Mudskipper genomes provide insights into the terrestrial adaptation of amphibious fishes

Abstract

Mudskippers are amphibious fishes that have developed morphological and physiological adaptations to match their unique lifestyles. Here we perform whole-genome sequencing of four representative mudskippers to elucidate the molecular mechanisms underlying these adaptations. We discover an expansion of innate immune system genes in the mudskippers that may provide defence against terrestrial pathogens. Several genes of the ammonia excretion pathway in the gills have experienced positive selection, suggesting their important roles in mudskippers' tolerance to environmental ammonia. Some vision-related genes are differentially lost or mutated, illustrating genomic changes associated with aerial vision. Transcriptomic analyses of mudskippers exposed to air highlight regulatory pathways that are up- or down-regulated in response to hypoxia. The present study provides a valuable resource for understanding the molecular mechanisms underlying water-to-land transition of vertebrates.

Figure 1. Habitats of the four sequenced mudskippers.

BP and SH are predominantly water dwelling, whereas PM and PS spend extended periods of time on land. Interestingly, the genome size decreases in the following order: BP>SH>PS>PM, which may be associated with their terrestrial affinity but unrelated to their body size (PS>BP>SH>PM).

Figure 2. The phylogenetic placement, demographic history and specific TLR13 expansion of mudskippers.

(a) A phylogenetic tree was constructed using fourfold degenerate sites of 1,913 genes from 12 vertebrate species. Blue numbers at the nodes represent divergence time between lineages. Red dots indicate the reference divergence times from the TimeTree (http://www.timetree.org/). (b) The population history of two representative mudskippers (BP and PM) was estimated. The red and blue lines represent the population size changes in BP and PM, respectively. The green and light blue lines, around the red and blue lines, are the PSMC estimates on 100 sequences randomly re-sampled from the original sequences. The orange line denotes the fluctuation of the global sea level. (c) Phylogeny of TLR13 family in ten representative vertebrates showing the expansion of TLR13 in mudskippers.

Figure 3. Differential ammonia excretion in the gills of mudskippers.

(a) An overview of ammonia excretion pathways in the gills illustrates the differential ammonia excretion in mudskippers. The core pathway comprises Na⁺–K⁺–Cl⁻ co-transporter (NKCC), Na⁺K⁺–ATPase (NKA), carbonic anhydrase (CA), cystic fibrosis transmembrane conductance regulator (CFTR), Na⁺/H⁺ exchanger (NHE) 3, H⁺–ATPase-V-type-B-subunit (H–ATPase), anion exchanger (AE), glycosylated Rhesus protein b (Rhbg) and c (Rhcg1 and Rhcg2). The black star represents genes with positive selection in both BP and PM, whereas the white and red stars indicate genes that are positively selected specifically in BP and PM, respectively. (b–d) Three-dimensional views of Rhcg1 proteins in BP (b), PM (c) and PS (d) highlight several PM- and PS-specific amino-acid substitutions. The red squares indicate the central pore of the channel for transporting NH₃, which includes the conserved Phe-Gate (F145, F250) and Twin-His (H200, H359). Three genetic variations around the central pore, Leu328Cys, Leu342Phe and Val361Met in PM and PS, may be related to a more-efficient NH₃ diffusion system in PM and PS suited for a land-dominant lifestyle.