Is there hybridization between diploid and tetraploid Euphrasia in a secondary contact zone?

PREMISE: Strong postzygotic reproductive isolating barriers are usually expected to limit the extent of natural hybridization between species with contrasting ploidy. However, genomic sequencing has revealed previously overlooked examples of natural cross-ploidy hybridization in some flowering plant genera, suggesting that the phenomenon may be more common than once thought. We investigated potential cross-ploidy hybridization in British eyebrights (Euphrasia, Orobanchaceae), a group from which 13 putative cross-ploidy hybrid combinations have been reported based on morphology. METHODS: We analyzed a contact zone between diploid Euphrasia rostkoviana and tetraploid E. arctica in Wales. We sequenced part of the internal transcribed spacer (ITS) of nuclear ribosomal DNA and used genotyping by sequencing (GBS) to look for evidence of cross-ploidy hybridization and introgression. RESULTS: Common variant sites in the ITS region were fixed between diploids and tetraploids, indicating a strong barrier to hybridization. Clustering analyses of 356 single-nucleotide polymorphisms (SNPs) generated using GBS clearly separated samples by ploidy and revealed strong genetic structure (FST = 0.44). However, the FST distribution across all SNPs was bimodal, indicating potential differential selection on loci between diploids and tetraploids. Demographic inference suggested potential gene flow, limited to around one or fewer migrants per generation. CONCLUSIONS: Our results suggest that recent cross-ploidy hybridization is rare or absent in a site of secondary contact in Euphrasia. While a strong ploidy barrier prevents hybridization over ecological timescales, such hybrids may form in stable populations over evolutionary timescales, potentially allowing cross-ploidy introgression to take place.

The Impact of Purifying and Background Selection on the Inference of Population History: Problems and Prospects.

Current procedures for inferring population history generally assume complete neutrality-that is, they neglect both direct selection and the effects of selection on linked sites. We here examine how the presence of direct purifying selection and background selection may bias demographic inference by evaluating two commonly-used methods (MSMC and fastsimcoal2), specifically studying how the underlying shape of the distribution of fitness effects and the fraction of directly selected sites interact with demographic parameter estimation. The results show that, even after masking functional genomic regions, background selection may cause the mis-inference of population growth under models of both constant population size and decline. This effect is amplified as the strength of purifying selection and the density of directly selected sites increases, as indicated by the distortion of the site frequency spectrum and levels of nucleotide diversity at linked neutral sites. We also show how simulated changes in background selection effects caused by population size changes can be predicted analytically. We propose a potential method for correcting for the mis-inference of population growth caused by selection. By treating the distribution of fitness effect as a nuisance parameter and averaging across all potential realizations, we demonstrate that even directly selected sites can be used to infer demographic histories with reasonable accuracy.

The nature of intraspecific and interspecific genome size variation in taxonomically complex eyebrights.

BACKGROUND AND AIMS: Genome size varies considerably across the diversity of plant life. Although genome size is, by definition, affected by genetic presence/absence variants, which are ubiquitous in population sequencing studies, genome size is often treated as an intrinsic property of a species. Here, we studied intra- and interspecific genome size variation in taxonomically complex British eyebrights (Euphrasia, Orobanchaceae). Our aim is to document genome size diversity and investigate underlying evolutionary processes shaping variation between individuals, populations and species. METHODS: We generated genome size data for 192 individuals of diploid and tetraploid Euphrasia and analysed genome size variation in relation to ploidy, taxonomy, population affiliation and geography. We further compared the genomic repeat content of 30 samples. KEY RESULTS: We found considerable intraspecific genome size variation, and observed isolation-by-distance for genome size in outcrossing diploids. Tetraploid Euphrasia showed contrasting patterns, with genome size increasing with latitude in outcrossing Euphrasia arctica, but with little genome size variation in the highly selfing Euphrasia micrantha. Interspecific differences in genome size and the genomic proportions of repeat sequences were small. CONCLUSIONS: We show the utility of treating genome size as the outcome of polygenic variation. Like other types of genetic variation, such as single nucleotide polymorphisms, genome size variation may be affected by ongoing hybridization and the extent of population subdivision. In addition to selection on associated traits, genome size is predicted to be affected indirectly by selection due to pleiotropy of the underlying presence/absence variants.

Remarkable variation of ribosomal DNA organization and copy number in gnetophytes, a distinct lineage of gymnosperms.

INTRODUCTION: Gnetophytes, comprising the genera Ephedra, Gnetum and Welwitschia, are an understudied, enigmatic lineage of gymnosperms with a controversial phylogenetic relationship to other seed plants. Here we examined the organization of ribosomal DNA (rDNA) across representative species. METHODS: We applied high-throughput sequencing approaches to isolate and reconstruct rDNA units and to determine their intragenomic homogeneity. In addition, fluorescent in situ hybridization and Southern blot hybridization techniques were used to reveal the chromosome and genomic organization of rDNA. KEY RESULTS: The 5S and 35S rRNA genes were separate (S-type) in Gnetum montanum, Gnetum gnemon and Welwitschia mirabilis and linked (L-type) in Ephedra altissima. There was considerable variability in 5S rDNA abundance, ranging from as few as ~4000 (W. mirabilis) to >100 000 (G. montanum) copies. A similar large variation was also observed in 5S rDNA locus numbers (two to 16 sites per diploid cell). 5S rRNA pseudogenes were interspersed between functional genes forming a single unit in E. altissima and G. montanum. Their copy number was comparable or even higher than that of functional 5S rRNA genes. In E. altissima internal transcribed spacers of 35S rDNA were long and intrinsically repetitive while in G. montanum and W. mirabilis they were short without the subrepeats. CONCLUSIONS: Gnetophytes are distinct from other gymnosperms and angiosperms as they display surprisingly large variability in rDNA organization and rDNA copy and locus numbers between genera, with no relationship between copy numbers and genome sizes apparent. Concerted evolution of 5S rDNA units seems to have led to the amplification of 5S pseudogenes in both G. montanum and E. altissima. Evolutionary patterns of rDNA show both gymnosperm and angiosperm features underlining the diversity of the group.

Astonishing 35S rDNA diversity in the gymnosperm species Cycas revoluta Thunb.

In all eukaryotes, the highly repeated 35S ribosomal DNA (rDNA) sequences encoding 18S-5.8S-26S ribosomal RNA (rRNA) typically show high levels of intragenomic uniformity due to homogenisation processes, leading to concerted evolution of 35S rDNA repeats. Here, we compared 35S rDNA divergence in several seed plants using next generation sequencing and a range of molecular and cytogenetic approaches. Most species showed similar 35S rDNA homogeneity indicating concerted evolution. However, Cycas revoluta exhibits an extraordinary diversity of rDNA repeats (nucleotide sequence divergence of different copies averaging 12 %), influencing both the coding and non-coding rDNA regions nearly equally. In contrast, its rRNA transcriptome was highly homogeneous suggesting that only a minority of genes (<20 %) encode functional rRNA. The most common SNPs were C > T substitutions located in symmetrical CG and CHG contexts which were also highly methylated. Both functional genes and pseudogenes appear to cluster on chromosomes. The extraordinary high levels of 35S rDNA diversity in C. revoluta, and probably other species of cycads, indicate that the frequency of repeat homogenisation has been much lower in this lineage, compared with all other land plant lineages studied. This has led to the accumulation of methylation-driven mutations and pseudogenisation. Potentially, the reduced homology between paralogs prevented their elimination by homologous recombination, resulting in long-term retention of rDNA pseudogenes in the genome.

Endogenous pararetrovirus sequences associated with 24 nt small RNAs at the centromeres of Fritillaria imperialis L. (Liliaceae), a species with a giant genome.

Endogenous pararetroviral sequences are the most commonly found virus sequences integrated into angiosperm genomes. We describe an endogenous pararetrovirus (EPRV) repeat in Fritillaria imperialis, a species that is under study as a result of its exceptionally large genome (1C = 42 096 Mbp, approximately 240 times bigger than Arabidopsis thaliana). The repeat (FriEPRV) was identified from Illumina reads using the RepeatExplorer pipeline, and exists in a complex genomic organization at the centromere of most, or all, chromosomes. The repeat was reconstructed into three consensus sequences that formed three interconnected loops, one of which carries sequence motifs expected of an EPRV (including the gag and pol domains). FriEPRV shows sequence similarity to members of the Caulimoviridae pararetrovirus family, with phylogenetic analysis indicating a close relationship to Petuvirus. It is possible that no complete EPRV sequence exists, although our data suggest an abundance that exceeds the genome size of Arabidopsis. Analysis of single nucleotide polymorphisms revealed elevated levels of C→T and G→A transitions, consistent with deamination of methylated cytosine. Bisulphite sequencing revealed high levels of methylation at CG and CHG motifs (up to 100%), and 15-20% methylation, on average, at CHH motifs. FriEPRV's centromeric location may suggest targeted insertion, perhaps associated with meiotic drive. We observed an abundance of 24 nt small RNAs that specifically target FriEPRV, potentially providing a signature of RNA-dependent DNA methylation. Such signatures of epigenetic regulation suggest that the huge genome of F. imperialis has not arisen as a consequence of a catastrophic breakdown in the regulation of repeat amplification.

Assembly-free quantification of vagrant DNA inserts.

Inserts of DNA from extranuclear sources, such as organelles and microbes, are common in eukaryote nuclear genomes. However, sequence similarity between the nuclear and extranuclear DNA, and a history of multiple insertions, make the assembly of these regions challenging. Consequently, the number, sequence and location of these vagrant DNAs cannot be reliably inferred from the genome assemblies of most organisms. We introduce two statistical methods to estimate the abundance of nuclear inserts even in the absence of a nuclear genome assembly. The first (intercept method) only requires low-coverage (<1×) sequencing data, as commonly generated for population studies of organellar and ribosomal DNAs. The second method additionally requires that a subset of the individuals carry extranuclear DNA with diverged genotypes. We validated our intercept method using simulations and by re-estimating the frequency of human NUMTs (nuclear mitochondrial inserts). We then applied it to the grasshopper Podisma pedestris, exceptional for both its large genome size and reports of numerous NUMT inserts, estimating that NUMTs make up 0.056% of the nuclear genome, equivalent to >500 times the mitochondrial genome size. We also re-analysed a museomics data set of the parrot Psephotellus varius, obtaining an estimate of only 0.0043%, in line with reports from other species of bird. Our study demonstrates the utility of low-coverage high-throughput sequencing data for the quantification of nuclear vagrant DNAs. Beyond quantifying organellar inserts, these methods could also be used on endosymbiont-derived sequences. We provide an R implementation of our methods called "vagrantDNA" and code to simulate test data sets.

Typing myalgic encephalomyelitis by infection at onset: A DecodeME study.

Background: People with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) experience core symptoms of post-exertional malaise, unrefreshing sleep, and cognitive impairment. Despite numbering 0.2-0.4% of the population, no laboratory test is available for their diagnosis, no effective therapy exists for their treatment, and no scientific breakthrough regarding pathogenesis has been made. It remains unknown, despite decades of small-scale studies, whether individuals experience different types of ME/CFS separated by onset-type, sex or age. Methods: DecodeME is a large population-based study of ME/CFS that recruited 17,074 participants in the first 3 months following full launch. Detailed questionnaire responses from UK-based participants who all reported being diagnosed with ME/CFS by a health professional provided an unparalleled opportunity to investigate, using logistic regression, whether ME/CFS severity or onset type is significantly associated with sex, age, illness duration, comorbid conditions or symptoms. Results: The well-established sex-bias among ME/CFS patients is evident in the initial DecodeME cohort: 83.5% of participants were females. What was not known previously was that females tend to have more comorbidities than males. Moreover, being female, being older and being over 10 years from ME/CFS onset are significantly associated with greater severity. Five different ME/CFS onset types were examined in the self-reported data: those with ME/CFS onset (i) after glandular fever (infectious mononucleosis); (ii) after COVID-19 infection; (iii) after other infections; (iv) without an infection at onset; and, (v) where the occurrence of an infection at or preceding onset is not known. Among other findings, ME/CFS onset with unknown infection status was significantly associated with active fibromyalgia. Conclusions: DecodeME participants differ in symptoms, comorbid conditions and/or illness severity when stratified by their sex-at-birth and/or infection around the time of ME/CFS onset.

Myalgic Encephalomyelitis / Chronic Fatigue Syndrome (ME/CFS) is a chronic disease that affects an estimated 250,000 people in the UK. Its defining symptom is post-exertional malaise, an excessive delayed worsening of symptoms following even minor physical or mental exertion. For those with it, ME/CFS means disability and poor quality of life. DecodeME is a research study which is looking for DNA differences between people with ME/CFS and people without any health problems. People with ME/CFS who take part in DecodeME complete a questionnaire that assesses their symptoms and whether they will then be invited to donate a DNA sample. This paper analyses the answers to this questionnaire; we will publish results of the DNA analysis separately. So far, more than 17 thousand people with ME/CFS have completed the DecodeME questionnaire. Their answers help us to address the question: "Are there different types of ME/CFS linked to different causes and how severe it becomes?" Results show that people with ME/CFS do not form a single group reporting similar symptoms and additional medical conditions. Instead, participants who had an infection at the start of their ME/CFS reported a different pattern of symptoms and conditions compared to those without an infection. It is well known that most people with ME/CFS are females. What was not clear previously was that females tend to have more additional health conditions. Also, being female, being older and being over 10 years from ME/CFS onset all make it more likely that someone is more severely affected by their ME/CFS. These findings could indicate that by studying people with different ME/CFS onset-types separately ­ rather than analysing all people with ME/CFS together ­ it will be easier to understand what is going wrong.

Measuring the Invisible: The Sequences Causal of Genome Size Differences in Eyebrights (Euphrasia) Revealed by k-mers.

Genome size variation within plant taxa is due to presence/absence variation, which may affect low-copy sequences or genomic repeats of various frequency classes. However, identifying the sequences underpinning genome size variation is challenging because genome assemblies commonly contain collapsed representations of repetitive sequences and because genome skimming studies by design miss low-copy number sequences. Here, we take a novel approach based on k-mers, short sub-sequences of equal length k, generated from whole-genome sequencing data of diploid eyebrights (Euphrasia), a group of plants that have considerable genome size variation within a ploidy level. We compare k-mer inventories within and between closely related species, and quantify the contribution of different copy number classes to genome size differences. We further match high-copy number k-mers to specific repeat types as retrieved from the RepeatExplorer2 pipeline. We find genome size differences of up to 230Mbp, equivalent to more than 20% genome size variation. The largest contributions to these differences come from rDNA sequences, a 145-nt genomic satellite and a repeat associated with an Angela transposable element. We also find size differences in the low-copy number class (copy number ≤ 10×) of up to 27 Mbp, possibly indicating differences in gene space between our samples. We demonstrate that it is possible to pinpoint the sequences causing genome size variation within species without the use of a reference genome. Such sequences can serve as targets for future cytogenetic studies. We also show that studies of genome size variation should go beyond repeats if they aim to characterise the full range of genomic variants. To allow future work with other taxonomic groups, we share our k-mer analysis pipeline, which is straightforward to run, relying largely on standard GNU command line tools.

Pervasive Phylogenomic Incongruence Underlies Evolutionary Relationships in Eyebrights (Euphrasia, Orobanchaceae).

Disentangling the phylogenetic relationships of taxonomically complex plant groups is often mired by challenges associated with recent speciation, hybridization, complex mating systems, and polyploidy. Here, we perform a phylogenomic analysis of eyebrights (Euphrasia), a group renowned for taxonomic complexity, with the aim of documenting the extent of phylogenetic discordance at both deep and at shallow phylogenetic scales. We generate whole-genome sequencing data and integrate this with prior genomic data to perform a comprehensive analysis of nuclear genomic, nuclear ribosomal (nrDNA), and complete plastid genomes from 57 individuals representing 36 Euphrasia species. The species tree analysis of 3,454 conserved nuclear scaffolds (46 Mb) reveals that at shallow phylogenetic scales postglacial colonization of North Western Europe occurred in multiple waves from discrete source populations, with most species not being monophyletic, and instead combining genomic variants from across clades. At a deeper phylogenetic scale, the Euphrasia phylogeny is structured by geography and ploidy, and partially by taxonomy. Comparative analyses show Southern Hemisphere tetraploids include a distinct subgenome indicative of independent polyploidy events from Northern Hemisphere taxa. In contrast to the nuclear genome analyses, the plastid genome phylogeny reveals limited geographic structure, while the nrDNA phylogeny is informative of some geographic and taxonomic affinities but more thorough phylogenetic inference is impeded by the retention of ancestral polymorphisms in the polyploids. Overall our results reveal extensive phylogenetic discordance at both deeper and shallower nodes, with broad-scale geographic structure of genomic variation but a lack of definitive taxonomic signal. This suggests that Euphrasia species either have polytopic origins or are maintained by narrow genomic regions in the face of extensive homogenizing gene flow. Moreover, these results suggest genome skimming will not be an effective extended barcode to identify species in groups such as Euphrasia, or many other postglacial species groups.

The impact of purifying and background selection on the inference of population history: problems and prospects.

Current procedures for inferring population history generally assume complete neutrality - that is, they neglect both direct selection and the effects of selection on linked sites. We here examine how the presence of direct purifying selection and background selection may bias demographic inference by evaluating two commonly-used methods (MSMC and fastsimcoal2 ), specifically studying how the underlying shape of the distribution of fitness effects (DFE) and the fraction of directly selected sites interact with demographic parameter estimation. The results show that, even after masking functional genomic regions, background selection may cause the mis-inference of population growth under models of both constant population size and decline. This effect is amplified as the strength of purifying selection and the density of directly selected sites increases, as indicated by the distortion of the site frequency spectrum and levels of nucleotide diversity at linked neutral sites. We also show how simulated changes in background selection effects caused by population size changes can be predicted analytically. We propose a potential method for correcting for the mis-inference of population growth caused by selection. By treating the DFE as a nuisance parameter and averaging across all potential realizations, we demonstrate that even directly selected sites can be used to infer demographic histories with reasonable accuracy.

A Draft Genome of the Ginger Species Alpinia nigra and New Insights into the Genetic Basis of Flexistyly.

Angiosperms possess various strategies to ensure reproductive success, such as stylar polymorphisms that encourage outcrossing. Here, we investigate the genetic basis of one such dimorphism that combines both temporal and spatial separation of sexual function, termed flexistyly. It is a floral strategy characterised by the presence of two morphs that differ in the timing of stylar movement. We performed a de novo assembly of the genome of Alpinia nigra using high-depth genomic sequencing. We then used Pool-seq to identify candidate regions for flexistyly based on allele frequency or coverage differences between pools of anaflexistylous and cataflexistylous morphs. The final genome assembly size was 2 Gb, and showed no evidence of recent polyploidy. The Pool-seq did not reveal large regions with high FST values, suggesting large structural chromosomal polymorphisms are unlikely to underlie differences between morphs. Similarly, no region had a 1:2 mapping depth ratio which would be indicative of hemizygosity. We propose that flexistyly is governed by a small genomic region that might be difficult to detect with Pool-seq, or a complex genomic region that proved difficult to assemble. Our genome will be a valuable resource for future studies of gingers, and provides the first steps towards characterising this complex floral phenotype.

Patterns of Genetic Variability in Genomic Regions with Low Rates of Recombination.

The amount of DNA sequence variability in a genomic region is often positively correlated with its rate of crossing over (CO) [1-3]. This pattern is caused by selection acting on linked sites, which reduces genetic variability and biases the frequency distribution of segregating variants toward more rare variants than are expected without selection (skew). These effects may involve the spread of beneficial mutations (selective sweeps [SSWs]), the elimination of deleterious mutations (background selection [BGS]), or both, and are expected to be stronger with lower CO rates [1-3]. However, in a recent study of human populations, the skew was reduced in the lowest CO regions compared with regions with somewhat higher CO rates [4]. A low skew in very low CO regions, compared with theoretical predictions, is seen in the population genomic studies of Drosophila simulans described here and in other Drosophila species. Here, we propose an explanation for lower than expected skew in low CO regions, and validate it using computer simulations; explanations for higher skew with higher CO rates, as in D. simulans, will be explored elsewhere. Partially recessive, linked deleterious mutations can increase neutral variability when the product of the effective population size (Ne) and the selection coefficient against homozygous carriers of mutations (s) is ≤1, i.e., there is associative overdominance (AOD) rather than BGS [5]. AOD can operate in low CO regions, producing a lower skew than in its absence. This opens up a new perspective on how selection affects patterns of variability at linked sites.

Molecular Phylogenetics and Micromorphology of Australasian Stipeae (Poaceae, Subfamily Pooideae), and the Interrelation of Whole-Genome Duplication and Evolutionary Radiations in This Grass Tribe.

The mainly Australian grass genus Austrostipa (tribe Stipeae) comprising approximately 64 species represents a remarkable example of an evolutionary radiation. To investigate aspects of diversification, macro- and micromorphological variation in this genus, we conducted molecular phylogenetic and scanning electron microscopy (SEM) analyses including representatives from most of Austrostipa's currently accepted subgenera. Because of its taxonomic significance in Stipeae, we studied the lemma epidermal pattern (LEP) in 34 representatives of Austrostipa. Plastid DNA variation within Austrostipa was low and only few lineages were resolved. Nuclear ITS and Acc1 yielded comparable groupings of taxa and resolved subgenera Arbuscula, Petaurista, and Bambusina in a common clade and as monophyletic. In most of the Austrostipa species studied, the LEP was relatively uniform (typical maize-like), but six species had a modified cellular structure. The species representing subgenera Lobatae, Petaurista, Bambusina as well as A. muelleri from subg. Tuberculatae were well-separated from all the other species included in the analysis. We suggest recognizing nine subgenera in Austrostipa (with number of species): Arbuscula (4), Aulax (2), Austrostipa (36), Bambusina (2), Falcatae (10), Lobatae (5), Longiaristatae (2), Petaurista (2) and the new subgenus Paucispiculatae (1) encompassing A. muelleri. Two paralogous sequence copies of Acc1, forming two distinct clades, were found in polyploid Austrostipa and Anemanthele. We found analogous patterns for our samples of Stipa s.str. with their Acc1 clades strongly separated from those of Austrostipa and Anemanthele. This underlines a previous hypothesis of Tzvelev (1977) that most extant Stipeae are of hybrid origin. We also prepared an up-to-date survey and reviewed the chromosome number variation for our molecularly studied taxa and the whole tribe Stipeae. The chromosome base number patterns as well as dysploidy and whole-genome duplication events were interpreted in a phylogenetic framework. The rather coherent picture of chromosome number variation underlines the enormous phylogenetic and evolutionary significance of this frequently ignored character.

Maintenance of Species Differences in Closely Related Tetraploid Parasitic Euphrasia (Orobanchaceae) on an Isolated Island.

Polyploidy is pervasive in angiosperm evolution and plays important roles in adaptation and speciation. However, polyploid groups are understudied due to complex sequence homology, challenging genome assembly, and taxonomic complexity. Here, we study adaptive divergence in taxonomically complex eyebrights (Euphrasia), where recent divergence, phenotypic plasticity, and hybridization blur species boundaries. We focus on three closely related tetraploid species with contrasting ecological preferences that are sympatric on Fair Isle, a small isolated island in the British Isles. Using a common garden experiment, we show a genetic component to the morphological differences present between these species. Using whole-genome sequencing and a novel k-mer approach we call "Tetmer", we demonstrate that the species are of allopolyploid origin, with a sub-genome divergence of approximately 5%. Using ∼2 million SNPs, we show sub-genome homology across species, with a very low sequence divergence characteristic of recent speciation. This genetic variation is broadly structured by species, with clear divergence of Fair Isle heathland Euphrasia micrantha, while grassland Euphrasia arctica and coastal Euphrasia foulaensis are more closely related. Overall, we show that tetraploid Euphrasia is a system of allopolyploids of postglacial species divergence, where adaptation to novel environments may be conferred by old variants rearranged into new genetic lineages.

Digest: Ancestry mosaics hint at selection and may provide an alternative to differentiation scans.

We now have a flood of genomic sequencing data available to study reproductive isolation and selection in action, but how are these data best analyzed? Usually, genetic differentiation is compared between two groups, scanning along genomes. This approach has several drawbacks, and has been criticized repeatedly. An alternative, truly genetic approach, based on blocks of common ancestry in a hybrid zone setting, is presented by Hvala et al. (2018) in this issue.

Karyotype evolution in Phalaris (Poaceae): The role of reductional dysploidy, polyploidy and chromosome alteration in a wide-spread and diverse genus.

Karyotype characteristics can provide valuable information on genome evolution and speciation, in particular in taxa with varying basic chromosome numbers and ploidy levels. Due to its worldwide distribution, remarkable variability in morphological traits and the fact that ploidy change plays a key role in its evolution, the canary grass genus Phalaris (Poaceae) is an excellent study system to investigate the role of chromosomal changes in species diversification and expansion. Phalaris comprises diploid species with two basic chromosome numbers of x = 6 and 7 as well as polyploids based on x = 7. To identify distinct karyotype structures and to trace chromosome evolution within the genus, we apply fluorescence in situ hybridisation (FISH) of 5S and 45S rDNA probes in four diploid and four tetraploid Phalaris species of both basic numbers. The data agree with a dysploid reduction from x = 7 to x = 6 as the result of reciprocal translocations between three chromosomes of an ancestor with a diploid chromosome complement of 2n = 14. We recognize three different genomes in the genus: (1) the exclusively Mediterranean genome A based on x = 6, (2) the cosmopolitan genome B based on x = 7 and (3) a genome C based on x = 7 and with a distribution in the Mediterranean and the Middle East. Both auto- and allopolyploidy of genomes B and C are suggested for the formation of tetraploids. The chromosomal divergence observed in Phalaris can be explained by the occurrence of dysploidy, the emergence of three different genomes, and the chromosome rearrangements accompanied by karyotype change and polyploidization. Mapping the recognized karyotypes on the existing phylogenetic tree suggests that genomes A and C are restricted to sections Phalaris and Bulbophalaris, respectively, while genome B occurs across all taxa with x = 7.

Correction: Karyotype evolution in Phalaris (Poaceae): The role of reductional dysploidy, polyploidy and chromosome alteration in a wide-spread and diverse genus.

[This corrects the article DOI: 10.1371/journal.pone.0192869.].

Angiosperms Are Unique among Land Plant Lineages in the Occurrence of Key Genes in the RNA-Directed DNA Methylation (RdDM) Pathway.

The RNA-directed DNA methylation (RdDM) pathway can be divided into three phases: 1) small interfering RNA biogenesis, 2) de novo methylation, and 3) chromatin modification. To determine the degree of conservation of this pathway we searched for key genes among land plants. We used OrthoMCL and the OrthoMCL Viridiplantae database to analyze proteomes of species in bryophytes, lycophytes, monilophytes, gymnosperms, and angiosperms. We also analyzed small RNA size categories and, in two gymnosperms, cytosine methylation in ribosomal DNA. Six proteins were restricted to angiosperms, these being NRPD4/NRPE4, RDM1, DMS3 (defective in meristem silencing 3), SHH1 (SAWADEE homeodomain homolog 1), KTF1, and SUVR2, although we failed to find the latter three proteins in Fritillaria persica, a species with a giant genome. Small RNAs of 24 nt in length were abundant only in angiosperms. Phylogenetic analyses of Dicer-like (DCL) proteins showed that DCL2 was restricted to seed plants, although it was absent in Gnetum gnemon and Welwitschia mirabilis. The data suggest that phases (1) and (2) of the RdDM pathway, described for model angiosperms, evolved with angiosperms. The absence of some features of RdDM in F. persica may be associated with its large genome. Phase (3) is probably the most conserved part of the pathway across land plants. DCL2, involved in virus defense and interaction with the canonical RdDM pathway to facilitate methylation of CHH, is absent outside seed plants. Its absence in G. gnemon, and W. mirabilis coupled with distinctive patterns of CHH methylation, suggest a secondary loss of DCL2 following the divergence of Gnetales.