Molecular Phylogenetic Taxonomy of Some Parrotfish Species (Perciformes, Scaridae) From the Red Sea Using -Actin Gene

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INTRODUCTION
Teleosts have developed unique features in the structure and physiology of muscles during their evolutionary history, Muscle growth in teleosts is signified by property phenomenon, the increase in fiber number as well as increase in fiber size, which is not found in other vertebrates (Kiessling et al. 2006 andJohnston et al. 2011).Such adaptive processes have appeared in modifications of the genetic pathways modulating muscle growth and functions in fish (Mudalige et al. 2007).Furthermore, metabolic and contractile characteristics of teleost muscles also, represent significant flexibility concern to environmental conditions as temperature and food supplying (Johnston 2006 andJohnston et al. 2008) and reproductive status as gonad maturation (Mathana et al. 2012).Thus, identification of genetic determinants for muscle-specific genes from fish represented an important basis to study the evolutionary and diversification of the musculature in the fish's lineage.
Actin is one of the major components of muscle tissues.Actins play important roles in maintaining cytoskeletal structure, cellular mobility, cell division and differentiation, intracellular movement, and contractile processes, which are associated with a wide range of physiological aspects in fish and all vertebrates, thus actins an evolutionarily conserved protein (Perrin andErvasti 2010 andLee et al. 2017).The genome of vertebrate species usually contains six different actin genes, four of these genes code for muscle isoforms (skeletal muscle, -cardiac muscle, vascular and -enteric muscles) and two other genes code for (the gama-and betacytoplasmic) types (Vandekerckhove andWeber, 1979, Kusakabe et al.,1997).In spite of Muscle actin gene has three isoforms (-skeletal, -cardiac, -vascular and -enteric), these isoforms share remarkably high sequence identity one another, each isoform exhibits distinct regulation pattern for its spatial and temporal expression (Adriane et al., 2007, Perrin and Ervasti 2010, Glasauer and Neuhauss 2014, and Lee et al. 2017).Order Perciformes, is comprises 156 recent families.Family Scaridae (parrotfish) is a distinctive group of order perciformes.The Scaridae is relatively small family, with 90 species in ten genera.It comprises small to large species with maximum adult sizes ranging from 110 to 1000 mm (Streelman et al., 2002).Scarid fishes are present in the Red Sea, tropical Atlantic and Indian and Pacific Oceans.Despite the economic importance of the family, the systematics of the Scaridae has been in a state of confusion for many years (David and Bellwood 1994) (Jordan and Gilbert 1882) designated Scarus psittaeus as the species of the genus Scarus.The Scaridae was first recognised as a distinct family by (Bleeker 1859b) who later, in 1862, provided detailed descriptions of the two families: the Scaroides (parrotfishes) and the Labroides.Prior to this classification, the two groups were both placed in a single family, the Labridae (Cuvier and Valenciennes, 1840;Kner, 1860;Gunther, 1862).However, the actual identity of this species was not resolved until 1978 (Randall and Ormond 1978).A number of recent studies have presented evidence which indicates that the Labridae, Odacidae and Scaridae represent a monophyletic assemblage (Liem andGreenwood, 1981, Stiassny andJensen 1987), and if the Scaridae represents a monophyletic group, then the immediate sister group of the Scaridae must be contained within the Labridae or Odacidae.In other studies, the genus Scarus appeared to be paraphyletic by (Bellwood 1986) and, subsequently, by (Bellwood andChoat 1990) and they were suggested that the genus Scarus was comprised of two distinct phyletic lineages.These two groups were identified as separate functional groups.There are numerous taxonomic problems associated with many groups of Perciformes (Nelson 2006).
Thus, it is obvious that much molecular taxonomy and phylogenetic research on Scaridae and other percoid families remain to be conducted.In this study, we focus on the Subfamily Scarinae discuss 6 species and compared to 14 representatives of other families of percoid fishes by molecular phylogenetic and taxonomic considerations using skeletal muscle actin sequence data.Two aims were considered: 1) whether the Subfamily Scarinea is monophyletic, and 2) whether the nucleotide diversity at skeletal muscle actin gene supports the currently accepted intra-family, intrasubfamily and intra-genus divisions.

MATERIAL AND METHODS a. Samples:
Six fish's species belonging to Subfamily Scarinea, Family Scaridae , Order Perciformes were collected from the Red Sea.First, the tested fish's species were identified morphologically according to (Randall, 1982) (Table 1).Skeletal muscles tissues were isolated from the collected fish samples and were preserved in -80ºC until used.b.DNA Isolation: Total genomic DNA was isolated from skeletal muscles of six species of Scarinea fishes using QIAamp DNA Mini kit (Qiagen, Hidden, Germany) following the manufacturer's protocol.

c. Primer and PCR Conditions:
To get the alpha actin gene sequence data of the six specimens.First, we used PCR to obtain alpha actin segments with the set of 2 primers (forward and reverse) Actin F1 (5'-GTA TTG TGC TGG ACT CTG GTG-3') Actin R1 (5'-GAA GCA CTT GCG GTG GAC GAT-3').The forward and reverse primers were designed from the -actin gene sequence described for the fish Ictalurus punctatus (Kim et al., 2000).PCR reactions were carried out with 12.5 µL PCR master mix (Qiagen, Hidden, Germany), 0.5 µL (10 pmoles/ µL) of each primer and ~100 ng of genomic DNA in a final reaction volume of 25 µL, the two primers that were used in this study.PCR reaction was performed as following, an initial denaturation: 95 ºC for 2 minutes, followed by 34 cycl 30s at 95 ºC, annealing: 55ºC for 30s and extension: 1 min at 72 °C plus a final 5 min extension at 72 °C.d.Gel Electrophoresis: The PCR products were electrophoresed in 1% agarose gel, stained with ethidium bromide and visualised under UV light.Electrophoresis analysis induced a single segment with each species that indicate the alpha actin gene segment, the fragments of the gene amplified is approximately 900 bp (Fig. 1), which is similar with the fragments length of the primer expect.
Table 1: Classification of understudying parrotfish and related percoid species were retrieved from the Gen Bank/ NCBI and references.When good PCR products were obtained, each sample was purified before sequencing.The purified products then were sent to Macrogen Company to make standard sequencing, using both forward and reverse -skeletal muscle actin gene (ACTA 1) segment of nuclear DNA were established for 23 fish.All analyses, starting with new DNA extraction, will be repeated for this specimen.Nucleotide sequences from the six parrotfish specimens were compared and aligned among themselves (Fig. 2) and in respect to other sequences present in databases using the Clustal W computer software package.The sequences were aligned using the multiple-alignment algorithm in Megalign (DNASTAR, window version 3.12e) and MEGA version 7.0 18 (Kumar et al., 2016), then the alignments were refined manually and all gaps were then deleted manually.During alignment, several sequences were removed from further analysis because of their low proximity to the bulk of the data, most sequences removed were those from GenBank that did not fit the length limit of our samples.The multiple aligned dataset generations involved both pair wise and progressive alignments based on a guide tree.Five approaches were used for alpha actin tree building, Neighbour-joining (NJ) (Saitou and Nei 1987), Minimum Evolution (ME) (Rzhetsky 1982), Maximum Parsimony (MP) (Fitch 1971), Maximum Likelihood (ML) (Takahashi and Nei 2000) and Bayesian inference method (BI) (Huelsenbeck and Ronquist, 2001), trees using Kimura 2-parameter distances were created to provide a graphical representation of the pattern of divergence between species, using Verify the robustness of the internal nodes, bootstrap analysis was carried out using 1,000 replicates (Felsenstein, 1985).RESULT alpha-actin gene was studied as a potential phylogenetic marker for six species of parrotfish from family scaridae, subfamily scarinea include three genus (Scarus, Chlorurus, and Hipposcarus).Two primers; the forward and reverse primers were used to amplify the particular target region of all the species.Alpha skeletal muscle actin genes (ACTA 1) are amplified to give the general view about the molecular genetic relationships among the understudying species and other related species of Order Perciformes depending on available sequence data from gene bank/NCBI.The genomic DNA of parrotfish (Hipposcarus harid, Scarus niger, Scarus fuscopurpureus, Scarus ferrugineus, Scarus psittacus and Chlorurus gibbus) were generated a single segment (Fig. 1), that indicate all the species showed different alpha nucleotide sequences and hence successfully barcode the region, the nucleated sequence lengths were of approximately (889, 902, 696, 905, 889 and 880bp respectively).No insertion/deletion or stops codon was found, supporting the view that all of the amplified sequences constitute functional alpha actin gene sequences.

Genetic distance :
For the sequences of six parrotfish species, the average nucleotide frequencies of thymine (T), cytosine (C), adenine (A) and guanine (G) were 22.2 %, 30.3 %, 23.9 %, and 23.1 % respectively, and were varied between 20.9 and 31.9 %.The average content of G+C (53.4 %) was higher than that of A+T (46.6 %) (Table 3).Pair wise genetic distances among the twenty percoid species and the three outgroups were estimated by MEGA 7 (Kumar et al., 2016) using the Kimura twoparameter model (Kimura 1980) with gamma correction (Table 4), that assumes transition mutations should occur more often than transversion.Pairwise genetic distances among and within the sex-species and the outgroup, Polyodon spathula, were estimated by MEGA (Kumar et al., 2004) using the K2P method with gamma correction.The P-distance between the understudying fishes was lowest between Scarus niger and Scarus fuscopurpureus (79.9%) and highest between Scarus psittacus with Chlorurus gibbus and also with Hipposcarus harid (99.8%).The results indicate also, the gene of Scarus ferrugineus genotype was the longest (905 bp), and the Scarus fuscopurpureus gene was the shortest (696 bp).The Scarus fuscopurpureus fish was AT rich, with A+T contents of 52.4%, however, the A+T contents of the rest species were quite similar which were (46.8, 45,8, 44.7, 45.2, 44.4 and 46.6%) (Table 3).

Phylogenetic Analysis:
Sequence data reported from sequencer followed by the corrected raw sequence (Fig 2 ), before it recorded for the first time in GenBank with accession numbers (MH203325, MH203326, MH203327, MH203328, MH203329 and MH203330) for the six understudying species (Hipposcarus harid, Scarus niger, Scarus fuscopurpureus, Scarus ferrugineus, Scarus psittacus and Chlorurus gibbus respectively).For phylogenetic purposes, the six scarus alpha-actin gene sequences were subjected to an analysis together with 14 of other percoid sequences representing all the available and appropriate species of four families (one species Scarus iseri of family scaridae, ten of family Labridae, eight of Labrus bergylta species, Thalassoma bifasciatum and Parajulis poecilepterus, two of Nototheniidae Notothenia coriiceps and Trematomus bernacchii and one of Serranidae Epinephelus coioides), that were taken for the comparative purposes and retrieved from Gen Bank/NCBI, in addition to 3 outgroup sequences from order Scoraeniformes (Teleosti) (Sebastes schlegelii Sebastes inermis Sebastiscus marmoratus) (Table 1).All 23 nucleotide sequences (including outgroups), are aligned (Fig. 2).The genetic distances and genetic divergences among all sequences of the six tested species of parrotfish with the available species of the order perciformes together with three species as outgroup are calculated (Table 4).
The molecular phylogenetic trees were constructed based on the alpha actin gene sequences of six parrotfish species and the fourteen available percoid species from Gene Bank/NCBI together with the out group species to determine the root of the trees.The dataset was analyzed with Neighbour-joining (NJ), Minimum evolution (ME) these analysis were performed by the MEGA version 7.0.18software, the, Maximumparsimony (MP) and Maximumlikelihood (ML) these analysis were performed by the MEGA version 7.0.18software and PAUP (version 4.0a150), ML analysis was based on the Akaike Information Criteria (AIC) test in MrModel test 2.3.Bayesian inference (BI) algorithms, phylogenetic analysis by BI utilising Monte Carlo Markov Chain (MCMC) analysis in MrModeltest 2.3.(Fig. 3-5).The robustness of the tree was corroborated with bootstrap analyses (Bootstrap value=1.000).The results showed, all phylogenetic trees obtained are widely based on the same topology with Scarus, Labrus and other percoid species.Bootstrap support ranges from > 50 to 100% under all tree-building methods.In tested scarinae species, the maximum genetic divergences have occurred between the Scarus niger and Scarus fuscopurpureus (22.5%) whereas the maximum p-distance was observed between the Scarus ferrugineus and the two species, Scarus psittacus and Scarus gibbus (99.8%) (Table 4).To establish the relationship between sex tested fish and other available perciforme species, the alpha actine gene sequences of scarid sprecies were aligned with the previously published sequences of 14 species of Order Perciformes, one species of family scaridae, S. iseri (  With simple exclusion to these data, it can be observed that tested species of family Scaridae form one distinct clade, Scarus fuscopurpureus is presente in all trees in a distinct separated clade with Labrus bergylta with weak support except strong support in (BI=95), without any interrelationship with scarus clade, it has a strong support with labrus species in BI tree and weak support with the rest trees that indicate it not fully resolved.According to the resulting phylogenetic trees, it is observed that both families Labridae and Nototheniidae and Serranidae are very close to each other.In all trees family Scaridae forming the basal clade to other families of order perciformes.Trees was drawn with amino acid sequences of selected α-actin isoforms using MEGA7 software (ver.7.0.18).including three species of order Scoraeniformes as outgroups.ach node was tested by bootstrap method (1000 replicates), and only bootstrap values of 50% or above were shown here.The present paper will help in support new paths to the future of systematic Ichthyology, and probably in resolving evolutionary relationships.Our study for alpha-actin gene was provided several quite long sequences for the gene alpha actin of the tested samples, which allowed us to make a molecular taxonomic and phylogenetic evaluation for tested fishes on this basis.However, taking into consideration the large diversity in Scarus fuscopurpureus species.Five different tree building method (NJ, ME and MP, ML and BI) constructed using Kimura 2-parameter distance shows Hipposcarus harid species being having the basic and longest branch length that it consideres the most ancestral of all species considered, while the

Notothenia coriiceps/
Trematomus bernacchii form one clad in the all trees that indicate they belong to a separate family that close to family Labridae than family Scaridae.To our knowledge, the monophyletic origin of most families in the order Perciformes has not yet been investigated thoroughly.DISCUSSION The present study compared six parrotfish samples obtained from the Red Sea in Egypt, both within the samples and with other previous studies by means of a sequence analysis of the alpha actin gene.Approximately 900 base pair (bp) fragments of the nuclear DNA alpha skeletal muscle acten gene were amplified and then sequenced from the tested samples, and 14 samples of other family of Order Perciformes and three outgroup from Order Scoraeniformes (Teleosti) were obtained from Gene Bake/NCBI to use in this study.The current study revealed that the A+T nucleotide ratio for the six species Hipposcarus harid, Scarus niger, Scarus fuscopurpureus, Scarus ferrugineus, Scarus psittacus and Chlorurus gibbus are (46.8, 45.8, 52.4, 44.7, 45.2, 44.4 and 46.6) respectively.Scarus fuscopurpureus that formed a complete separate clade far from the rest of scarus species showed a high A+T bias that may be confirms a common phenomenon in fish, a high in an A+T nucleotide bias, when present this tends to accumulate in hyper variable sites (Simon, 1991 andÇiftci 2013).

Phylogenetic Analysis:
Our results In agreement to the known phylogenetic positions of perciformes, the phylogenetic relationships of the species in this order are still controversial (Kaufman and Liem, 1982, Bellwood 1986, Stiassny and Jensen 1987, Smith et al., 2008  On the other hand, In BI tree only Scarus psittacus formed a basal clade with Hipposcarus harid clad with strong support (BI=100) this finding in agreement with (Smith et al., 2008) who reported Scarus psittacus is the sister to the rest of the Scarus clade in tree, and it is one of the most broadly distributed species among all parrotfish, occurring from the the Red Sea to South Africa, all samples of our study were collected from the Red Sea.Furthermore, all our trees pattern revealed Scarus fuscopurpureus is fused with Labrus bergylta In a separate clade far from Scarus species, that indicate Scarus fuscopurpureus is very close to family Labridae than family Scaridae, this result in support that reported by (Kaufman &Liem, 1982 andStiassny &Jensen, 1987) those authors reported, it is widely accepted that the Labridae and Scaridae represent a monophyletic assemblage.Some workers followed (Kaufman and Liem 1982) included the Scaridae in the Labridae, eg, (Stiassny and Jensen 1987).Others questioned this decision.On the other hand, (Richards and Leis 1984) cautioned against the fusion of the two families, based on observations on the early life history characters of the two families.(Bruce and Randall 1985) retained the Scaridae arguing that they are unique.Other studies suggested, the genus Scarus appeared to be paraphyletic, and it was suggested that the genus was comprised of two distinct phyletic lineages.These two groups were identified as separate functional groups by (Bellwood, 1986) and, subsequently, by (Bellwood andChoat 1990), this distinction also appeared to have a phylogenetic basis (Stiassny and Jensen 1987).To our knowledge, the monophyletic origin of most families in the order Perciformes has not yet been investigated thoroughly, there is a need for further examination of this question (Smith and Craig 2007).
Our results support the previous studies that suggested that The high degree of similarity among the nucleotide sequence and amino acid residues of the actin gene of scaride species and those of other percoid species is, unsurprisingly, not only a consequence of the narrow evolutionary divergence between several species but is also due to the origin of the actin genes.It seems that actin isoforms are encoded by a set of structurally related genes that developed as a consequence of gene duplication followed by functional divergence (Hightower and Meagher, 1986), resulting in highly conserved proteins.

Conclusion:
The present study strongly supports the monophyly of the Scaridae and indicate that the traditional characters are inadequate to recent classification.To our knowledge, the monophyletic origin of most families in the order Perciformes has not yet been investigated thoroughly.The actin gene proved to be a valuable phylogenetic marker, thus the investigation of more genes e.g.actin gene is required for the further development of a natural classification of the perciformes.In five resulting trees (Figs 3-5) Hipposcarus harid clade is a distinct clade which basal to all other subfamily scarinea and they are resolved as a monophyletic clade.Scarid clad is represented in all trees as a distance separated clad without interrelationships with other clads and have a strong support.Chlorurus gibbus is fused with scarus species in one clade of all trees that indicate it close to Scarus species and we prefer the name is Scarus gibbus than Chlorurus gibbus as named previously.
The species Scarus fuscopurpureus are closely related to Labrid species and well separated from the Scarid species, this gives an indicator that Scarus fuscopurpureus share Labrid species in more characters than Scarus species.

Fig. 1 .
Fig. 1.Electrophoretic analysis of PCR products of the alpha skeletal muscle actin fragments.Lanes 1 -6 PCR products for tested fish samples (Hipposcarus harid,Scarus niger, S. fuscopurpureus, S. ferrugineus, S. psittacus and S. gibbus respectively) and M, molecular weight marker .e. Sequencing of PCR Products: Alignment report -Actin Gene nucleotide sequences of the six parrotfish species, Photo.(a, b, and c).Dots indicate identical nucleotides and A,T,C and G indicate the difference nucleotides f.Sequence Alignments: BI tree (Fig. 5) exhibit Polyphyletic group of the tested scaridae species (Scarus niger and Chlorurus gibbus formed the sister clade, whereas Scarus psittacus formed the basal clade to the Scarid species, Hipposcarus harid formed the basal clade for the species (Scarus niger and Chlorurus gibbus and Scarus ferrugineus).Scarus fuscopurpureus formed a separate basal clade together Labrus bergylta clade with strong support 95%.The non-members of the family Scaridae as family Labridae (members of the genera labrus, Thalassoma and Parajulis), family Nototheniidae (members of genera Notothenia and Trematomus) and family Serranidae (genus Epinephelus) in Figs 3 (a and b) exhibit a polytomy (9-way polytomy) while and Fig. 4 (a and b) and (Fig 5) formed 10 polytomy.These polytomy contain some resolved relationships of acceptable to strong support values as follwing: Thalassoma/ Parajulis (NJ=69, ME= 51, Mp=100, ML= 68 and BI=94), genus Notothenia coriiceps/ Trematomus bernacchii with strong support (NJ=99,ME=99, MP=100, ML=97 and BI=94) and Epinephelus coioides/ Labrus with somewhat strong support (NJ=65, ME=71 and MP=100 and ML=70) and in BI tree Epinephelus coioides is separate and formed a basal clade to other percoid species.Weak support values which observed in the some clades may represent an indicator that the relation among or within these clades either not fully resolved or more information is needed.

Table 4 .Fig. 3 .
Fig. 3. Phylogenetic trees showing the genetic relationships among members of the Scaridae sequences of the -actin gene (ACTA 1) and with other 14 percoid species (a) Neighbor-Joining (NJ) and (b) Minimum Evolution (ME).Trees were drawn with amino acid sequences of selected α-actin using MEGA7 software (ver.7.0.18).including three species of order Scoraeniformes as outgroups.Each node was tested by bootstrap method (1000 replicates), and only bootstrap values of 50% or above were shown here.

Fig. 4 .
Fig. 4. Phylogenetic trees showing the genetic relationships among members of the Scaridae sequences of the -actin gene (ACTA 1) and with other 14 percoid species (a) Maximum Parsimony (MP) and (b) Maximum-Likelihood (ML) method.Trees was drawn with amino acid sequences of selected α-actin isoforms using MEGA7 software (ver.7.0.18).including three species of order Scoraeniformes as outgroups.ach node was tested by bootstrap method (1000 replicates), and only bootstrap values of 50% or above were shown here.

Fig. 5 .
Fig. 5. Phylogenetic relationships among members of the Scaridae inferred from actin gene (ACTA 1) following analysis using Bayesian Inference(BI) method.The numbers above or below branches indicate bootstrapping values (1000 Replications).The percentage of replicate trees are indicated for all nodes.
Scarus fuscopurpureus forms completely separate clade indicating this species has highly diverged from the rest of the scarus species (Fig 3-5) and may share advanced character with Labrus species and may be primitive characters with Scarus species.Our own data show that the investigated representatives of the family Scaridae formed a monophyletic clade.The BI tree showed the difference compared to the NJ, ME, MP and ML trees mostly in somewhat there is deference in the position of Scarus psittacus and Hipposcarus harid in the Scarus clade.Nevertheless, all trees showed 4 major properties: (1) a separate cluster for outgroup species, (2) Scarus clade forming basal clade for the rest families of perciformes.3) Scarus fuscopurpureus formed a separate clade with Labrus bergylta far from scarus species that indicat Scarus fuscopurpureus more close to genus Labrus than genus Scarus.4) Epinephelus coioides also, form a clade with Labrus bergylta that indicate it close to genus Labrus.
Epinephelus coioides (AY 735013.1)togetherwiththreeout group (Table2).For comparative analysis, sequences from the GenBank were combined with the sequences of the present study and used to infer the phylogenetic relationship of scarinae species of the Red Sea.For the analysis, three representatives of order Scoraeniformes was used as the outgroup, Sebastes schlegelii, Sebastes inermis and Sebastiscus marmoratus with accession no.(JN 226152.1,JN226153.1  and HQ 906886.1)respectively.

Table 2 :
Species and accession numbers of Actin gene sequences of understudying species (6 parrotfish species) and that obtained from gene bank (13 percoid species) together with outgroup species (3 of Order Scoraeniformes, Teleosti).S. muscle = skeletal muscle C. muscle = Cardiac muscle

Table 3 .
Average of base composition (A, T, C and G) percentage in 6 parrotfish species for alpha actin sequence.