Practical implications of variant-calling strategies in giga-genome species: A case study in the imperiled conifer, Chamaecyparis thyoides

Giga-genome species are the next frontier for high throughput, next-generation sequencing analysis. While non-model conservation genomics strategies have become increasingly widespread, the complexity of giga-genomes has remained a barrier for numerous species of conservation concern. Until recently, next-generation sequencing projects for giga-genome species have largely been limited to economically important species, e.g., pines (Pinus spp.), spruce (Picea spp.), and wheat (Triticum spp.). Here, we present a case study in the imperiled, non-model conifer, Atlantic white-cedar (Chamaecyparis thyoides), to show that reduced representation sequencing approaches (here, genotyping-by-sequencing) are a viable and economical strategy for evaluating giga-genome species. By comparing variant calling results using three reference genomes–a de novo reference genome of C. thyoides, a phylogenetically distant reference genome from western red-cedar, Thuja plicata, and a congeneric reference genome from Hinoki cypress, C. obtusa–we identify the benefits of a closely related reference genome over de novo and distantly related references. We also discuss the statistical and architectural assumptions that are made about the genome of study organisms that lack closely related references, as well as ways to address these challenges. Specifically, we show that each variant calling strategy impacts the inference of population structure, population summary statistics, and the identification of genotype-environment associations in the presence of subspecies variation, and we discuss the importance of conservative interpretations of population summary statistics among variant calling strategies. Our analysis also reveals signatures of regional environmental adaptation and patterns of inter-subspecies hybridization in the Gulf Coastal Plain, which clarify taxonomic ambiguities in C. thyoides and will improve conservation planning and restoration efforts for the species more broadly.