Multiple hybridization events and repeated evolution of homoeologue expression bias in parthenogenetic, polyploid New Zealand stick insects.

During hybrid speciation, homoeologues combine in a single genome. Homoeologue expression bias (HEB) occurs when one homoeologue has higher gene expression than another. HEB has been well characterized in plants but rarely investigated in animals, especially invertebrates. Consequently, we have little idea as to the role that HEB plays in allopolyploid invertebrate genomes. If HEB is constrained by features of the parental genomes, then we predict repeated evolution of similar HEB patterns among hybrid genomes formed from the same parental lineages. To address this, we reconstructed the history of hybridization between the New Zealand stick insect genera Acanthoxyla and Clitarchus using a high-quality genome assembly from Clitarchus hookeri to call variants and phase alleles. These analyses revealed the formation of three independent diploid and triploid hybrid lineages between these genera. RNA sequencing revealed a similar magnitude and direction of HEB among these hybrid lineages, and we observed that many enriched functions and pathways were also shared among lineages, consistent with repeated evolution due to parental genome constraints. In most hybrid lineages, a slight majority of the genes involved in mitochondrial function showed HEB towards the maternal homoeologues, consistent with only weak effects of mitonuclear incompatibility. We also observed a proteasome functional enrichment in most lineages and hypothesize this may result from the need to maintain proteostasis in hybrid genomes. Reference bias was a pervasive problem, and we caution against relying on HEB estimates from a single parental reference genome.

An exploration of assembly strategies and quality metrics on the accuracy of the rewarewa (Knightia excelsa) genome.

We used long read sequencing data generated from Knightia excelsa, a nectar-producing Proteaceae tree endemic to Aotearoa (New Zealand), to explore how sequencing data type, volume and workflows can impact final assembly accuracy and chromosome reconstruction. Establishing a high-quality genome for this species has specific cultural importance to Maori and commercial importance to honey producers in Aotearoa. Assemblies were produced by five long read assemblers using data subsampled based on read lengths, two polishing strategies and two Hi-C mapping methods. Our results from subsampling the data by read length showed that each assembler tested performed differently depending on the coverage and the read length of the data. Subsampling highlighted that input data with longer read lengths but perhaps lower coverage constructed more contiguous, kmers and gene-complete assemblies than short read length input data with higher coverage. The final genome assembly was constructed into 14 pseudochromosomes using an initial flye long read assembly, a racon/medaka/pilon combined polishing strategy, salsa2 and allhic scaffolding, juicebox curation, and Macadamia linkage map validation. We highlighted the importance of developing assembly workflows based on the volume and read length of sequencing data and established a robust set of quality metrics for generating high-quality assemblies. Scaffolding analyses highlighted that problems found in the initial assemblies could not be resolved accurately by Hi-C data and that assembly scaffolding was more successful when the underlying contig assembly was of higher accuracy. These findings provide insight into how quality assessment tools can be implemented throughout genome assembly pipelines to inform the de novo reconstruction of a high-quality genome assembly for nonmodel organisms.