Differentiation across the Podisma pedestris hybrid zone inferred from high-throughput sequencing data

PhDHybrid zones are regions where genetically differentiated forms come together and exchange genes through hybrid offspring. The study of characters gradually changing across such zones (clines) can give insight into evolutionary processes, providing exceptionally sensitive estimates of the intensity of selection, and allowing the detection of loci that might be involved in reproductive isolation and speciation. The Alpine grasshopper Podisma pedestris has a hybrid zone in Southern France where two populations meet. They differ in their sex chromosome system, and strong selection against hybrids is observed. These distinct populations likely have split and re-joined several times during the Quaternary glacial cycles. A model explaining the selection observed against hybrids postulates hundreds of loci of small effect spread over two differentiated genomes meeting in secondary contact. Yet, over 50 years of study to-date non have been discovered. However, so far the study of P. pedestris has not made use of high-throughput sequencing data which provides an unprecedented resolution of molecular markers. I am aiming to close the gap with this thesis. I assemble the grasshopper’s mitochondrial genome sequence and infer what proportion of its genome is made up by mitochondrial inserts (Numts). Using transcriptome data from two individuals, I then go on to fit demographic models, finding the populations split approximately 400 000 years ago and that the current-day population sizes are considerably smaller than the ancestral one. The final data chapter explores the genetic architecture of the hybrid zone using data from a targeted sequence capture of hundreds of loci covering some 10 000 polymorphic sites. Only two loci under selection are identified, which is surprising given the power of the analysis. Both loci are located on the X chromosome and are subject to weak selection (0.3% and 0.03%). This shows the power of hybrid zone analysis to infer targets of selection. The results are discussed in light of a theoretical chapter on the ‘inexorable spread’ phenomenon and lead to the proposal for further research into the causes of the reproductive isolation observed between the grasshopper populations.EU’s Leonardo-da-Vinci programme Queen Mary’s School of Biological and Chemical Sciences PhD studentship Genetics Societ

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

Is there hybridisation between diploid and tetraploid euphrasia in a secondary contact zone?

• Premise of the study: Hybridisation between species with contrasting ploidy is usually considered rare in nature due to strong ploidy related postzygotic reproductive isolating barriers. However, genomic sequencing has revealed previously overlooked examples of natural cross-ploidy hybridisation, suggesting this phenomenon may be more common than once thought. Here, we investigate potential cross-ploidy hybridisation in British eyebrights (Euphrasia, Orobanchaceae), a group where thirteen putative cross-ploidy hybrid combinations have been reported based on morphology. • Methods: We analysed a contact zone between diploid E. rostkoviana and tetraploid E. arctica in Wales. We sequenced part of the internal transcribed spacer of nuclear ribosomal DNA (ITS1) and used Genotyping by Sequencing (GBS) to look for evidence of cross-ploidy hybridisation and introgression. • Key results: All variant sites in the ITS1 region were fixed between diploids and tetraploids, indicating a strong barrier to hybridisation. Clustering analyses of 356 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 with dadI suggested potential gene flow – with this limited to around one or fewer migrants per generation. • Conclusions: Our results suggest recent cross-ploidy hybridisation is rare or absent in a site of secondary contact in Euphrasia. While a strong ploidy barrier prevents hybridisation over ecological time-scales, such hybrids may form in stable populations over evolutionary time-scales and may allow for cross-ploidy introgression to take place.Funding provided by: Natural Environment Research CouncilCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100000270Award Number: NE/L011336/1Funding provided by: Natural Environment Research CouncilCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100000270Award Number: NE/N006739/1Funding provided by: Natural Environment Research CouncilCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100000270Award Number: NE/R010609/1Genotyping by sequencing data were generated for diploid and tetraploid Euphrasia species using the enzyme ApeKI. Sanger sequencing was also performed, amplifying the ITS2 region

Horticultural protocols for experimental studies of eyebrights (Euphrasia, Orobanchaceae):Student Project

Parasitic plants are particularly challenging to cultivate as the growth conditions must be suitable for the parasite, the host, and their interaction. Here, we review our progress growing British native eyebrights (Euphrasia), a group of hemiparasitic plants found in diverse habitats in Britain and Ireland. We consider the protocols required to grow them under a range of conditions, including the laboratory, in pot trials, in cultivated fields, and in the wild. We highlight the need to use seed stratification to break seed dormancy, to replicate planting to overcome low seed viability, and to manage host plants to avoid competition. While Euphrasia can be successfully grown in different environments more work is required to develop reliable horticultural protocols for growing plants under natural conditions

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

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. Key words: demographic inference, background selection, distribution of fitness effects, MSMC, fastsimcoal2, approximate Bayesian computation (ABC)

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