Introduction

Salinity is an important environmental factor and plays a major role in growth, survival, reproduction, respiratory metabolism, and immune defense in decapod shrimps (Rozas and Minello 2011; Lin et al. 2012b; Ponce-Palafox et al. 1997; Li et al. 2007). In decapod shrimps, osmoregulation is a fundamental and dynamic process that is crucial for the maintenance of ion and water balance. Decapod shrimps exhibit a wide range of osmoregulatory capabilities and patterns. From those restricted to marine environments, to their estuarine and freshwater counterparts, decapod shrimps have exploited virtually different adaptation abilities (Mcnamara and Faria 2012).

Decapod shrimps contribute millions of tons to food production worldwide every year, which renders them the most valuable internationally traded commodity in aquaculture (Gillett 2008). However, change in salinity can trigger disease outbreaks by affecting immune resistance of shrimps, and ultimately results in higher rates of mortality (Prayitno and Latchford 1995; Wang and Chen 2005). Therefore, osmoregulatory physiology of decapod shrimps has long aroused scientific curiosity, and research has often focused on their osmoregulatory patterns (Mcnamara and Faria 2012). Litopenaeus vannamei, Fenneropenaeus chinensis, Penaeus monodon, and Macrobrachium rosenbergii are the four major economically important aquacultured decapod shrimps in China. Unlike shrimps (e.g., M. rosenbergii) living in freshwater (FW), many decapod shrimps have adapted to seawater (SW) environments. Additionally, there are euryhaline species that can thrive across a wide range of salinities (e.g., L. vannamei thrives in a salinity range of 1–50 ppt and F. chinensis and P. monodon in 5–45 ppt), whereas the FW shrimp M. rosenbergii can only live in the 0–25 ppt range (Perez-Velazquez et al. 2007; Cheng and Chen 2000), and Neocaridina denticulata acclimate in slight saline condition (5–10 ppt) (FAH and Christianus 2013). Hyperosmotic and Hypoosmotic conditions contribute to the different osmoregulatory capacity between SW and FW shrimps.

The osmoregulation mechanisms of decapod shrimps have also received substantial attention, mainly with respect to ion movement, particularly that of Na+, Cl−, and Ca2+ (Freire et al. 2008; Ahearn et al. 2004; Kirschner 2004). In general, the regulation of salt and water balance through ion pumps, ion transporters, and ion exchangers in decapod gills and other tissues is the primary osmoregulatory mechanism in decapod shrimps (Towle et al. 2011; Wheatly and Gao 2004). In previous research, several osmoregulatory molecular has been identified as responsible for osmoregulation in decapods, including Na+/K+–ATPase (NKA), Na+/K+/2Cl− cotransporter (NKCC), carbonic anhydrase (CA), cystic fibrosis transmembrane regulator (CFTR), H+–ATPase (HAT) Aquaporin 3 (AQP3), and Rho GTPase (RHOGTP8) (Mcnamara and Faria 2012; Havird et al. 2013b; Defaveri et al. 2011; Cutler et al. 2007; TJM et al. 1993). As the primary ion pump, NKA is a crucial component in osmotic/ionic regulation and is responsible for establishing electrochemical gradients across the cell membrane. NKCC can transport ions into gill cells from the external environment (Nilsen et al. 2007). RHOGTP8 is an important ion transporter that plays a key role in the active secretion of ions across the gills (Defaveri et al. 2011).

General features of the osmoregulatory physiology of decapod shrimp tolerance to salinity have been investigated (Mcnamara and Faria 2012; Charmantier and Anger 2011). However, the genetic basis of these physiological adaptations has not been investigated. By adapting to environments with differing salinities, SW and FW shrimps have likely evolved different salinity control mechanisms, and those might have generated a cluster of genes with these adaptive features. Commonly, the coding sequence and gene expression level are thought to be the two aspects that are shaped by natural selection to provide adaptive characteristics (Marra et al. 2014; Nielsen 2005). Regarding gene expression, previous research has investigated differential gene expression (DGE) in shrimps after salinity stress (Gao et al. 2012; Shekhar et al. 2013; Hu et al. 2015). However, few studies have focused on the adaptive evolution of the coding sequences in SW and FW shrimps.

Adaptive evolution analysis has been widely used to identify positively selected genes under natural selection (Olson-Manning et al. 2012). Phenotypic convergence, shaped by convergent evolution, has been connected to parallel substitutions of single nonsynonymous amino acid sites of positively selected genes occurring independently in unrelated taxa (Stewart et al. 1987; Foote et al. 2015). Therefore, in this study, we performed adaptive evolution analysis on all the potential protein-coding genes of six decapod shrimp species based on transcriptome data. We collected all the positively selected genes among SW and FW shrimps. Finally, the adaptive and convergent evolution events of decapod shrimps were investigated.

Materials and Methods

Sources of Transcriptome Data

All the Illumina paired-end transcriptome data of six decapod shrimps, F. chinensis, L. vannamei, P. monodon, Pandalus latirostris, M. rosenbergii, and N. denticulata, were collected from our group and the SRA database of NCBI (Table 1, accession numbers: SRR1039534, SRR653437, SRR346404, SRR1460493, SRR1460494, SRR1460495, SRR1460504, SRR1460505, SRR388222, SRR388207, SRR388221, SRR345611, SRR345610, SRR345609, DRR001118, DRR001119, DRR001120, DRR001121, and SRR1185328). The RNA samples in the aforementioned research were extracted from different development stages and various tissue samples (Wei et al. 2014; Li et al. 2012), which ensured that the assembled unigenes provided sufficient materials for the analysis. Among the six shrimp species, the first four are SW shrimps, and the last two, M. rosenbergii and N. denticulata, inhabit freshwater.

Table 1 Transcriptome data of six decapod shrimps
Full size table

As the only completely sequenced crustacean, Daphnia pulex was used as the reference for the comparative analysis. The sequences of the full protein-coding genes of D. pulex were downloaded from the JGI Genome Portal (http://genome.jgi-psf.org/).

Transcriptome Sequence Assembly and Filtering

All the paired end Illumina reads were trimmed to remove the low-quality data and adaptor sequences. Low-quality data were filtered using the NGS QC Toolkit IlluQC_PRLL.pl with the parameter of “2 A” (Patel and Jain 2012). Reads with 10 bp or more overlapping any adaptor were removed. Next, using Trinity (Haas et al. 2013, Grabherr et al. 2011), all the clean data were de novo assembled with the default assembly parameters.

All the assembled contigs were clustered to remove redundant sequences. Isoforms and incomplete short gene segments are two major redundant sequences in the contigs assembled by Trinity. To filter gene isoforms, TIGR Gene Indices clustering tools (TGICL) was used for clustering isoforms with default settings (Pertea et al. 2003). Next, a stricter filtering method combining BLAST and BLAT algorithm was applied (Kent 2002). BLAST was utilized to obtain the identity value of homologous sequences, while BLAT was used to obtain their match length. When the match length exceeded 70% of the length of two homologous sequences, and their identity value was greater than 98%, the shorter one was excluded from the gene dataset. Next, BLASTx searches were performed for all the left contigs against the GenBank non-redundant protein database (Nr) and followed by conjoining fragmental alignments using SOLAR (Yu et al. 2006). Thus, the partial or full open reading frame (ORF) of each contig was obtained and translated into amino acid sequences. Because short sequences were useless for the following analyses, we collected unigenes whose correspondent protein-coding sequences were longer than 30 aa.

Gene Ontology Classification

The unigenes of SW and FW shrimps were collected for the gene ontology (GO) classification. We used Blast2GO software suite to predict functions of individual unigenes and assign GO terms (Gotz et al. 2008). Gene enrichment analysis of each GO term was performed through using Web Gene Ontology Annotation Plotting (WEGO) (Ye et al. 2006).

Ortholog Assignment

Ortholog assignment is an important procedure to perform before adaptive evolution analysis. A pairwise BLASTx alignment was performed to align the unigenes of the six shrimps to the genes of D. pulex with an e-value cutoff of 1E-05, and the pair of sequences that had the pairwise best hit in the BLASTx results was selected. For each pairwise best hit sequence pair, the fragmental alignments were conjoined with SOLAR, and the sequence pairs whose match length was longer than 60% of the correspondent gene of D. pulex were selected. To filter out pseudogenes, we cut the sequence of unigenes based on the results of SOLAR and translated it to amino acid sequences. When a stop codon was involved in the sequence, the correspondent sequence was eliminated.

TreeFam method was used for clustering orthologous gene families (Schreiber et al. 2014). In the beginning, a pairwise BLASTp alignment was performed to align all-to-all with an e-value cutoff of 1E-07. We assigned a connection between two genes when the aligned regions covered more than 1/3 of the genes. An H-score between two genes (G1, G2), defined as “score (G1, G2)/max (score (G1, G1), score (G2, G2))”, was used to weigh the similarity of them. Then, Hcluster_sg was used to construct gene families (Li et al. 2006). Finally, we selected all the single copy gene families as orthologous groups for the following analysis.

Phylogenetic Analysis

In each orthologous group, there were seven orthologous sequences, including the unigenes of the six shrimps and the orthologous gene of D. pulex. In order to perform phylogenetic and adaptive evolution analysis, the shared region of these orthologous genes was extracted. The nucleotide and deduced amino acid sequences of each gene were aligned using ClustalW (Thompson et al. 2002). When there was an indel in the alignment, the corresponding sites of amino acid and codons of the other orthologous sequences were also removed.

The maximum likelihood (ML) and the Bayesian inference (BI) methods were used for phylogenetic tree construction. For ML-tree construction, sequence alignments were performed using MUSCLE 3.6 (Edgar 2004). The substitution models that best fit observed alignment data were estimated with the program jModelTest 2 (Darriba et al. 2012). Using PhyML (Guindon and Gascuel 2003), we performed ML analysis with the substitution model HKY87 + gamma + Inv. One thousand bootstraps were conducted to produce the branch support values (Li et al. 2011; Zhu et al. 2011). Based on the alignment results above, Bayesian phylogenetic inference was made with the help of the program Mrbayes 3.2.1 (Ronquist and Huelsenbeck 2003). In the BI analysis, two independent runs, each with four chains, were tested for millions of generations until the standard deviation of split frequencies converged towards zero. The first 25% of sampled trees was discarded as burn-in.

Adaptive Evolution Analysis

Adaptive evolution was checked by comparing the nonsynonymous/synonymous substitution ratios (ω = dN/dS) with ω = 1, ω < 1, and ω > 1, indicating neutral evolution, purifying selection, and positive selection, respectively. All the positively selected genes and amino acid sites were predicted using the branch-site model of PAML (null hypothesis: model = 2, Nssites = 2, fix_omega = 1, omega = 1; alternative hypothesis: model = 2, Nssites = 2) (Zhang et al. 2005). Positive selection was indicated in each test when a significant difference between the tested and null models was observed using a likelihood ratio test (LRT). To assess significance, we obtained a p value for each LRT from the Chi-square distribution. The amino acid sites under positive selection were identified using Bayes empirical Bayes approach (BEB). p values were calculated using the BEB corrected for multiple testing by the false discovery rate (FDR) method. Positively selected sites were inferred at p ≧ 95%.

Results

Orthologous Genes in Six Decapod Shrimp Species

After trimming and filtering, all the transcriptome data for the six decapod shrimp species were well assembled into contigs with median length greater than 1.2 Kb (Supplementary materials Table S1). All these contigs were clustered to remove isoforms and blasted against the nr database, leaving only annotated contigs, which were assigned as the unigenes of the six shrimps. There were approximately 12,000 annotated unigenes for each shrimps (Table 1), and the distribution patterns of the number of unigenes in each GO term among the six shrimps tended to be similar (Supplementary materials Fig. S1). Therefore, it appeared that the annotated unigenes of the six shrimps were similar and sufficient for the following comparative analysis.

When the annotated unigenes of the six shrimps were compared, the number of reciprocal best-hit unigene pairs was biased from 1973 (between F. chinensis and P. latirostris) to 3331 (between L. vannamei and P. monodon). To select the orthologous groups of the six shrimps, we compared them against the full genes of D. pulex and clustered them into groups of gene families. Finally, 1471 groups of orthologous genes were collected after selecting single copy gene families among them. Based on these 1471 groups of orthologous genes, a consensus phylogenetic tree was constructed using ML and BI methods (Supplementary materials Fig. S2). The phylogenetic tree was consistent with most of the trees from previous research (Bracken et al. 2009; Lin et al. 2012a) and was used as the reference for the adaptive evolution analysis.

We determined the similarity of orthologous genes in each group by analyzing the distribution of the identity values in pairwise BLAST results (Fig. 1). As F. chinensis, L. vannamei, and P. monodon stem from the same family, Penaeidae, the similarities among them were typically greater than between the other shrimps. Then, we compared the identity deference of amino acid sequences to that of nucleotide sequences in each pair of orthologous genes (Fig. 1). In general, the identity values of amino acid sequences were higher than those of nucleotide sequences (the identity deference >0 in the box plot of Fig. 1), indicating that synonymous substitutions were commonly occurring among these orthologous genes. However, there were still some orthologous genes with relative higher identity values for nucleotide sequences than those of amino acid sequences, which suggested that these genes may have more non-synonymous than synonymous substitutions (Fig. 1).

Fig. 1
figure 1

Identity distribution of the orthologous genes. The histogram showed the distribution of the identities of the nucleotide (in red color) and amino acid (in light blue color) sequences of the orthologous genes between any two shrimps. The box plot showed the distribution of the identity deference between amino acid and nucleotide sequences of the orthologous genes in different range of identities (60 ∼ 70%, 70 ∼ 80%, 80 ∼ 90%, 90 ∼ 100%)

Full size image

Positive Selected Genes in SW and FW Shrimp Species

Based on the branch-site test, we identified many positively selected genes which had ω > 1 for a proportion of their sites (BEB test p ≧ 0.95, FDR ≦ 0.1), for the SW and FW shrimp lineages, indicating significant evidence of positive selection. In the SW shrimp lineage, 275 genes were identified as positively selected, whereas in the FW shrimp lineage, there were 234 positively selected genes. Among these genes, 75 were shared by these two groups. Therefore, 434 candidate positively selected genes were identified in the two shrimp lineages.

Functional enrichment analysis assigned these positively selected genes into different GO terms. We found that the gene distributions in the different GO terms were similar between two shrimp lineages (pairwise t test, p > 0.05). Most of the genes were enriched in GO terms of membrane-bound organelles, ion binding, and many metabolic processes (Table 2), which were primarily related to water environment adaptation. However, there were some differences between the two groups of positively selected genes (Supplementary materials Fig. S3). In the GO term of nucleobase, nucleoside, and nucleotide metabolic process (GO:0055086) and transmembrane transporter activity (GO:0022857), many positively selected genes were enriched in SW shrimps, but few in FW shrimps. Furthermore, the positively selected genes of FW shrimps were specifically enriched in GO terms of transferase activity that transferred one-carbon groups (GO:0016741).

Table 2 Gene ontology distribution of the positively selected genes in SW and FW shrimps
Full size table

Osmoregulation-Related Genes under Positive Selection

Among these positively selected genes, it was luckily found that there were several genes that might play essential roles in osmoregulation. In SW shrimps, five positively selected genes were detected to be related to osmoregulation (Table 3). The likelihood ratio test for the five genes (CaCC, BEST2, GPDH, NKA, and Integrin) was significant at the level α = 5%. Calcium-activated chloride channel (CaCC) and sodium potassium-transporting ATPase (NKA) are two critical components in osmotic/ionic regulation for establishing electrochemical gradients across the membrane of the gills (Henry et al. 2012; Havird et al. 2013a; Ismailov et al. 1996). Bestrophin-2 (BEST2) is known to participate in regulation of chloride ion secretion, especially regulating NKCC, which is a kind of ion pumps like NKA (Lorin-Nebel Catherine et al. 2006; Bruno Guinand et al. 2015). Integrin participates in regulation of the activation of the NKCC (Hwang and Lee 2007; Jablonski et al. 2014; Havird et al. 2013a). Glycerol-3-phosphate dehydrogenase (GDPH) plays important role in osmotolerance that is essential for growth under osmotic tress (J Albertyn et al. 1994; A Blomberg and Adler 1989). Therefore, the five genes of SW shrimps were reported to have positive effects on osmoregulation.

Table 3 Positively selected osmoregulation-related genes in SW and FW shrimps
Full size table

Among the positively selected genes of FW shrimps, there were four genes (RasGAP, RhoGDI, CNK3, and ODC) identified to participate in osmoregulation (Table 3). The likelihood-ratio test for these genes was significant, except for RasGAP, but a positively selected site was detected on it (Table 4, BEB p ≧ 0.95). Ras GTPases act as ion transporters for the regulation of ion channels (Pochynyuk et al. 2007). However, Ras GTPase-activating protein (RasGAP) can negatively regulate the conserved RAS/MAPK signaling pathway and shift the balance from the active GTP-bound towards the inactive GDP-bound state (Stetak et al. 2008). Similar to RasGAP, Rho GDP dissociation inhibitor (RhoGDI) plays a role in modulating the cycling of Rho GTPases between active GTP-bound and inactive GDP-bound states, which makes it a key negative regulator of Rho GTPases (Rivero et al. 2002; Eggermont et al. 2001). Connector enhancer of kinase suppressor of ras 3 (CNK3) is a molecular scaffold that participates in the formation of a multiprotein epithelial sodium channel (ENaC) regulatory complex and hence regulates Na+ homeostasis (Soundararajan et al. 2012). Ornithine decarboxylase (ODC) has been reported to be responsible for gill polyamine homeostasis, which are a family of low molecular weight organic cations that is critical for low salinity acclimation (Guan et al. 2016; Henry and Watts 2001). Therefore, the two positively selected osmoregulation-related genes of FW shrimps, RasGAP and RhoGDI, seem to cause negative effects on osmoregulation through regulating the activity of Rho or Ras GTPases. CNK3 and ODC play central roles in cation homeostasis in osmoregulation.

Table 4 Positively selected amino acid sites of the osmoregulation-related genes
Full size table

To further test the results we detected, we performed adaptive evolution analysis on these nine osmoregulation-related genes with two more crabs involved, a FW crab Erocheir sinensis and a SW crab Portunus trituberculatus. Although only six orthologous genes of the nine osmoregulation-related genes were identified in these two crabs, the adaptive evolution analysis indicated that these genes were all positively selected. The positively selected sites were similar to those identified in decapod shrimps, and several new positively selected sites were also detected (Table 4, Supplementary materials Table S2). Therefore, the nine osmoregulation-related genes may be not only positively selected in decapod shrimps, but also positively selected in other species of Decapoda.

Positively Selected Sites of the Osmoregulation-Related Genes

In branch-site tests, amino acid sites under positive selection were further identified using the BEB approach. Along the nine positively selected osmoregulation-related genes in SW and FW shrimps, 40 sites were identified as positively selected at the significance level of p ≧ 0.95 by the BEB test (Table 4). As expected, the background species shared the same sites, whereas the sites from the foreground species were biased with each other. For the positively selected sites of the five osmoregulation-related genes of SW shrimps (CaCC, BEST2, GPDH, NKA, and Integrin), the background species, the two FW shrimps (M. rosenbergii and N. denticulata), had the same amino acid sites, whereas the foreground SW shrimps displayed different amino acid changes among these sites (Table 4). Similar results were observed in the four positively selected genes of FW shrimps.

However, parallel substitutions occurred at site 464 (S) of Integrin, 43 (C) of RasGAP, and site 237(T) of ODC, where identical amino acid changes were detected in the foreground lineages. Parallel substitutions are characterized by parallel nonsynonymous amino acid replacements between at least two related but distinct species with a common ancestor (Erler et al. 2014). The intersection genes, those between the genes with parallel substitutions and genes under positive selection, were inferred to have undergone convergent selection (Foote et al. 2015). In addition to the two sites mentioned above, parallel substitutions also occurred at other sites on these positively selected genes. A total number of 26 sites within eight genes (except GPDH) were detected as parallel substitutions (Supplementary materials Table S3). Therefore, parallel substitutions commonly occurred on positively selected osmoregulation-related genes, which indicated that these genes probably have undergone convergent selection. Thus, we reconstructed the phylogenetic tree of these seven species based on the 40 positively selected amino acid sites of the nine osmoregulation-related genes (Fig. 2). Surprisingly, the phylogenetic tree grouped the two FW shrimps (M. rosenbergii and N. denticulata) together (Table 1). The SW shrimp P. latirostris is phylogenetically closer to M. rosenbergii because they both belong to the infraoder Caridea, but the phylogenetic tree grouped it with the other three SW shrimp species. Therefore, it appears that convergent selection may have happened on SW and FW shrimp species.

Fig. 2
figure 2

Phylogenetic tree of the seven species. a The phylogenetic tree constructed based on all the full sequences of the nine positively selected osmoregulation-related genes. b The phylogenetic tree constructed based on 32 positively selected sites of the 9 genes. The support values of ML and BI analysis were displayed on each branch

Full size image

The functional significance of the positively selected sites was investigated by locating them along the genes and comparing them against the conserved functional protein domains. Almost all the positively selected sites were located in the functional protein domains, except for one site (2D) of BEST2 (Fig. 3). In addition, it was found that many sites were located in the key functional domains of the correspondent proteins, including the CLCA_N domain of CaCC, which is in the family of calcium-activated chloride channels that have four or five transmembrane regions; the Bestrophin of BEST2, which is a basolateral plasma membrane protein expressed in retinal pigment epithelial cells; the NAD_Gly3p_dh_N of GPDH, which is the N-terminal NAD-binding domain; the Na_K-ATPase domain of NKA, which encodes Na+/K+ ATPase beta subunit in eukaryotes; the C2_SynGAP_like domain of RasGAP, which is the C2 domain present in the RasGAP family that are primarily Ca+-dependent membrane-targeting modules; the Rho_GDI domain of RhoGDI, which is the consensus superfamily of RhoGDI; and the LysA of ODC, which catalyzes the transformation of ornithine into putrescine. Furthermore, there were two domains (the DUF1973 domain of CaCC and the Integrin_B_tail domain of Integrin) with several positively selected sites distributed at a relatively high density (Fig. 3). The DUF1973 domain is a functionally uncharacterized domain that is found in various eukaryotic calcium-dependent channels. Among the three domains detected on Integrin, Integrin_B_tail is the one that encodes the tail domain of the Integrin protein involved in cell-extracellular matrix interactions.

Fig. 3
figure 3

The distribution of the positively selected sites along the nine osmoregulation-related genes. The protein domains along each gene were assigned in different colors

Full size image

Discussion

Distinct Convergent Evolution of the Decapod Shrimps Living in Different Aquatic Environments

Natural selection may be particularly strong in extreme environments, which can place selective pressures on the adaptations to different environments (Marra et al. 2014; Nielsen 2005). Because they have lived in different aquatic environments over a long evolutionary history, SW and FW shrimps may both have adaptively evolved under selective pressures. L. vannamei can live in 1–50 ppt, which is a broader range than that of FW shrimps. However, FW shrimps have to maintain water and salt balance for cells in hypoosmotic aquatic environments. In this study, nine osmoregulation-related genes under positive selection were identified in SW and FW shrimps, indicating that adaptive evolution appeared to occur in the osmoregulation system of decapod shrimps.

There are substantial levels of adaptive evolution in many species, and more than 25% of all amino acid substitutions are estimated to be the consequence of positive adaptive evolution (Strasburg et al. 2011; Olson-Manning et al. 2012; Castellano et al. 2015). However, parallel substitutions of amino acids between at least two related but distinct species with a common ancestor are rare and are considered a consequence of parallel evolution (Erler et al. 2014). Parallel amino acid substitutions were hypothesized to be possible indicators of convergent evolution if the correspondent gene was under positive selection (Foote et al. 2015). Additionally, phenotypic convergence shaped by convergent evolution has been connected to parallel substitutions of nonsynonymous amino acid sites of the positively selected genes occurring independently in unrelated taxa (Stewart et al. 1987; Foote et al. 2015). Among the six shrimp species analyzed in this study, P. latirostris shared a similar environment with the three Penaeus shrimps, even though P. latirostris and M. rosenbergii stemmed from the same infraorder (Caridea). Unrelated shrimps living in similar environments might have undergone convergent evolution in their osmoregulation systems.

Four lines of evidence support the hypothesis that convergent evolution of shrimp osmoregulation has occurred. First, the adaptive evolution analysis showed that the positively selected genes identified in SW and FW shrimps were enriched in GO terms of ion binding and membrane-bound organelles, suggesting improvement on water environment adaptation. Second, the five positively selected osmoregulation-related genes detected in SW shrimps all appeared to have positive effects on ion transportation, whereas the four positively selected osmoregulation-related genes detected in FW shrimps primarily had negative effects on ion transportation or sustain cation homeostasis for low salinity acclimation. Third, parallel substitutions of amino acid sites have been detected on the positively selected osmoregulation-related genes of the SW and FW shrimps. In particular, three sites with parallel substitutions were also detected to be the positively selected sites of the correspondent genes. Lastly, the phylogenetic tree, constructed by positively selected sites of these osmoregulation-related genes, grouped SW and FW shrimps although they were from different infraorders. Therefore, parallel substitutions were detected on the osmoregulation-related genes under positive selection for SW and FW shrimps, indicating that distinct convergent evolution may occur in the genetic sequences of these two kinds of shrimps.

Differences of Osmoregulatory Mechanisms Between SW and FW Shrimps

The osmoregulatory physiology of the decapod shrimps has long aroused scientific curiosity regarding patterns of osmoregulatory ability and body fluid composition and concentration (Mcnamara and Faria 2012). Living in aquatic environment, both SW and FW shrimps have to establish osmotic and ionic balance against the environmental gradients (Charmantier and Anger 2011). In SW, decapod shrimps have the osmoregulatory mechanisms to maintain cell structure by replacing water lost through osmosis to ambient water and to discharge absorbed salt. In contrast to SW shrimps, FW shrimps need to replace salt lost through diffusion to the ambient water and to eliminate excess absorbed water (Park et al. 2012; Mcnamara and Faria 2012). For SW shrimps, transfer from SW to FW environments induces changes in osmotic plasma parameters and activates osmoregulatory system (Mancera et al. 2002), whereas for FW shrimps, general opinions indicated that the ancestor of decapods is presumably marine species, which suggested that adaptive evolution more probably happened on osmoregulation system of FW shrimps. Thus, the osmoregulatory mechanisms of SW and FW shrimps will be different under different levels of salinity stress.

Generally, Na+, Ca2+, Mg2+, and Cl− are the four major ions that are used for maintaining ionic gradients, which may be essential for the mechanism of decapod osmoregulation (Evans et al. 1999; Faria et al. 2011; Mcnamara and Faria 2012). In terms of transbranchial ion movements in osmoregulation, some of the specific ion pumps, such as NKA and V-ATPase, have been identified to be essential for ion uptake and secretion. Beside ion pumps, ion transporters and ion exchangers in decapod gills and other osmoregulatory tissues, such as CaCC and BEST2, are also important for osmoregulation. Generally, ion transporters and the proteins participating in intracellular signaling pathways are two major molecules involved in osmoregulation, and most of these genes have been identified to be significantly differentially expressed under different levels of salinity stress, including NKA, Integrin, GPDH, CNK3, and ODC (Gao et al. 2012; Shekhar et al. 2013; Hu et al. 2015; Guan et al. 2016; Yu et al. 2006; A Blomberg and Adler 1989).

Among the five positively selected osmoregulation-related genes in SW shrimps, four of them (NKA, CaCC, BEST2, and Integrin) are directly related to ion pumps or ion transporters, and one of them (GPDH) plays important role in osmotolerance that is essential for growth under osmotic tress (J Albertyn et al. 1994; A Blomberg and Adler 1989). All the five positively selected genes appear to cause positive effects on osmoregulation and can improve ion movements. In addition, some of the genes, such as NKA and Integrin, have been identified to be significantly upregulated in L. vannamei and P. monodon after being exposed to low salinity environment (Gao et al. 2012; Shekhar et al. 2013; Hu et al. 2015). Therefore, the positive selection of these genes in SW shrimps in the present study suggests enhanced capacity for ion transportation during osmoregulation.

Ion channels can be regulated by Ras, Rho, and Rab GTPases, which belong to the family of GTPase, which play an important role in physiological control of ion channel function (Pochynyuk et al. 2007). The GTPase switch can be turned on by GEFs (such as RhoGEF), which stimulates dissociation of the tightly bound GDP, and turned off by GAPs (such as RasGAP), which shift the balance from the active GTP-bound towards the inactive GDP-bound state (Stetak et al. 2008; Cherfils and Zeghouf 2013). For Ras and Rho GTPases, this switch incorporates a membrane/cytosol alternation regulated by GDIs (such as RhoGDI), which can modulate the cycling of GTPases between active GTP-bound and inactive GDP-bound states (Cherfils and Zeghouf 2013). Among the four osmoregulation-related genes under positive selection in FW shrimps, two of them (RhoGDI and RasGAP) appear to cause negative effects on osmoregulation that turns off the GTPase switches. RhoGDI and RasGAP are two genes included in intracellular signaling pathways involved in balancing ions through adjusting the signaling pathway under salinity challenge. Furthermore, the other two genes, CNK3 and ODC, are also important for the cation homeostasis in osmoregulation. CNK3 physically interacts with ENaC-regulatory complex and hence plays essential roles in Na+ homeostasis (Soundararajan et al. 2012). CNK3 is also reported to be involved in MAPK pathway regulation and is required for the maintenance of transepithelial sodium transport in the kidney (Ziera et al. 2009). As a family of low molecular weight organic cations, polyamines are produced by the coordinated actions of arginase II and ODC, which can blunt the hypotonic inhibition of NaCl secretion and may lead to early apoptosis of SW ionocytes and their replacement by FW-type ionocytes (Guan et al. 2016; Henry and Watts 2001). Besides, in blue crabs, low salinity acclimation is dependent of ODC activity (Henry and Watts 2001). FW shrimps generally adapt to a narrow range of salinity, because maintenance of water and salt balance is essential for survival in a hyperosmotic environment. Therefore, the adaptive selection of these four genes may benefit for regulation of water and salt balance for FW shrimps.