Molecular evolution and role of aquaporins in the water-to-land transitions of amphibious fishes

Abstract

Aquaporins (AQPs) or major intrinsic proteins (MIPs) form an ancient family of transporters for water and small solute across biological membranes. They constitute a highly diverse protein superfamily, and their evolutionary history and functions have been relatively well studied in vertebrates and plants. For instance, in vertebrates four well defined clusters are described: AQP1-like (water-selective classical aquaporins), AQP8- like (ammonia channels), AQP3-like (aquaglyceroporins), and AQP11-like (unorthodox or super aquaporins). In land vertebrates (Tetrapoda), an exclusive clade of MIPs/AQPs—which is clustered within the classical aquaporins and includes three different orthologues—appears to have been important for their process of colonisation of terrestrial environments. MIPs are broadly present across the eukaryotic tree of life suggesting both a more complex evolutionary history and a larger set of functions than previously thought. Here, we studied the diversity of MIP proteins in the entire eukaryotic lineage by setting a general phylogenetic context for understanding their evolution. Besides, considering the importance of aquaporins in the water-to-land transition of sarcopterygian vertebrates (emergence of tetrapods), we studied the molecular evolution of these proteins in several amphibious fishes of the actinopterygian branch in order to investigate possible new duplication events or adaptive modifications at the sequence level that could be related with their acquisition of an amphibious lifestyle. Finally, we provide a robust bioinformatic workflow and pipeline for gene isolation from large-scale genomic data and phylogenetic analyses that enabled the achievement of the molecular evolution goals. Taking advantage of genomic and transcriptomic data from publicly available eukaryotic databases plus datasets compiled from previous studies, we managed to ascertain a comprehensive catalogue of MIPs within Eukaryotes. In agreement with previous studies, we reported a highly diverse repertoire of MIPs in unicellular eukaryotes suggesting a complex catalogue in the last eukaryotic common ancestor (LECA). In this sense, we reported three MIP clades that likely have deep evolutionary origins. However, whether the origin of these clades occurred during the transition from the first eukaryotic common ancestor (FECA) to LECA, or whether they were already present in FECA is still under investigation. Regarding AQPs/MIPs evolution in amphibious fishes, we obtained a final catalogue of 356 aquaporin sequences from 22 amphibious fish genomes (plus four lungfish sequences). Unlike sarcopterygians, we found no evidence of the emergence of new AQP classes that can be related to the water-to-land transition in the studied species of actinopterygian amphibious fishes. Instead, we detected signatures of adaptive selection in 19 AQP branches (including AQP1, AQP3, AQP8, AQP10, AQP11, and AQP12 classes) in 13 different amphibious fish lineages, and spotted specific sequence changes in 13 of such branches. Some of these changes are located in, or close to, important motifs of the AQP sequence involved in pore formation or substrate selectivity (such as the NPA motif or the ar/R selectivity filter) suggesting a change in protein structure, function or regulation. Of all our results, those of AQP11 orthologs suggest these could constitute the most promising candidates for further research. Up to 15 positively-selected sequence positions (one shared among three lineages) correspond to six different AQP11 branches. Furthermore, it is remarkable the case of the adaptive selection detected in the AQP11b stem branch of the Gobiidae clade, which, in our dataset, is represented by the amphibious mudskippers and two fully-aquatic relatives. In these, we identified a change at the NPA motif where the canonical N (asparagine) is substituted by an S (serine). The sequence modification of this AQP11b occurred before the evolution of the mudskippers lineage and it could represent a possible case of exaptation in Gobiidae. We conclude that, due to the importance of aquaporins in osmoregulation in fishes, the sequence positions found under adaptive selection may have led to modifications in the structure or function of these proteins that could have played a role in the water-to-land transitions of the studied amphibious fish.