We then analysed the data using phylogenetic mixed models. KEY RESULTS: The concentration of UV-B protectives increased with UV-B doses estimated from hemispheric photographs of the sky above sample collection sites, but the increase was more rapid in species with monocentric chromosomes. Endopolyploidy index increased with UV-B doses and with concentrations of UV-B-absorbing compounds only in species with monocentric chromosomes, while holocentric species responded negligibly. CONCLUSIONS: Endopolyploidy seems to be a common response to increased UV-B in monocentric plants. Low sensitivity to UV-B in holocentric species might relate to their success in high-UV-stressed habitats and corroborates the hypothesized role of holocentric chromosomes in plant terrestrialization.

Pest Arthropods with Holocentric Chromosomes are More Resistant to Sterilizing Ionizing Radiation.

It has been hypothesized that species with holocentric chromosomes have a selective evolutionary advantage for developmental and reproductive success because holocentric chromosomes are less susceptible to chromosome breakage than monocentric chromosomes. We analyzed data on sterilizing doses of ionizing radiation for more than 250 species of arthropods to test whether the minimal dose for reproductive sterilization is higher for species with holocentric chromosomes than for species with monocentric chromosomes. Using linear mixed models that account for phylogeny, we show that holocentric arthropods are more tolerant of sterilizing radiation than monocentrics. Moreover, higher dose rates correlate with lower sterilizing doses in monocentrics, but not in holocentrics, which is a novel finding that may be of importance for radiosanitation practice. Under the dose rate of 1 Gy/min, holocentric arthropods are sterilized on average with a 2.9 times higher minimal dose than monocentrics. Life stage and sex have significant but considerably weaker effects on sterilizing dose than chromosome type. Adults and males require 1.2 and 1.4 times higher sterilizing doses than juveniles and females, respectively. These results support the hypothesis that holocentric lineages may originate and thrive better in times of increased exposure to chromosome-breaking factors.

Holocentric chromosomes: from tolerance to fragmentation to colonization of the land.

Background: The dispersed occurrence of holocentric chromosomes across eukaryotes implies they are adaptive, but the conditions under which they confer an advantage over monocentric chromosomes remain unclear. Due to their extended kinetochore and the attachment of spindle microtubules along their entire length, holocentric chromosomes tolerate fragmentation; hence, they may be advantageous in times of exposure to factors that cause chromosomal fragmentation (clastogens). Scope: It is shown that holocentric organisms may, indeed, thrive better than monocentric organisms under clastogenic conditions and that such conditions of various duration and intensity have occurred many times throughout the history of Earth's biota. One of the most important clastogenic events in eukaryotic history, in which holocentric chromosomes may have played the key role, was the colonization of land by plants and animals half a billion years ago. In addition to arguments supporting the anticlastogenic hypothesis of holocentric chromosomes and a discussion of its evolutionary consequences, experiments and analyses are proposed to explore this hypothesis in more depth. Conclusions: It is argued that the tolerance to clastogens explains the origin of holocentric lineages and may also have far-reaching consequences for eukaryotic evolution in general as exemplified by the potential role of holocentric chromosomes in terrestrialization.

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

This corrects the article DOI: 10.1038/srep27161.

Evolution of genome size and genomic GC content in carnivorous holokinetics (Droseraceae).

BACKGROUND AND AIMS: Studies in the carnivorous family Lentibulariaceae in the last years resulted in the discovery of the smallest plant genomes and an unusual pattern of genomic GC content evolution. However, scarcity of genomic data in other carnivorous clades still prevents a generalization of the observed patterns. Here the aim was to fill this gap by mapping genome evolution in the second largest carnivorous family, Droseraceae, where this evolution may be affected by chromosomal holokinetism in Drosera METHODS: The genome size and genomic GC content of 71 Droseraceae species were measured by flow cytometry. A dated phylogeny was constructed, and the evolution of both genomic parameters and their relationship to species climatic niches were tested using phylogeny-based statistics. KEY RESULTS: The 2C genome size of Droseraceae varied between 488 and 10 927 Mbp, and the GC content ranged between 37·1 and 44·7 %. The genome sizes and genomic GC content of carnivorous and holocentric species did not differ from those of their non-carnivorous and monocentric relatives. The genomic GC content positively correlated with genome size and annual temperature fluctuations. The genome size and chromosome numbers were inversely correlated in the Australian clade of Drosera CONCLUSIONS: Our results indicate that neither carnivory (nutrient scarcity) nor the holokinetism have a prominent effect on size and DNA base composition of Droseraceae genomes. However, the holokinetic drive seems to affect karyotype evolution in one of the major clades of Drosera Our survey confirmed that the evolution of GC content is tightly connected with the evolution of genome size and also with environmental conditions.

Absence of positive selection on CenH3 in Luzula suggests that holokinetic chromosomes may suppress centromere drive.

BACKGROUND AND AIMS: The centromere drive theory explains diversity of eukaryotic centromeres as a consequence of the recurrent conflict between centromeric repeats and centromeric histone H3 (CenH3), in which selfish centromeres exploit meiotic asymmetry and CenH3 evolves adaptively to counterbalance deleterious consequences of driving centromeres. Accordingly, adaptively evolving CenH3 has so far been observed only in eukaryotes with asymmetric meiosis. However, if such evolution is a consequence of centromere drive, it should depend not only on meiotic asymmetry but also on monocentric or holokinetic chromosomal structure. Selective pressures acting on CenH3 have never been investigated in organisms with holokinetic meiosis despite the fact that holokinetic chromosomes have been hypothesized to suppress centromere drive. Therefore, the present study evaluates selective pressures acting on the CenH3 gene in holokinetic organisms for the first time, specifically in the representatives of the plant genus Luzula (Juncaceae), in which the kinetochore formation is not co-localized with any type of centromeric repeat. METHODS: PCR, cloning and sequencing, and database searches were used to obtain coding CenH3 sequences from Luzula species. Codon substitution models were employed to infer selective regimes acting on CenH3 in Luzula KEY RESULTS: In addition to the two previously published CenH3 sequences from L. nivea, 16 new CenH3 sequences have been isolated from 12 Luzula species. Two CenH3 isoforms in Luzula that originated by a duplication event prior to the divergence of analysed species were found. No signs of positive selection acting on CenH3 in Luzula were detected. Instead, evidence was found that selection on CenH3 of Luzula might have been relaxed. CONCLUSIONS: The results indicate that holokinetism itself may suppress centromere drive and, therefore, holokinetic chromosomes might have evolved as a defence against centromere drive.

CenH3 evolution reflects meiotic symmetry as predicted by the centromere drive model.

The centromere drive model explaining rapid evolution of eukaryotic centromeres predicts higher frequency of positive selection acting on centromeric histone H3 (CenH3) in clades with asymmetric meiosis compared to the clades with only symmetric meiosis. However, despite the impression one might get from the literature, this key prediction of the centromere drive model has not only never been confirmed, but it has never been tested, because all the previous studies dealt only with the presence or absence instead of the frequency of positive selection. To provide evidence for or against different frequencies of positively selected CenH3 in asymmetrics and symmetrics, we have inferred the selective pressures acting on CenH3 in seventeen eukaryotic clades, including plants, animals, fungi, ciliates and apicomplexa, using codon-substitution models, and compared the inferred frequencies between asymmetrics and symmetrics in a quantitative manner. We have found that CenH3 has been evolving adaptively much more frequently in clades with asymmetric meiosis compared with clades displaying only symmetric meiosis which confirms the prediction of centromere drive model. Our findings indicate that the evolution of asymmetric meiosis required CenH3 to evolve adaptively more often to counterbalance the negative consequences of centromere drive.

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.

Holokinetic drive: centromere drive in chromosomes without centromeres.

Similar to how the model of centromere drive explains the size and complexity of centromeres in monocentrics (organisms with localized centromeres), our model of holokinetic drive is consistent with the divergent evolution of chromosomal size and number in holocentrics (organisms with nonlocalized centromeres) exhibiting holokinetic meiosis (holokinetics). Holokinetic drive is proposed to facilitate chromosomal fission and/or repetitive DNA removal (or any segmental deletion) when smaller homologous chromosomes are preferentially inherited or chromosomal fusion and/or repetitive DNA proliferation (or any segmental duplication) when larger homologs are preferred. The hypothesis of holokinetic drive is supported primarily by the negative correlation between chromosome number and genome size that is documented in holokinetic lineages. The supporting value of two older cross-experiments on holokinetic structural heterozygotes (the rush Luzula elegans and butterflies of the genus Antheraea) that indicate the presence of size-preferential homolog transmission via female meiosis for holokinetic drive is discussed, along with the further potential consequences of holokinetic drive in comparison with centromere drive.

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.

Evidence for centromere drive in the holocentric chromosomes of Caenorhabditis.

In monocentric organisms with asymmetric meiosis, the kinetochore proteins, such as CENH3 and CENP-C, evolve adaptively to counterbalance the deleterious effects of centromere drive, which is caused by the expansion of centromeric satellite repeats. The selection regimes that act on CENH3 and CENP-C genes have not been analyzed in organisms with holocentric chromosomes, although holocentrism is speculated to have evolved to suppress centromere drive. We tested both CENH3 and CENP-C for positive selection in several species of the holocentric genus Caenorhabditis using the maximum likelihood approach and sliding-window analysis. Although CENP-C did not show any signs of positive selection, positive selection has been detected in the case of CENH3. These results support the hypothesis that centromere drive occurs in Nematoda, at least in the telokinetic meiosis of Caenorhabditis.

Prognostic impact of p53 aberrations for R-CHOP-treated patients with diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most frequent lymphoma in adults. There are specific alterations that appear repeatedly in DLBCL cases and play a role in lymphomagenesis or progression of the disease. Some aberrations were used as prognostic markers in the pre-rituximab era. Addition of rituximab to the classical anthracycline-based chemotherapy significantly increased the survival rate in DLBCL. Only few prognostic factors have been re-evaluated for patients treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone). We performed complex analysis of the p53 tumor suppressor in collection of 75 DLBCL cases. Fifty-four patients were de novo cases, twenty-one cases developed into DLBCL by transformation from less aggressive disease. We determined functional status by analysis of separated alleles in yeast (FASAY) and analyzed the p53 mutations by cDNA sequencing. We assessed the level of the p53 protein by immunoblot analysis. We used FISH to analyze loss of the p53 and ATM (ataxia telangiectasia mutated) gene deletions. We detected 16 p53 mutations (21.3%) including the mutation activating non-sense-mediated RNA decay pathway. Deletion of the p53 allele was more common in cases with p53 mutation. Mutations and/or deletions of p53 had statistically significant negative impact on progression-free survival and tended to decrease also overall survival in 46 de novo DLBCL patients treated with R-CHOP. p53 aberrations are negative predictors for survival of DLBCL patients treated with R-CHOP.