Diversity and evolutionary history of RNA viruses among different horseshoe crab species.

Horseshoe crabs (Xiphosura: Limulidae) are the sole surviving species of the class Merostomata, with only four extant species remaining today. Recent advances in metagenomic next-generation sequencing have unveiled a vast diversity of RNA viruses and non-retroviral endogenous RNA viral elements (nrEVEs) in invertebrates. This raises intriguing questions about the RNA virome and nrEVEs in horseshoe crabs as "living fossils," potentially offering insights into the evolutionary relationships between RNA viruses and these ancient organisms. In this study, 22 novel RNA viruses were identified across the four horseshoe crab species by screening 117 data sets, including picornaviruses, totiviruses, a flavivirus, a rhabdovirus, as well as a plant-associated tombusvirus and a fungi-associated narnavirus. Additionally, 20 nrEVEs were identified in the genomes of the four horseshoe crab species (hcEVEs), with most sharing homology with the viral family ( = 11), supporting the hypothesis that modern negative-sense RNA viruses may trace their origins to ancient oceanic chuviruses. A time-scaled phylogenetic tree based on hcEVEs suggests that at least two independent ancient chuvirus infections and genome integration events occurred in the common ancestor of horseshoe crab species. Interestingly, transcriptional analyses indicated that hcEVE-containing transcripts display typical exon-intron structures in the three Asian horseshoe crab species, suggesting that these hcEVEs may have been co-opted by horseshoe crabs during coevolution. These findings advance our understanding of the RNA viruses associated with horseshoe crabs and shed light on the potential role of RNA viruses in shaping the evolutionary history of this "living fossil" arthropod host.IMPORTANCERecent studies have discovered abundant RNA viruses in invertebrates, revealing that viral genomes may integrate into host genomes, creating a genetic record of past infections. In this study, we explored the evolutionary relationship between RNA viruses and the four extant horseshoe crab species-the last representatives of the class Merostomata, often termed "living fossils"-by analyzing viral sequences embedded in their genomes. The presence of chuvirus-like sequences in the genomes of these horseshoe crabs suggests that modern negative-sense RNA viruses may trace their origins back to ancient chuviruses from the ocean. Furthermore, we identified at least two independent ancient integrations of chuviruses in the evolutionary history of horseshoe crabs, with one orthologous gene containing a chuvirus-derived G protein gene/coding sequence potentially inherited from a common ancestor of the three Asian species before their divergence. Our findings contribute to a deeper understanding of the long-term coevolution between RNA viruses and their arthropod hosts.