Buckley, ThomasChoi, Seung-Sub2023-05-162023-05-162023https://hdl.handle.net/2292/64031Hybridization is the cross breeding of two closely related species that produce offspring with an intermediate genomic structure. Hybridization is often linked to asexual reproduction and polyploidy and thought to help generate evolutionary diversity through rapid speciation. However, there is controversy on the importance of hybridization as hybrid F1 offspring tend to express lower fitness and survival due to hybrid breakdown caused by Dobzhansky–Muller incompatibilities. On the other hand, some hybrids have avoided hybrid breakdown and show heterosis, where they have greater fitness and adaptation to harsher environments compared to their parental species. Despite these debates, more and more species have been identified to have originated from hybridization and the occurrence of hybridization has increased due to destruction of natural habitat. The genomic impact of hybridization is not well understood. Stick insects express hybridization, parthenogenesis and polyploidy, and the latter two phenomena are rare in the animal kingdom. For this reason, stick insects have previously been chosen as a model organism to study these phenomena. The New Zealand endemic stick genus Acanthoxyla contains four species and demonstrates all these processes. The genus contains both diploid and triploid species and while most of the species are obligate parthenogens. The species, Acanthoxyla geisovii, Acanthoxyla prasina, and Acanthoxyla inermis, are hypothesized to have originated from hybridization either between ancient sexual Acanthoxyla species or between male C. hookeri and an unknown female species. To infer the pattern of hybridization in Acanthoxyla and its genomic impact, as well as the effects of asexual reproduction, and polyploidy on genomic structure, a high-quality reference genome was needed. Thus, we utilized four different next generation sequencing (NGS) platforms (Illumina short read, Chromium 10X, Oxford Nanopore long reads and Hi-C) to perform hybrid de novo assembly using C. hookeri as a model species. The resulting assembly was a significant improvement over a previous short read assembly in terms of correctness, completeness, and accuracy. By aligning whole genome sequence (WGS) reads to the reference assembly, we estimated the ploidy levels of different Acanthoxyla lineages. We then used assembled whole mitochondrial genomes to reconstruct maternal phylogenetic relationships among Acanthoxyla species and related genera, in agreement with previous findings. The deleterious substitution patterns revealed that species that are parthenogenetic or have restricted geographic distributions tend to accumulate deleterious substitutions in the homozygous state whereas species having undergone recent hybridization have higher proportions of heterozygous deleterious substitutions. With the addition of hybridization inference from D-statistics and a phylogenetic network from phased BUSCO gene trees, we have confirmed C. hookeri as a paternal ancestor of A. inermis and A. prasina. We also infer that A. geisovii, or a close relative of it, might be the maternal ancestor of A. prasina. Acanthoxyla geisovii itself might also have originated via hybridization between ancient sexual Acanthoxyla species. Homoeolog expression bias (HEB) using RNAseq was performed in two populations of A. prasina and A. inermis using C. hookeri and A. geisovii as parental species. HEB analysis demonstrated that all hybrid populations express a dominance expression model preferring the C. hookeri subgenome despite differences in ploidy levels between the two hybrid species. However, only A. inermis, which is a diploid species, showed a weak positive correlation between non-synonymous substitution rate with the degree of homoeolog expression bias suggesting that differences in protein sequence between allelic products in hybrid genomes may be driving HEB due to protein co-adaptation. This effect was only significant in the diploid A. inermis, also suggesting that the signal may be harder to detect in triploids. Gene ontology enrichment on C. hookeri biased genes showed enrichment of pathways involving the ribosome, mitochondria, unfolded protein binding and heat shock proteins. While some of the genes in these pathways were biased towards the Acanthoxyla subgenome, the genes that are essential to these pathways were biased towards C. hookeri suggesting a preference for the C. hookeri subgenome. The observation of HEB towards the paternal C. hookeri subgenome in nuclear-encoded mitochondrial genes contradicts expectations of the mitonuclear incompatibility hypothesis, as the maternal mitochondrially-encoded genes were expressed in all hybrid Acanthoxyla populations.Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated.https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htmhttp://creativecommons.org/licenses/by-nc-sa/3.0/nz/Thesis2023-03-08Copyright: The authorhttp://purl.org/eprint/accessRights/OpenAccess