3.6 Expansion of opsin genes in ray-finned fishes
To test whether the diversity of opsin genes is useful for behavioral functions, we investigated copy number of opsin genes in 13 well-assembled genomes of ray-finned fishes, representing several visual types, and C. milli as an outgroup (Fig. 6B). For example,L. maculates usually rely on eye development and the visual system for prey capture (Shao et al. 2018). B. pectinirostris , an amphibious fish adapted to terrestrial environments, is adapted for aerial vision in order to avoid terrestrial predators (You et al. 2014). In contrast, S. anshuiensis is a cavefish, and its eyes are completely lost (Yang et al. 2016). Opsin genes in the zebrafish genome were used as a sequence reference. We finally identified 122 opsin genes (Table S11), including 117 complete genes, 5 incomplete genes (missing DNA segments less than 100 bp in four, and inserting DNA fragments of 480 bps in one), and sorted into five subfamilies. The LWS2 and Rh2-1 genes were lost in these fishes. Genes in the Rh1 subfamily were lost in G. aculeatus ; the Rh1-2 gene was only found in S. anshuiensis . Rh2-2 and three genes were found only in three species. The SWS1 gene was lost in S. melops , T. rubripes , C. semilaevis , and B. pectinirostris . Species lost opsin genes that were not related to that species’ visual sensitivity variation.
Synteny analyses of opsin genes, together with evolutionary trees, showed LWS1 as a single ancient opsin, and duplicated an immediately adjacent component of SWS2 in evolutionary origins (Fig. 7A), which was a result of the second run of whole-genome duplication events (2R) (Lin et al. 2017). A duplicate of the SWS2- LWS1 complexity in different chromosomes of S. anshuiensiswas believed to be a result of the 4R that occurred in the common ancestor of Cypriniformes (Lin et al. 2017). The SWS2 gene has two duplicated copies in B. pectinirostris , L. maculates , and O. niloticus, and up to four adjacent copies inC. undulatus , which could improve visual acuity of these species by gene duplications to adapt to dim-light environments (Yokoyama & Jia 2020). SWS2 is a single-gene duplication that often acts as a springboard for adaptive diversification in a genomic region (~ 30 kb), where two additional duplication events occurred to produce up to three SWS2 genes in some percomorph fish lineages (Cortesi et al. 2015). Interestingly, we observed the retention of up to four SWS2 genes in C. undulatus , suggesting an unexpected expansion of the SWS2 gene in the percomorph group. However, the SWS2 gene in L. maculatesshowed divergence due to an inserted fragment, and a gene that was located between SWS2 and LWS1 could be diversified from the SWS2 gene in O. latipes (Fig. 7A). Our results provide evidence that SWS2 could diversify after gene duplication, subsequently causing gene functional changes (Porath-Krause et al. 2016).
Surprisingly, the LWS1 gene has displayed single-gene duplications to produce four copies, and a retrotransposed duplicate inC. undulatus , whereas other species showed either one LWS1gene, or one LWS1 gene plus a retrotransposed duplicate, such asB. pectinirostris and L. bergylta (Fig. 7A). Traditionally, expansion events of the LWS1 gene have not been observed in approximately 100 ray-finned fish genomes (Cortesi et al. 2015; Lin et al. 2017; Phillips et al. 2016; Rennisonet al. 2012). However, an insect species, Xenos vesparum , with compound eyes possessed five unique LWS opsin genes, and these LWS duplications were used to restore the longer-wavelength sensitivity due to SWS gene loss (Sharkey et al. 2017).LWS1 expansions were notably important in C. undulatus , with behaviors often guided by visual cues. C. undulatusjuveniles inhabit sandy inshore regions, shallow reefs, and murky outer river areas to capture zooplankton (Sadovy et al. 2003), and theLWS1 gene is required for the prevalent wavelengths under shallow water conditions, such as depths less than 30 m (Lin et al.2017). Therefore, duplication of the LWS1 gene could increase the ability of juveniles to detect prey against a light background.LWS1 gene duplications indicated an independent opsin expansion event, rather than whole-genome duplication. C. undulatus has 48 chromosomes (Huo et al. 2009), suggesting that this species underwent the 3R, a fish-specific genome duplication, similar to that of most percomorph fishes (Cortesi et al. 2015). We inferred thatLWS1 expansion occurred in C. undulatus after 3R with very few changes in copy number, except for one copy translocated to another chromosome (Fig. 7A).
Synteny analyses of Rh2 showed that gene expansions have occurred to produce three to five tandem copies in the branches composed of Labridae fishes, including C. undulatus , S. melops , andL. bergylta (Fig. 7B). One copy was translocated to another chromosome in L. bergylta , and one copy was diversified to acquire a novel function different from the opsin gene in S. melops . In contrast, other species displayed no more than threeRh2 copies in tandem arrays, except zebrafish. C. undulatus has five Rh2 copies, the most reported of any fish (Phillips et al. 2016). Rh2 duplications may coincide with eye evolution in which they have played an important role in expanding the photoreceptive capabilities of organisms by opsin copies (Davieset al. 2007). Labridae fish are more commonly observed inhabiting offshore habitats along steep outer reef slopes, reef flats, and lagoon reefs to depths of up to 60 m (Sadovy et al. 2003). It is reported that marine fish below 50 m possess more Rh2 genes than those living above 30 m (Lin et al. 2017). Green-sensitive Rh2 helps vertebrate species to better discriminate wavelengths in this environment (Yokoyama & Jia 2020). Multiple Rh2 genes imply good visual adaptation in predating sea hares, boxfish, and starfish, which employ color disguises, similar to reef environments. In contrast, the Rh2 gene was reduced to one in C. semilaevis and A. percula (Fig. 7B), suggesting that these species likely adapted alternative mechanisms instead of gene copy to contribute to visual sensitivity, such as the known opsin sequence tuning sites (Phillips et al. 2016).
To detect duplication events in the SWS2 , LWS1 , andRh2 genes, we constructed phylogenetic trees of these genes. Four duplications of the SWS1 gene were present in C. undulatus , and one duplication in L. maculates (Fig. S3). As divergence after gene duplication, B. pectinirostris and O. niloticus did not show SWS1 gene duplication, although synteny analyses revealed two adjacent genes in one chromosome (Fig. 7A). In theLWS1 gene tree, gene duplications were observed in two species (Fig. S4). Due to 4R, the S. anshuiensis genome showedLWS1 gene duplications, whereas four gene duplications plus a retrotransposed duplicate was found in C. undulatus (Fig. 7A, Fig. S4). Phylogenetic analysis was applied to infer Rh2 gene duplication, and many species showed duplication events (Fig. S5). In the C. undulatus genome, the Rh2 gene showed one duplication and two copies were separated into different closed clades, which was regarded as diversification after gene duplication. To our knowledge, it is not known whether opsin genes, such as SWS1 ,LWS1 , and Rh2 , expanded their copy numbers in one species. Opsin genes can change copies by genome duplication, gene duplication, and gene conversion (Sawyer 1989). Interestingly, C. undulatusshowed unexpected opsin copies, revealing multiple genetic mechanisms of opsin expansion.