Comparative Hi-C of northern Australian Gehyra: do chromosomal rearrangements restrict gene flow in sympatric species?

The gecko genus Gehyra is a diverse radiation of Australasian lizards, and the subject of studies on chromosome change, speciation and adaptive evolution for over 40 years (King 1979, 1983; Moritz 1984, 1986; Heinicke et al. 2011; Gamble et al. 2015; Ashman et al. 2018; Moritz et al. 2018; Oliver et al. 2019). Recent
phylogenomic and taxonomic studies have resolved species boundaries and relationships among the 62 lineages (50 recognised species within Australia) (Doughty et al. 2012, 2018; Sistrom et al. 2013; Hutchinson et al. 2014; Oliver et al. 2016, 2020; Bourke et al. 2017; Kealley et al. 2018; Moritz et al. 2018), near doubling the total number of described Gehyra species.
As one of the most chromosomally variable vertebrate radiations known in Australia, there has been longstanding interest in whether Robertsonian fusions and inversions influence speciation in Gehyra (King 1979, 1983; Moritz 1984, 1986). For many of recently described Gehyra species, chromosome numbers or
rearrangements are currently unknown. Chromosomal inversions ortranslocations may reduce the likelihood of gene flow between species, acting as a mechanism for speciation via postzygotic reproductive isolation(White 1978; Potter et al. 2017). Previous studies of chromosomal rearrangements in Gehyra have been
limited to cytological karyotyping approaches, which are labour intensive and lack sensitivity (i.e., low resolution).
Chromosome conformation capture (Hi-C) sequencing is a novel approach that enables reconstruction of the three-dimensional structure of the genome using contact probabilities between all pairs of loci across the genome (Lieberman-Aiden et al. 2009). High-coverage, single sample Hi-C sequencing is emerging as a standard approach to scaffolding fragmented genomes to chromosome-level assembly (e.g. Burton et al. 2013). More recently, a small number of studies have examined the use comparative Hi-C to detect chromosomal rearrangements at high-resolution in human tumour cells (Harewood et al. 2017), and across
multiple species (Himmelbach et al. 2018; Mudd et al. 2020). These initial studies indicate that Hi-C could be a powerful tool for comparative analysis of structural genomic variation across evolutionary radiations;however, it is currently unclear what sequencing depth is required as a minimum to detect megabase-scale rearrangements in complex animal genomes.
Using the chromosomally variable Gehyra radiation as a model system, we propose to test the application of low-coverage Hi-C in detecting megabase-scale rearrangements across 16 species, and to determine the
role of chromosomal rearrangements as barriers to gene flow among closely related sympatric (geographically overlapping) species. Our proposal focuses on 16 species across three clades in the Gehyraradiation: the australis group (four species); the koira group (five species); and the nana group (seven
species). These groups show varying degrees of sympatry throughout their northern Australian ranges.
Preliminary data (estimated admixture from DArTseq data) suggest that introgression levels across species boundaries are relatively high within the nana group, but low in the koira and australis groups. If chromosomal rearrangements act as barriers to gene flow in Gehyra, we expect to observe the most Centre for Biodiversity Analysis | Ignition Grant application 2significant chromosomal rearrangements among species in the australis and koira groups, where gene flow is limited, and more conserved chromosomal structure across species the nana group, where gene flow is higher.
Specifically, we aim to:
1) Validate the accuracy of Hi-C by comparing chromosome conformation maps with existing cytological karyotype data (Moritz 1984, 1986) in Gehyra. For a subset of recently described taxa, establish cell lines from freshly collected material to generate karyotype data for validation
2) Determine the minimum Hi-C sequencing coverage required to detect megabase-scale chromosomal rearrangements in animal genomes
3) Assess the utility of museum-preserved tissue up to ~40 years old for Hi-C sequencing
4) Obtain Hi-C chromosome maps across 16 species from three Gehyra groups, detecting species- or clade-specific rearrangements relative to existing, high-quality reference genomes (these four highquality Gehyra references genomes are generated through the AusARG project, G. moritzi, G.
lapistola, G. paranana and G. purpurascens)
5) Using Hi-C maps for each species, contrast chromosomal rearrangements with estimates of introgression from reduced-representation data (DArTseq), to determine whether chromosomal rearrangements limit gene flow in sympatric species of Gehyra
This project has the potential to establish low-coverage, comparative Hi-C as a powerful technique for studying chromosomal rearrangements across species-rich evolutionary radiations, as well as to disentangle the
relationship between structural genomic variation, gene flow, reproductive isolation, and speciation. Comparative Hi-C could also inform population translocations and genetic rescue of threatened species, by screening for chromosomal incompatibilities that may contribute to outbreeding depression in mixed populations.