The Utility of Otx1B Gene Sequence in Evaluating Some Epinephelus species Evolutionary Variations Compared with Other Ray-finned Fishes

Because of extremely high species diversity, fishes are attractive for the study of many biological problems, especially those related to taxonomy and evolution (Volff, 2004).
       
Fish species identification is the core of fishery management which constitutes fishery conservation around the world (Lleonart et al., 2006; Cawthorn et al., 2012; Kim et al., 2017; Lee and Kim, 2020).
       
Coral reef fish (those living amongst or in close relation to coral reefs) communities were found throughout the tropics, including the Red Sea. Epinephelus species are economically important and grasp high price in fish markets. Juveniles of Epinephelus species are heavily harvested for aquaculture from their nursery grounds in several Asian countries (Hobbs et al., 2013; Yu et al., 2018).
       
Some coral reef fishes such as Epinephelus species are endangered or vulnerable to overfishing because they are long-lived and late maturing. Due to numerous taxa and large distribution, the true Epinephelus species relationships have long been poorly understood (Matthew and Philip, 2007). A good management plan for fish genetic resources should ideally involve a continuum of activities including documentation, characterization and utilization of fish genetic resources (Eknath, 1995). Therefore the application of more molecular identification techniques is required for developing informative molecular markers to identify Epinephelus species. Besides, analyses of genetic markers can provide the information needed for the sound management of wild fish stocks (Saad et al., 2012).
       
Several factors should be considered during the choice of molecular markers such as the techniques must be inexpensive and simple (Vignal et al., 2002).
       
The use of molecular methodology in fisheries research has increased over the past decade because of the increased obtainability of such approaches (Park et al., 1993; Alam et al., 2020). Also, such markers will be useful for understanding the genome evolution in aquatic organisms (Chen et al., 2016; Manorama et al., 2017; Ornjira et al., 2018).

These informative markers will play a role in the detection of fish speciation and evolution to design an innovative program for reducing the risk of the extinction of economic biological resources. Characterization of fish taxa via molecular genetic markers was applied using many techniques such as RAD, ISSR, AFLP, SSR and DNA sequencing (Wang et_al2002; Saad et al., 2012; Sun et al., 2015; Yu et al., 2018).

       
This study aimed to evaluate the utility of the Otx1B gene sequence variations for the identification of some Red Sea Epinephelus species (E. areolatus, E. malabaricus, E. summana, E. radiatus and E. chlorostigma) compared with other ray-finned fishes.

Sample collection, DNA extraction and amplification
 
The Epinephelus species (E. areolatus, E. malabaricus, E. summana, E. radiatus and E. chlorostigma) samples were procured from the natural habitats, Yanbu (Red Sea port at western KSA). Fish samples were classified based on the morphological variations in the field (FAO, 1993). 
       
A total of ten fish samples from each sampled fish species were placed on ice in the field and small pieces of caudal fins were preserved in 95% ethanol.
       
Total DNA was extracted from caudal fins tissues from the fish samples (Hillis et al., 1996).
 
Otx1B gene fragments amplification
 
PCR reactions were performed separately in a reaction volume containing 25 μl of 2X Green Go Taq (Promega, USA) master mix, 1 μl forward primer Otx1B-F1 TCC AAG CAG TCT GTG TGG TGT TAA A, 1 μl reverse primer Otx1B_R1: TTT GGT TCA AGA ACC GGA GG, 1 μl of template DNA (0.5 μg/50 μl) and nuclease-free water up to 50 μl (Smith et al., 2008). 
       
PCR amplifications were produced using (Labnet multigrodiet Thermo Cycler). PCR (Labnet multigrodiet Thermo Cycler) amplifications were performed with denaturation for 2 min at 95°C; 30 cycles of 95°C for 1 min, 55°C for 1min, 72°C for 90 sec. and a final extension at 72°C for 5 min. The PCR products were purified using a QIAGEN PCR purification kit then ligated to pGem-TTA cloning kit (Promega, A1360).
 
Purification of plasmids and DNA sequencing 
 
Plasmid ligation and purification steps were carried out as described in Kit (Promega) technical manual. The purified plasmids were introduced for sequencing (Macrogen Inc., Republic of Korea) using a T7 universal primer. The revealed DNA fragment sequences were analyzed. Some of the DNA sequences were submitted to the National Center for Biotechnology Information (NCBI).
 
Analysis of DNA sequences
 
Sequences were aligned using the Clustal Omega program for Multiple Sequence Alignment (https://www.ebi.ac.uk/Tools/msa/clustalo/).
       
Otx1B gene fragment sequences (obtained from NCBI) from some fish genera (Chlorurus, Scarus, Bodianus, Cirrhilabrus, Paracheilinus, Pseudolabrus, Oxycheilinus, Coris and Pseudocheilinus) were comparatively analyzed with sequenced Epinephelus samples (Table 1).
 

       
DNA polymorphism and nucleotide contents were calculated using DNAsp.Ver.5.10.01. (http://www.ub.edu/dnasp/DnaSP_OS.html).
       
The phylogenetic relationship among the evaluated fish species was reconstructed using two methods (Maximum Likelihood and Neighbor-Joining methods). The final data were presented using the maximum likelihood method.
       
The evolutionary distances were computed using the maximum composite likelihood method. All evolutionary analyses were conducted in MEGA6 (Tamura et al., 2013).

General DNA polymorphisms
 
The PCR products (approximately 700 bp fragments of Otx1B gene) were visualized on the agarose gel (Fig 1).
 

       
After sequence trimming, the 663 bp fragment sequences of the Otx1B have been identified in five Red Sea Epinephelus species (E. areolatus, E. malabaricus, E. summana, E.radiatus and E.chlorostigma). These results were compared with other Otx1B gene sequences from different fish species (sequences were obtained from NCBI). All evaluated fish species, accession numbers and codes were presented in Table 1.
       
The sequences were aligned for detecting the variable positions among the evaluated fishes.
       
A total of 62 Otx1B gene fragment sequences from 56 fish species belonging to 10 fish genera (Epinephelus, Chlorurus, Scarus, Bodianus, Cirrhilabrus, Paracheilinus, Pseudolabrus, Oxycheilinus, Coris and Pseudocheilinus) were analyzed. The DNA polymorphisms among all evaluated fish species were detected (Table 2).
 

       
The average values of GC (0.644), GC₂ (0.626) and GC₃ (0.789) were calculated. The number of haplotypes (nh=56), single nucleotide polymorphism (SNPs=140), estimates of haplotype diversity (hd=0.997), nucleotide diversity (Pi=0.063), theta from polymorphic sites (θ=0.051), average number of nucleotide differences (ka=41.86), conservation threshold (CT=0.8) and sequence conservation value (SC=0.787) were calculated for overall evaluated fish species.
       
The consensus sequence for each fish genus was detected. The variable nucleotide sites (108) of Otx1B consensus sequences (663 bp) in all evaluated fish genera were presented in Fig 2.
 

 
DNA polymorphisms in the estimated fish genera
 
The number of haplotypes (nh), single nucleotide polymorphism (SNPs), estimates of haplotype diversity (hd), nucleotide diversity (Pi), theta from polymorphic sites (θ), average number of nucleotide differences (Ka), conservation threshold (CT) and sequence conservation value (SC) were calculated in each estimated fish genus (Table 2). Also, the results showed that the GC₂ values were not informative to differentiate among evaluated fish genera. On the other hand, GC and GC₃ values were variables. The lowest GC and GC₃ values were detected in Paracheilinus (g). The highest GC and GC₃ values were detected in Bodianus (e). Both GC and GC₃ values detected in Epinephelus (a) were higher than those detected in Cirrhilabrus (f), Pseudocheilinus (k), Coris (j), Oxycheilinus (i), Paracheilinus (g) and Pseudolabrus (h) fish genera. On the other hand, these values in (a) were lower than GC and GC₃ in the other evaluated fish genera (b), (c) and (e).
       
The numbers of haplotypes (nh) and theta from site (θ) values were variables in most of the evaluated fish genera. Both calculated (pi) and (ka) values in Epinephelus were higher than pi and ka values in genera Chlorurus (b), Scarus (c), Cirrhilabrus (f), Paracheilinus (g), Pseudolabrus (h), Oxycheilinus (I) and Coris (j). Both pi and Ka values in Bodianus (e) and Oxycheilinus (k) fish genera were higher than pi and ka in (a). The highest SNPs value was calculated in (e). A high genetic distance value was calculated within each Pseudocheilinus (k) and Bodianus (e) while the lowest genetic distance was calculated in Coris (j) relatively. The sequence conservation values were ranged from 0.967 (in Bodianus or e) to 0.997 (in Coris or j). The same Conservation threshold (CT) was detected in all evaluated fish genera.
 
Divergence among the evaluated Epinephelus species
 
The phylogenetic relationships among evaluated fish species are presented in Fig 3 and 4. The genetic distance values among the evaluated fish genera based on Otx1B gene consensus sequence variations are presented in Table 3. In addition, the constructed tree reflects the genetic distance among the estimated fish species. The analysis showed that fish samples are clustered into unique branches.
 

 

 

       
Concerning the Epinephelus species, the E. summana is closely related to E.  malabaricus. E. areolatus is distantly related to the cluster formed by E. chlorostigma and E. radiatus. The same genetic distance was calculated between E. summana and both E. radiatus and E. chlorostigma. The distance between E. areolatus and E. malabaricus is higher than the distance between E. areolatus and E. summana. The same distance was detected between E. malabaricus and both E. radiatus and E. chlorostigma.
 
Divergence among evaluated fish genera
 
The Epinephelus (a) is distantly related to the group formed by Chlorurus (b), Scarus (c) and Oxycheilinus (i). On the other hand, a low genetic distance value is calculated between Epinephelus (a) and Bodianus (e). The same distance value was detected between Epinephelus (a) and three fish genera Cirrhilabrus (f), Paracheilinus (g) and Pseudocheilinus (k). The distance value between Epinephelus (a) and Pseudolabrus (h) is lower than the distance value between Epinephelus (a) and Oxycheilinus (i). The lowest distance value was detected between Chlorurus (b) and Scarus (c). Oxycheilinus (i) is closer to Chlorurus (b) than to Scarus (c). The distance between Pseudocheilinus (k) and Cirrhilabrus (f) is higher than the distance between Pseudocheilinus (k) and Paracheilinus (g).
Epinephelus species were attractive for the study of taxonomy and evolution comparatively with the other ray-finned fishes (Liu et al., 2013; Yu et al., 2018).
       
In the present study, the utility of Otx1B gene sequence variations for evaluating the evolutionary variations in some Red Sea, Epinephelus species compared with some other fish taxa were evaluated.
       
A group of studies confirmed that the analysis of regulatory loci (such as Otx1B gene) sequence variations was informative for calculating the genetic distance values and exploring the evolutionary variations among fish taxa (Santini and Bernardi 2005). Indication exists of main variations in the regulatory genes (Bmp4, Otx1 and Dlx2) that could be produced vital evolutionary variations in body plan and morphology (Smith et al., 2008).
       
Analysis of Otx1B gene sequence variations was efficient for detecting and exploring the evolution among all evaluated fish taxa (56 fish species).
       
The estimated DNA polymorphisms explored the genetic differences in each evaluated fish genus. The GC, GC₂ and GC₃ were calculated due to their values in predicting the mutational forces in the genus Epinephelus comparatively with the other evaluated fish genera. The calculation of such parameters for exploring the evolutionary variations among different animal taxa was recommended by many authors (Popovic and Stevanovic, 2009; Saad and El-Sebaie, 2017; Redwan et al., 2018).
       
As revealed from our results, nucleotide changes at the third codon position were higher than the second and first codon position in all estimated genera. The same observation was confirmed during barcoding of the Australian marine fishes using COI as a universal animal barcoding system (Ward et al., 2005). We found that the calculation of GC and GC₃ values were informative for detecting the molecular variations within each evaluated genus comparatively with the other fish genera. The GC₃ values were high in all evaluated fish genera. This finding supported that this nuclear gene is highly transcript and reflecting the vital biological role in all evaluated fish taxa.
       
All evaluated fish genera were varied in averages of single nucleotide polymorphism, nucleotide diversity, the average number of nucleotide differences. Most applied DNA polymorphism parameters were informative in exploring the molecular diversity in the evaluated fish genera. This finding was confirmed during the evaluation of the molecular diversity in some marine Crustacean species (Saad and El-Sebaie, 2017). The calculation of such parameters was also recommended for exploring the molecular variability among some camel populations (Redwan et al., 2018).
       
The phylogenetic relationship among the evaluated fish species was reconstructed using two methods (Maximum Likelihood and Neighbor-Joining methods). No topology variations were observed between the revealed trees. Some other methods such as Maximum parsimony and UPGMA methods were widely used for the reconstruction of the phylogenetic relations among fish taxa. The Maximum parsimony, UPGMA and Neighbor-Joining methods were applied for exploring the phylogenetic relations of the Prickly shark. The three methods gave the same tree topology also (Giacomo and Dennis, 1992).
       
The genetic divergences among the evaluated Epinephelus species were reflected by the calculated distance values on the reconstructed phylogenetic tree.
       
The phylogenetic relations among some Epinephelus species were constructed based on isozyme polymorphism (Deepti et al., 2014). The estimated fish species were clustered into two branches. The first branch included E. coioides, E. malabaricus and E. tauvina. The E. angularis, E. bleekeri, E. chlorostigma and E. longispinis constituted the second branch. This study separated E. chlorostigma from E. malabaricus. This result is similar to our findings based on Otx1B sequence variations. In addition, the same distance was detected between E. malabaricus and both E. radiatus and E. chlorostigma.
 
The resolution of the phylogeny between E. areolatus and the other fishes were visualized by the variation in the Otx1B data set within the genera.
 
Some fish genera were deeper divergence than others. Each of Epinephelus and Bodianus is distantly related to all the other evaluated fish genera. On the other hand, a low genetic distance value is calculated between Epinephelus and Bodianus.
       
The lowest divergence was detected between the two genera Chlorurus and Scarus. This finding was supported before using other different dominant and co-dominant molecular markers (Smith et al., 2008; Saad et al., 2013). The genus Oxycheilinus constitute a sister branch with the group formed by the two genera Chlorurus and Scarus.
       
Mutations in regulatory loci lead to made evolutionary differences in the body of organisms (Smith et al., 2008).
       
The analyses of our results are providing a suitable system for studying evolution and speciation among and within the evaluated fish taxa.

The utility of the Otx1B gene sequence variations for the identification of some Red Sea Epinephelus species (E. areolatus, E. malabaricus, E. summana, E. radiatus and E. chlorostigma) comparatively with other ray-finned fishes was evaluated. The Otx1B gene can be easily amplified for studying fish characterization and evolution purposes. All identified fish species can be differentiated by Otx1B gene sequences. The results confirmed that analysis of Otx1B gene sequence variations was a suitable tool to both estimate levels of genetic diversity and to detect genetic polymorphism in the evaluated fishes. This study leads to new inferences regarding the management and conservation of the evaluated fish species. Comparison of data generated in the current study will make it possible to understand the evolutionary variations in these fish genetic resources comparatively with other fishes.
       
Identification of more SNPs in Epinephelus genomes will promote the advancement of marker-assisted selection (MAS) in such economic fishes. Also, these molecular markers will be the basic principle for quality improvement in such fishes in the future. 

This project was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, under grant No. (40-130-35-RG). The authors, therefore, acknowledge with thanks DSR for technical and financial support.