Unexpected complex horizontal gene transfer in teleost fish.

Horizontal gene transfer (HGT) is a common occurrence across all domains of life. However, most HGT events were reported between single-celled organisms or parasites and hosts (Van Etten and Bhattacharya 2020). A type II antifreeze protein (AFP) gene was the first and sole evidence of HGT direct vertebrate-to-vertebrate DNA transmission. AFP is only found in 3 widely separated branches of teleost fishes (herring, sea raven, and smelts), sharing amino acid similarity up to 80% (Graham et al. 2008). Therefore, HGT is the most acceptable mechanism to explain this high gene similarity (Graham et al. 2008). However, there are debates on the direction of AFP horizontal transfer between Atlantic herring and smelts (Japanese smelt and rainbow smelt). Previous phylogenetic analysis concluded the direction of transfer from smelts to Atlantic herring (Sorhannus 2012). In contrast, recent comparative analysis of transposable elements between Atlantic herring and smelts supported the opposite transfer direction (Graham and Davies 2021). Nowadays, the accumulated genome and transcriptome data provide chances for a deep study of the HGT between herring and smelts. In addition, the existence of AFP in Japanese surf smelt Hypomesus japonicus was previously confirmed by SDS-PAGE technology (Kamijima et al. 2013). These information should offer novel clues to explore the time frames, details, and even the numbers of HGT events of teleost fish species. In the present study, AFP genes were searched in 4 species from subfamily Clupeinae including Atlantic herring, Pacific herring, related sardine species (Sardina pilchardus and Sardinops melanostictus), and 8 species from order Osmeriformes covering 4 genera of family Osmeridae and 1 closely related family Plecoglossidae. Besides, AFP genes were also searched in other 47 related marine fish species by using genomic and transcriptomic data. Detailed materials and methods are described in Supplementary Materials. Excluding the previous reported species containing AFP, AFP genes were newly discovered in Pacific herring, European smelt Osmerus eperlanus, and delta smelt Hypomesus transpacificus through Blastp and Tblastn algorithms. The phylogenetic analysis of AFP gene indicated at least 3 HGT events (Figure 1). According to our phylogenetic topology, the latest HGT was from herrings to genus Osmerus (rainbow smelt and European smelt), and the time frame for this HGT was estimated after the divergence of genera Spirinchus and Thaleichthys from genus Osmerus (20.8–6.9 million years ago, Mya). According to the gene tree topology, the first earlier HGT event was from the sea raven Hemitripterus americanus—sea poacher Brachyopsis segaliensis group to genus Hypomesus (Japanese smelt, delta smelt, and Japanese surf smelt). Thus, the previous presumptive HGT from sea raven to Atlantic herring was not supported with the basal placement of Japanese smelt and delta smelt (Figure 1B). Considering the topology of family Osmeridae and the absence of AFP gene in genera Spirinchus and Thaleichthys, this first ancient HGT event might occur after the divergence of family Osmeridae (31.9 Mya) and before the split of genus Hypomesus (24.2 Mya). Even with no direct evidence, however, given the confirmed ancient and latest HGT events, there should exist an independent HGT event from genus Hypomesus to herrings to complete the AFP transfer route. From this perspective, our gene tree topology also revealed certain evidence for this intermediate HGT event (Figure 1B). Species in genus Hypomesus, not sea raven–sea poacher group, were possible donors of the AFP gene to herrings. This gene must have entered in herrings before the upper time of the latest HGT event (24.2–6.9 Mya). The phylogenetic AFP tree indicated complex HGT scenarios and at least 3 independent HGT events among these 3 species groups, including 2 separately HGT events between herrings and 2 smelts genera (Hypomesus and Osmerus). This result confirmed and revised the recently published conclusion about the transfer AFP from herring to 2 smelts (Graham and Davies 2021). Based on the currently available data, it is not possible to estimate the precise time points for these HGT events. Further studies are needed to introduce more key node species to describe a precise scenario and decipher the underlying molecular mechanisms of the AFP–HGT events among teleost species.