Fig 1.
Phylogenetic tree of Rho GTPases.
Construction of the phylogenetic tree was based on the amino acid sequences of Rho GTPases from selected species of mammals, amphibians, fish, drosophila, nematodes, cnidarians and mollusks using the Maximum-Likelihood (ML) algorithm in MEGA 6.0. The detailed accession numbers of the protein sequences are presented in S1 Table. The topological stability of the ML tree was evaluated by 1000 bootstrapping replications, and bootstrapping values higher than 60 are indicated by numbers at the nodes. Rho GTPases from Crassostrea gigas, Patinopecten yessoensis, and Chlamys farreri are marked with blue, red and green triangles, respectively.
Fig 2.
Detailed phylogenetic tree of Bivalvia Rho GTPases and the corresponding amino acid similarity heat map.
An unrooted phylogenetic tree (A) was constructed using Rho sequences from C. gigas, P. yessoensis, and C. farreri with the same protocol as in Fig 1. The summarized amino acid similarity of bivalve Rho proteins is presented using a heat map (B) generated by heatmap.2 in R. The detailed accession numbers of the protein sequences are shown in S1 Table. The Rho genes duplicated in bivalves are marked with red lines.
Fig 3.
Gene structure of Rho GTPase genes.
Exons in the ORF (open reading frame) and UTRs are shown separately as dark-green and light-green boxes, and introns are shown as folded lines; the exon numbers are marked. CgRho-1 and CgRho-2 are repeated in tandem. The detailed accession numbers for the protein sequences are shown in S1 Table.
Fig 4.
Analysis of the protein sequences and structures of Rho GTPases.
(A) Schematic representation of atypical Rho GTPases. Boxes in color are characteristic structures: the Rho GTPase domain (blue), CAAX box (orange), BTB domains (green), EFH domains (pink) and second GTPase domain in Miro (light blue). The first GTPase domain resembles Rho GTPases, and the second is more related to the Rab family of small GTPases [21]. (B) Sequence alignment of the Rho family. The amino acid sequences of Rho GTPases were aligned using ClustalW. The highlighted (cyan) residues are important for GTPase activity. The characteristic structures are marked. The amino acid sequences of RhoBTB and Miro proteins are truncated at the C-terminus. Red shaded and red letters indicate identical and similar amino acids, respectively.
Table 1.
Sequence attributes of Rho GTPase genes of Bivalvia.
Table 2.
Rho GTPases in different species.
Fig 5.
Interspecies comparison of Rho subfamily proteins.
Species abbreviations: Hm, Hydra magnipapillata; Nv, Nematostella vectensis; Sd, Suberites domuncula; Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Cg, C. gigas; Py, P. yessoensis; Cf, C. farreri; Sp, Strongylocentrotus purpuratus; Bf, Branchiostoma floridae; Ci, Ciona intestinalis; Dr, Danio rerio; Gg, Gallus gallus; Xt, Xenopus tropicalis; Mm, Mus musculus; Hs, Homo sapiens. The white bars indicate the total number of Rho genes; the black bars indicate Rho gene members.
Fig 6.
Expression analysis of Rho genes in bivalves.
(A) Heat map summarizing the expression of Rho GTPase genes during embryonic and larval developmental stages and in different adult tissues. RPKM values were modified by Log10 transformation. (B) Cumulative expression of Rho GTPase genes in different development stages and adult tissues of P. yessoensis (Py), C. farreri (Cf) and C. gigas (Cg).
Fig 7.
Spatiotemporal expression of duplicated genes in C. gigas.