Genetic loci associated with natural variation in hypoxia tolerance in the copepod Tigriopus californicus revealed by bulked segregant analysis

The copepod Tigriopus californicus is among crustaceans that have recently been shown to have lost key genes encoding the master regulators of the hypoxia-inducible factor (HIF) pathway, thought to be essential for oxygen homeostasis in animals. This species inhabits supralittoral pools that impart hours of hypoxic stress daily, frequently reaching anoxia. Despite lacking the canonical HIF transcription factor, T. californicus exhibits one of the highest levels of tolerance to hypoxia recorded in aquatic animals. The genetic mechanisms involved in hypoxia response in taxa lacking HIF components is almost entirely unknown. We took advantage of geographic variation in tolerance in T. californicus to identify quantitative trait loci (QTL) using recombinant hybrids between a more tolerant California population and a less tolerant Oregon population. After quantifying individual-level tolerance in nearly 800 hybrid copepods using P crit , we performed Pool-seq and bulked segregant analysis to identify QTLs. Our analysis revealed seven QTLs across five chromosomes, harboring a total of 43,648 SNPs. SNPs within these QTLs represented 721 genes enriched for stress response, sugar metabolism, neuronal signaling, and mitochondrial functions, and included genes implicated in exoskeletal and membrane modifications. Many genes in the QTLs were known in T. californicus to show differential expression in response to experimental hypoxic events, suggesting that some genetic variation explaining differences in P crit among populations is associated with loci that are a part of acute transcriptomic responses. Most SNPs in QTLs were found in introns or within 1000 bp upstream of genes, indicating a bias toward variants with potential regulatory impacts in the QTLs. QTL genes with known transcriptional response tended to have more SNPs in these regions compared to genes that do not participate in acute responses to hypoxia. SNPs annotated as high impact, including stop lost or stop gained variants, affected over a dozen genes including an important antioxidant regulatory gene. These results provide a first glimpse at the genetic architecture underlying natural variation in hypoxia tolerance in a species lacking the key oxygen-sensing regulatory gene and highlight candidate genes of diverse biological pathways that may contribute to stress resilience in extreme environments.