Genome sequence and genetic diversity of European ash trees.

Ash trees (genus Fraxinus, family Oleaceae) are widespread throughout the Northern Hemisphere, but are being devastated in Europe by the fungus Hymenoscyphus fraxineus, causing ash dieback, and in North America by the herbivorous beetle Agrilus planipennis. Here we sequence the genome of a low-heterozygosity Fraxinus excelsior tree from Gloucestershire, UK, annotating 38,852 protein-coding genes of which 25% appear ash specific when compared with the genomes of ten other plant species. Analyses of paralogous genes suggest a whole-genome duplication shared with olive (Olea europaea, Oleaceae). We also re-sequence 37 F. excelsior trees from Europe, finding evidence for apparent long-term decline in effective population size. Using our reference sequence, we re-analyse association transcriptomic data, yielding improved markers for reduced susceptibility to ash dieback. Surveys of these markers in British populations suggest that reduced susceptibility to ash dieback may be more widespread in Great Britain than in Denmark. We also present evidence that susceptibility of trees to H. fraxineus is associated with their iridoid glycoside levels. This rapid, integrated, multidisciplinary research response to an emerging health threat in a non-model organism opens the way for mitigation of the epidemic.

Resolving phylogeny and polyploid parentage using genus-wide genome-wide sequence data from birch trees.

Numerous plant genera have a history including frequent hybridisation and polyploidisation (allopolyploidisation), which means that their phylogeny is a network of reticulate evolution that cannot be accurately depicted as a bifurcating tree with a single tip per species. The genus Betula, which contains many ecologically important tree species, is a case in point. We generated genome-wide sequence reads for 27 diploid and 36 polyploid Betula species or subspecies using restriction site associated DNA (RAD) sequences. These reads were assembled into contigs with a mean length of 675 bp. We reconstructed the evolutionary relationships among diploid Betula species using both supermatrix (concatenation) and species tree methods. We identified the closest diploid relatives of the polyploids according to the relative rates at which reads from polyploids mapped to contigs from different diploid species within a concatenated reference sequence. By mapping reads from allopolyploids to their different putative diploid relatives we assembled contigs from the putative sub-genomes of allopolyploid taxa. We used these to build new phylogenies that included allopolyploid sub-genomes as separate tips. This approach yielded a highly evidenced phylogenetic hypothesis for the genus Betula, including the complex reticulate origins of the majority of its polyploid taxa. Our phylogeny divides the genus into two well supported clades, which, interestingly, differ in their seed-wing morphology. We therefore propose to split Betula into two subgenera.

Do modals identify better models? A comparison of signal detection and probabilistic models of inductive reasoning.

The nature of the relationship between deductive and inductive reasoning is a hotly debated topic. A key question is whether there is a single dimension of evidence underlying both deductive and inductive judgments. Following Rips (2001), Rotello and Heit (2009) and Heit and Rotello (2010) implemented one- and two-dimensional models grounded in signal detection theory to assess predictions for receiver operating characteristic data (ROCs), and concluded in favor of the two-dimensional model. Recently, Lassiter and Goodman (2015) proposed a different type of one-dimensional model, the Probability Threshold Model (PTM), that they concluded offered a good account of data collected over a range of decision modals (e.g., How likely, possible, or necessary is the argument conclusion?). Here, we apply the PTM and the signal detection models to ROCs from 3 large experiments in which participants made judgments about arguments varying in terms of modals introduced by Lassiter and Goodman (2015). Two independent variables that are theoretically important for the study of inductive reasoning, namely premise-conclusion similarity (as utilized in Heit & Rotello, 2010) and number of premises (as utilized in Rotello & Heit, 2009), are also varied in Experiments 1 and 2, respectively. In all cases, the PTM provides the poorest fit both quantitatively and qualitatively; the two-dimensional signal detection model is preferred.

Genomic repeat abundances contain phylogenetic signal.

A large proportion of genomic information, particularly repetitive elements, is usually ignored when researchers are using next-generation sequencing. Here we demonstrate the usefulness of this repetitive fraction in phylogenetic analyses, utilizing comparative graph-based clustering of next-generation sequence reads, which results in abundance estimates of different classes of genomic repeats. Phylogenetic trees are then inferred based on the genome-wide abundance of different repeat types treated as continuously varying characters; such repeats are scattered across chromosomes and in angiosperms can constitute a majority of nuclear genomic DNA. In six diverse examples, five angiosperms and one insect, this method provides generally well-supported relationships at interspecific and intergeneric levels that agree with results from more standard phylogenetic analyses of commonly used markers. We propose that this methodology may prove especially useful in groups where there is little genetic differentiation in standard phylogenetic markers. At the same time as providing data for phylogenetic inference, this method additionally yields a wealth of data for comparative studies of genome evolution.

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.

Analysis of the giant genomes of Fritillaria (Liliaceae) indicates that a lack of DNA removal characterizes extreme expansions in genome size.

Plants exhibit an extraordinary range of genome sizes, varying by > 2000-fold between the smallest and largest recorded values. In the absence of polyploidy, changes in the amount of repetitive DNA (transposable elements and tandem repeats) are primarily responsible for genome size differences between species. However, there is ongoing debate regarding the relative importance of amplification of repetitive DNA versus its deletion in governing genome size. Using data from 454 sequencing, we analysed the most repetitive fraction of some of the largest known genomes for diploid plant species, from members of Fritillaria. We revealed that genomic expansion has not resulted from the recent massive amplification of just a handful of repeat families, as shown in species with smaller genomes. Instead, the bulk of these immense genomes is composed of highly heterogeneous, relatively low-abundance repeat-derived DNA, supporting a scenario where amplified repeats continually accumulate due to infrequent DNA removal. Our results indicate that a lack of deletion and low turnover of repetitive DNA are major contributors to the evolution of extremely large genomes and show that their size cannot simply be accounted for by the activity of a small number of high-abundance repeat families.

The effect of polyploidy and hybridization on the evolution of floral colour in Nicotiana (Solanaceae).

BACKGROUND AND AIMS: Speciation in angiosperms can be accompanied by changes in floral colour that may influence pollinator preference and reproductive isolation. This study investigates whether changes in floral colour can accompany polyploid and homoploid hybridization, important processes in angiosperm evolution. METHODS: Spectral reflectance of corolla tissue was examined for 60 Nicotiana (Solanaceae) accessions (41 taxa) based on spectral shape (corresponding to pigmentation) as well as bee and hummingbird colour perception in order to assess patterns of floral colour evolution. Polyploid and homoploid hybrid spectra were compared with those of their progenitors to evaluate whether hybridization has resulted in floral colour shifts. KEY RESULTS: Floral colour categories in Nicotiana seem to have arisen multiple times independently during the evolution of the genus. Most younger polyploids displayed an unexpected floral colour, considering those of their progenitors, in the colour perception of at least one pollinator type, whereas older polyploids tended to resemble one or both of their progenitors. CONCLUSIONS: Floral colour evolution in Nicotiana is weakly constrained by phylogeny, and colour shifts do occur in association with both polyploid and homoploid hybrid divergence. Transgressive floral colour in N. tabacum has arisen by inheritance of anthocyanin pigmentation from its paternal progenitor while having a plastid phenotype like its maternal progenitor. Potentially, floral colour evolution has been driven by, or resulted in, pollinator shifts. However, those polyploids that are not sympatric (on a regional scale) with their progenitor lineages are typically not divergent in floral colour from them, perhaps because of a lack of competition for pollinators.

Diploidization and genome size change in allopolyploids is associated with differential dynamics of low- and high-copy sequences.

Recent advances have highlighted the ubiquity of whole-genome duplication (polyploidy) in angiosperms, although subsequent genome size change and diploidization (returning to a diploid-like condition) are poorly understood. An excellent system to assess these processes is provided by Nicotiana section Repandae, which arose via allopolyploidy (approximately 5 million years ago) involving relatives of Nicotiana sylvestris and Nicotiana obtusifolia. Subsequent speciation in Repandae has resulted in allotetraploids with divergent genome sizes, including Nicotiana repanda and Nicotiana nudicaulis studied here, which have an estimated 23.6% genome expansion and 19.2% genome contraction from the early polyploid, respectively. Graph-based clustering of next-generation sequence data enabled assessment of the global genome composition of these allotetraploids and their diploid progenitors. Unexpectedly, in both allotetraploids, over 85% of sequence clusters (repetitive DNA families) had a lower abundance than predicted from their diploid relatives; a trend seen particularly in low-copy repeats. The loss of high-copy sequences predominantly accounts for the genome downsizing in N. nudicaulis. In contrast, N. repanda shows expansion of clusters already inherited in high copy number (mostly chromovirus-like Ty3/Gypsy retroelements and some low-complexity sequences), leading to much of the genome upsizing predicted. We suggest that the differential dynamics of low- and high-copy sequences reveal two genomic processes that occur subsequent to allopolyploidy. The loss of low-copy sequences, common to both allopolyploids, may reflect genome diploidization, a process that also involves loss of duplicate copies of genes and upstream regulators. In contrast, genome size divergence between allopolyploids is manifested through differential accumulation and/or deletion of high-copy-number sequences.

A universe of dwarfs and giants: genome size and chromosome evolution in the monocot family Melanthiaceae.

• Since the occurrence of giant genomes in angiosperms is restricted to just a few lineages, identifying where shifts towards genome obesity have occurred is essential for understanding the evolutionary mechanisms triggering this process. • Genome sizes were assessed using flow cytometry in 79 species and new chromosome numbers were obtained. Phylogenetically based statistical methods were applied to infer ancestral character reconstructions of chromosome numbers and nuclear DNA contents. • Melanthiaceae are the most diverse family in terms of genome size, with C-values ranging more than 230-fold. Our data confirmed that giant genomes are restricted to tribe Parideae, with most extant species in the family characterized by small genomes. Ancestral genome size reconstruction revealed that the most recent common ancestor (MRCA) for the family had a relatively small genome (1C = 5.37 pg). Chromosome losses and polyploidy are recovered as the main evolutionary mechanisms generating chromosome number change. • Genome evolution in Melanthiaceae has been characterized by a trend towards genome size reduction, with just one episode of dramatic DNA accumulation in Parideae. Such extreme contrasting profiles of genome size evolution illustrate the key role of transposable elements and chromosome rearrangements in driving the evolution of plant genomes.

Evolutionary relationships in the medicinally important genus Fritillaria L. (Liliaceae).

Fritillaria (Liliaceae) is a genus of approximately 140 species of bulbous perennial plants that includes taxa of both horticultural and medicinal importance. As well as being commercially valuable, Fritillaria species have attracted attention because of their exceptionally large genome sizes, with all values recorded to date in excess of 30Gb. Despite such interest in the genus, phylogenetic relationships between the majority of species have remained untested. Here we present the first phylogenetic reconstruction of relationships to encompass most of the currently recognised species diversity in the genus. Three regions of the plastid genome were sequenced in 117 individuals of Fritillaria, representing 92 species (c. 66% of the genus) and in representatives of nine other genera of Liliaceae. Eleven low-copy nuclear gene regions were also screened in selected species for their potential utility. Phylogenetic analysis of a combined plastid dataset using maximum parsimony and Bayesian inference provided support for the monophyly of the majority of currently recognised subgenera. However, subgenus Fritillaria, which is by far the largest of the subgenera and includes the most important species used in traditional Chinese medicine, is found to be polyphyletic. Moreover, several taxa that were represented by multiple individuals show evidence of species non-monophyly. The Japanese endemic subgenus Japonica, which contains the species with the largest recorded genome size for any diploid plant, is resolved as sister to the predominantly Middle Eastern and Central Asian subgenus Rhinopetalum. Whilst relationships between most of the major Fritillaria lineages can now be resolved, our results also highlight the need for data from additional independently evolving loci; an endeavour that may be particularly challenging in light of the huge nuclear genomes found in these plants.

Exploring giant plant genomes with next-generation sequencing technology.

Genome size in plants is characterised by its extraordinary range. Although it appears that the majority of plants have small genomes, in several lineages genome size has reached giant proportions. The recent advent of next-generation sequencing (NGS) methods has for the first time made detailed analysis of even the largest of plant genomes a possibility. In this review, we highlight investigations that have utilised NGS for the study of plants with large genomes, as well as describing ongoing work that aims to harness the power of these technologies to gain insights into their evolution. In addition, we emphasise some areas of research where the use of NGS has the potential to generate significant advances in our current understanding of how plant genomes evolve. Finally, we discuss some of the future developments in sequencing technology that may further improve our ability to explore the content and evolutionary dynamics of the very largest genomes.

A high-quality reference genome for Fraxinus pennsylvanica for ash species restoration and research.

Green ash (Fraxinus pennsylvanica) is the most widely distributed ash tree in North America. Once common, it has experienced high mortality from the non-native invasive emerald ash borer (EAB; Agrilus planipennis). A small percentage of native green ash trees that remain healthy in long-infested areas, termed "lingering ash," display partial resistance to the insect, indicating that breeding and propagating populations with higher resistance to EAB may be possible. To assist in ash breeding, ecology and evolution studies, we report the first chromosome-level assembly from the genus Fraxinus for F. pennsylvanica with over 99% of bases anchored to 23 haploid chromosomes, spanning 757 Mb in total, composed of 49.43% repetitive DNA, and containing 35,470 high-confidence gene models assigned to 22,976 Asterid orthogroups. We also present results of range-wide genetic variation studies, the identification of candidate genes for important traits including potential EAB-resistance genes, and an investigation of comparative genome organization among Asterids based on this reference genome platform. Residual duplicated regions within the genome probably resulting from a recent whole genome duplication event in Oleaceae were visualized in relation to wild olive (Olea europaea var. sylvestris). We used our F. pennsylvanica chromosome assembly to construct reference-guided assemblies of 27 previously sequenced Fraxinus taxa, including F. excelsior. Thus, we present a significant step forward in genomic resources for research and protection of Fraxinus species.

Intragenic recombination events and evidence for hybrid speciation in Nicotiana (Solanaceae).

Reticulate evolution may function both at the species level, through homoploid and polyploid hybridization, and below the species level, through inter and intragenic recombination. These processes represent challenges for the reconstruction of evolutionary relationships between species, because they cannot be represented adequately with bifurcating trees. We use data from low-copy nuclear genes to evaluate long-standing hypotheses of homoploid (interspecific) hybrid speciation in Nicotiana (Solanaceae) and reconstruct a complex series of reticulation events that have been important in the evolutionary history of this genus. Hybrid origins for three diploid species (Nicotiana glauca, N. linearis, and N. spegazzinii) are inferred on the basis of gene tree incongruence, evidence for interallelic recombination between likely parental alleles, and support for incompatible splits in Lento plots. Phylogenetic analysis of recombinant gene sequences illustrates that recombinants may be resolved with one of their progenitor lineages with a high posterior probability under Bayesian inference, and thus there is no indication of the conflict between phylogenetic signals that results from reticulation. Our results illustrate the importance of hybridization in shaping evolution in Nicotiana and also show that intragenic recombination may be relatively common. This finding demonstrates that it is important to investigate the possibility of recombination when aiming to detect hybrids from DNA-sequence data and reconstruct patterns of reticulate evolution between species.

DNA barcoding of lichenized fungi demonstrates high identification success in a floristic context.

• Efforts are currently underway to establish a standard DNA barcode region for fungi; we tested the utility of the internal transcribed spacer (ITS) of nuclear ribosomal DNA for DNA barcoding in lichen-forming fungi by sampling diverse species across eight orders. • Amplification of the ITS region (ITS1-5.8S-ITS2) was conducted for 351 samples, encompassing 107, 55 and 28 species, genera and families, respectively, of lichenized fungi. We assessed the ability of the entire ITS vs the ITS2 alone to discriminate between species in a taxonomic dataset (members of the genus Usnea) and a floristic dataset. • In the floristic dataset, 96.3% of sequenced samples could be assigned to the correct species using ITS or ITS2; a barcode gap for ITS is present in 92.1% of species. Although fewer species have a barcode gap in the taxonomic dataset (73.3% with ITS and 68.8% with ITS2), up to 94.1% of samples were assigned to the correct species using BLAST. • While discrimination between the most closely related species will remain challenging, our results demonstrate the potential to identify a high percentage of specimens to the correct species, and the remainder to the correct genus, when using DNA barcoding in a floristic context.

Nuclear glutamine synthetase evolution in Nicotiana: phylogenetics and the origins of allotetraploid and homoploid (diploid) hybrids.

Interspecies relationships in Nicotiana (Solanaceae) are complex because 40 species are diploid (two sets of chromosomes) and 35 species are allotetraploid (four sets of chromosomes, two from each progenitor diploid species). We sequenced a fragment (containing four introns) of the nuclear gene 'chloroplast-expressed glutamine synthetase' (ncpGS) in 65 species of Nicotiana. Here we present the first phylogenetic analysis based on a low-copy nuclear gene for this well studied and important genus. Diploid species have a single-copy of ncpGS, and allotetraploids as expected have two homeologous copies, each derived from their progenitor diploid. Results were particularly useful for determining the paternal lineage of previously enigmatic taxa (for which our previous analyses had revealed only the maternal progenitors). In particular, we were able to shed light on the origins of the two oldest and largest allotetraploid sections, N. sects. Suaveolentes and Repandae. All homeologues have an intact reading frame and apparently similar rates of divergence, suggesting both remain functional. Difficulties in fitting certain diploid species into the sectional classification of Nicotiana on morphological grounds, coupled with discordance between the ncpGS data and previous trees (i.e. plastid, nuclear ribosomal DNA), indicate a number of homoploid (diploid) hybrids in the genus. We have evidence for Nicotiana glutinosa and Nicotiana linearis being of hybrid origin and patterns of intra-allelic recombination also indicate the possibility of reticulate origins for other diploid species.

Repeat-sequence turnover shifts fundamentally in species with large genomes.

Given the 2,400-fold range of genome sizes (0.06-148.9 Gbp (gigabase pair)) of seed plants (angiosperms and gymnosperms) with a broadly similar gene content (amounting to approximately 0.03 Gbp), the repeat-sequence content of the genome might be expected to increase with genome size, resulting in the largest genomes consisting almost entirely of repetitive sequences. Here we test this prediction, using the same bioinformatic approach for 101 species to ensure consistency in what constitutes a repeat. We reveal a fundamental change in repeat turnover in genomes above around 10 Gbp, such that species with the largest genomes are only about 55% repetitive. Given that genome size influences many plant traits, habits and life strategies, this fundamental shift in repeat dynamics is likely to affect the evolutionary trajectory of species lineages.

Convergent molecular evolution among ash species resistant to the emerald ash borer.

Recent studies show that molecular convergence plays an unexpectedly common role in the evolution of convergent phenotypes. We exploited this phenomenon to find candidate loci underlying resistance to the emerald ash borer (EAB, Agrilus planipennis), the United States' most costly invasive forest insect to date, within the pan-genome of ash trees (the genus Fraxinus). We show that EAB-resistant taxa occur within three independent phylogenetic lineages. In genomes from these resistant lineages, we detect 53 genes with evidence of convergent amino acid evolution. Gene-tree reconstruction indicates that, for 48 of these candidates, the convergent amino acids are more likely to have arisen via independent evolution than by another process such as hybridization or incomplete lineage sorting. Seven of the candidate genes have putative roles connected to the phenylpropanoid biosynthesis pathway and 17 relate to herbivore recognition, defence signalling or programmed cell death. Evidence for loss-of-function mutations among these candidates is more frequent in susceptible species than in resistant ones. Our results on evolutionary relationships, variability in resistance, and candidate genes for defence response within the ash genus could inform breeding for EAB resistance, facilitating ecological restoration in areas invaded by this beetle.

Genomic basis of European ash tree resistance to ash dieback fungus.

Populations of European ash trees (Fraxinus excelsior) are being devastated by the invasive alien fungus Hymenoscyphus fraxineus, which causes ash dieback. We sequenced whole genomic DNA from 1,250 ash trees in 31 DNA pools, each pool containing trees with the same ash dieback damage status in a screening trial and from the same seed-source zone. A genome-wide association study identified 3,149 single nucleotide polymorphisms (SNPs) associated with low versus high ash dieback damage. Sixty-one of the 192 most significant SNPs were in, or close to, genes with putative homologues already known to be involved in pathogen responses in other plant species. We also used the pooled sequence data to train a genomic prediction model, cross-validated using individual whole genome sequence data generated for 75 healthy and 75 damaged trees from a single seed source. The model's genomic estimated breeding values (GEBVs) allocated these 150 trees to their observed health statuses with 67% accuracy using 10,000 SNPs. Using the top 20% of GEBVs from just 200 SNPs, we could predict observed tree health with over 90% accuracy. We infer that ash dieback resistance in F. excelsior is a polygenic trait that should respond well to both natural selection and breeding, which could be accelerated using genomic prediction.

A genome for gnetophytes and early evolution of seed plants.

Gnetophytes are an enigmatic gymnosperm lineage comprising three genera, Gnetum, Welwitschia and Ephedra, which are morphologically distinct from all other seed plants. Their distinctiveness has triggered much debate as to their origin, evolution and phylogenetic placement among seed plants. To increase our understanding of the evolution of gnetophytes, and their relation to other seed plants, we report here a high-quality draft genome sequence for Gnetum montanum, the first for any gnetophyte. By using a novel genome assembly strategy to deal with high levels of heterozygosity, we assembled >4 Gb of sequence encoding 27,491 protein-coding genes. Comparative analysis of the G. montanum genome with other gymnosperm genomes unveiled some remarkable and distinctive genomic features, such as a diverse assemblage of retrotransposons with evidence for elevated frequencies of elimination rather than accumulation, considerable differences in intron architecture, including both length distribution and proportions of (retro) transposon elements, and distinctive patterns of proliferation of functional protein domains. Furthermore, a few gene families showed Gnetum-specific copy number expansions (for example, cellulose synthase) or contractions (for example, Late Embryogenesis Abundant protein), which could be connected with Gnetum's distinctive morphological innovations associated with their adaptation to warm, mesic environments. Overall, the G. montanum genome enables a better resolution of ancestral genomic features within seed plants, and the identification of genomic characters that distinguish Gnetum from other gymnosperms.