V.A. Iswarya Deepti, K.V.L. Shrikanya & K. Sujatha* Indian Journal of Geo-Marine Sciences Vol. 43(12), December 2014, pp.

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1 Indian Journal of Geo-Marine Sciences Vol. 43(12), December 2014, pp Taxonomic studies and phylogenetic relationship of seven spotted groupers species of genus Epinephelus (Pisces: Serranidae) off Visakhapatnam, middle east coast of India V.A. Iswarya Deepti, K.V.L. Shrikanya & K. Sujatha* Department of Marine Living Resources, Andhra University, Visakhapatnam India. * [E.mail: sujatha.mlr@gmail.com] Received 21 November 2013; revised 25 February 2014 Present study consists the meristic, morphometric characters and colour pattern that are used to identify seven spotted grouper species: Epinephelus coioides, E. malabaricus, E. tauvina, E. angularis, E. bleekeri, E. chlorostigma, E. longispinis that are represented in the catches of Visakhapatnam. Biometric measurements are subjected to multivariate analysis (Principal Component Analysis) to distinguish each species and to infer relationship of the closely related species. In addition to traditional taxonomic work, allozyme based survey of genetic variation in these seven species has also been carried out. Eleven enzyme systems screened yielded twenty six scorable loci. Average heterozygosity ranged from to The results yielded species specific diagnostic markers in the above species. UPGMA dendrogram revealed two main branches where Epinephelus coioides, E. malabaricus and E. tauvina formed one cluster and E. angularis, E. bleekeri, E. chlorostigma and E. longispinis formed another cluster. [Key words: Seven species of Genus Epinephelus, biometric measurements, multivariate analysis, allozyme electrophoretic studies] Introduction It is essential for proper management of fishery resources to correctly identify the species concerned. Some grouper species are commercially cultivated in Thailand, Malaysia, Hongkong, Singapore, Philippines, Taiwan and Kuwait 1,2. For any culture practice, taxonomy is the basis as misidentification leads to collapse of the entire culture system. Phylogenetic relationships among grouper species were studied and an aggressive reorganization of genera within groupers was proposed 3. They stated that reticulated groupers (E. fasciatus species group) form a monophyletic group whose affinities have been discussed based largely on colour pattern. In India studies on taxonomic relationships were carried out using RAPD markers among seven species of groupers belonging to genus Epinephelus 4 and allozyme variation in four species of groupers of genus Epinephelus was done 5. Although some of the recent studies have helped to clear the status of some nominal species, confusion still surrounds the identification of many common species of groupers. A qualitative analysis adds greatly to the precision of a description 6. To deal with more than two variables, multivariate data analysis like Principal Component Analysis (PCA) must be employed to closely related species 7. Hence Principal Component Analysis (PCA) has been carried out to identify the influential variables that aid in distinguishing closely related species of groupers. The present paper deals with meristic and morphometric characters and colour pattern that has been utilized to identify seven spotted grouper species represented in the catches of Visakhapatnam. In the present study biometric measurements were subjected to multivariate analysis (Principal Component Analysis) in closely related species of genus Epinephelus. Understanding the present genetic makeup of wild grouper populations has a significant practical value particularly for fishery management and conservation of stocks. So far no attempt has been made to analyse the genetic structure of groupers of this region. Hence it was thought worthwhile to carryout allozyme based survey of genetic variation in seven species of groupers in addition to traditional taxonomic work. Material and Methods A total of 289 individuals of all size groups belonging to seven species of spotted groupers of genus Epinephelus: E. coioides, E. malabaricus, E. tauvina, E. bleekeri, E. angularis, E. chlorostigma and E. longispinis were collected from traditional fish landing centres, local markets and fishing harbour of Visakhapatnam

2 ISWARYA DEEPTI et al.: TAXONOMY AND PHYLOGENETIC RELATIONSHIP OF SEVEN GROUPER SPECIES 2255 (lat17 44ˊN; long 83 23ˊE). The colour of the specimens was noted in fresh condition and morphometric and meristic data of specimens of all size groups represented in the catches is taken to study any existing variation in biometric characters. Methodology for taking morphometric and meristic data follows standard procedures 8. Based on a combination of number of rows of teeth on upper jaw, number of dorsal fin spines, number of pectoral fin rays and colour, the seven spotted grouper species can be distinguished into two groups. Group I - E. coioides, E. malabaricus and E. tauvina; Group II - E. angularis, E. bleekeri, E. chlorostigma and E. longispinis. A qualitative multivariate data analysis, Principal Component Analysis (PCA) is employed to distinguish the closely related species of above two groups. Based on 26 morphometric and six meristic characters, PCA was performed. Because of the variation in size of fish, morphometric data was statistically adjusted to permit comparative analyses in terms of shape independent of size 9. This allometric adjustment effectively removes size variation from the data examined 10. Meristic characters were log transformed. A PCA for the allometrically adjusted and log transformed measurements factoring correlation matrix was performed using SPSS version 13.0 software. Tables showing factor loadings resulting from PCA followed by results of Tukey test only for those characters that became significantly different for morphometric and meristic characters are given. For allozyme electrophoretic studies, after identifying the species, total length, weight and sex of each specimen is noted and muscle tissue is immediately brought to the laboratory in an insulated ice box with crushed ice inside. Methodology for allozyme extraction, casting of gels follows standard procedures 11. For separation of allozymes at different enzyme loci vertical native PAGE gels were used and electrophoresis was done at a constant voltage of 4 C. A total of eleven enzyme systems were examined in 147 individuals of seven grouper species. Visualization of different alleles of enzymes such as Acid phosphatase (ACP, E.C.no ), Aspartate aminotransferase (AAT, E.C.no ), Alcohol dehydrogenase (ADH, E.C.no ), Esterase (EST, E.C.no ), Glucose -6 Phosphate dehydrogenase (G6PDH, E.C. no ), Glutamate dehydrogenase (GDH E.C.no ), Glutamine synthetase (GLNS E.C.no ), Lactate dehydrogenase (LDH E.C.no ), Malate dehydrogenase (MDH, E.C.no ), Phosphogluco Mutase (PGM, ) and Superoxide dismutase (SOD, E.C.no ) was done by histochemical staining 12. Nomenclature of loci and alleles follow standard procedures 13. At all the loci, most common allele was designated as 100. Alternate alleles designated as per their mobility in relation to the most common allele. Calculation of allele frequencies and test for conformity to Hardy- Weinberg expectations (probability test) were undertaken using GENEPOP version 3.4 software 14. Cluster analysis was performed and dendrogram plotted based on pairwise genetic distance estimated using the Unweighted Pair Group Method with arithmetic mean (UPGMA) 15 modified from NEIGHBOUR procedure of PHYLIP version 3.5 using POPGENE version Results Brief description and colour of seven species of groupers of genus Epinephelus provided below. Frequency distribution of meristic characters of the seven species in pooled samples given in Table 1. Morphometric data for the three species Epinephelus coioides, E. malabaricus, E. tauvina (Fig. 1,2,3) given in Table 2 and for four species E. angularis, E. bleekeri, E. chlorostigma and E. longispinis (Fig. 4,5,6,7) given in Table 5. Epinephelus coioides (Hamilton, 1822) - Orangespotted grouper: Dorsal Rays: 14-15; Pectoral rays: 17-20; Maxilla reaches beyond the posterior border of the eye; maxillary scaled; Two rows of teeth on upper jaw; Post orbital % in HL; Preopercle having notch at its angle; Pyloric caeca Colour: Head and body tan dorsally, shading to whitish ventrally. Body with five irregular bars which tends to bifurcate, ventrally bars may be broken into series of two large blotches. Orange spot become poorly defined and darker with growth. First dark bar below anterior dorsal spine; second bar below seventh to ninth dorsal spine; third bar below the origin of soft dorsal ray to anus; fourth bar start from ninth dorsal ray to anal ray; fifth bar on caudal peduncle. Two dark spots on interopercle and one dark spot at the junction of sub and interopercle. Head covered with dark brown spots below the orbit and on lower side of

3 2256 INDIAN J. MAR. SCI., VOL. 43 No.12 DECEMBER 2014 operculum. Upper margin of maxilla with dark brown lining. Orange spots present at the base of dorsal fin, second dorsal edges black in colour, pectoral in grey with irregular dark brown spots. Anal and caudal light brown with dark brownish spots and edges black in colour. Pelvic fin dark brown in colour. In large specimen body tan dorsally shading white ventrally with small orange spots, light brown bars present which bifurcate ventrally. In juvenile specimens the orange colour spots are larger. Epinephelus malabaricus (Bloch and Schneider, 1801) - Malabar grouper: Dorsal 14-16; Pectoral 17-20; Maxilla reaches beyond the posterior border of the eye; maxillary scaled; Three rows of teeth on upper jaw; Post orbital distance 63.5% in HL; Pyloric caeca Colour: Head and body brownish in colour with dark brown spots. On head spots present on maxilla, lower jaw, snout and operculum. Spots present on chin larger than spots on operculum. Dorsal fin brownish with dark brown spots. Anal fin brown in colour with dark brown spots. Pectoral dusky with dark brown spots on twothird of length from its base. Pelvic fin light brown in colour with dark brown spots, edges black in colour. Caudal fin brown in colour with dark brown spots, edges black in colour. Ventrally no spots present behind chin to pelvic fin origin. In the specimen measuring 1530 mm TL, head and body uniformly brownish grey without any spots, dorsal, pectoral, anal and caudal fins with small black spots while pelvic fin with no spots. Epinephelus tauvina (Forsskål, 1775) - Greasy grouper: Dorsal 15; Pectoral 18-19; Maxilla reaches beyond the posterior border of the eye; maxillary scaled; Two rows of teeth on upper jaw; Post orbital distance 57.33% in HL ; Pyloric caeca Colour: Body light brown with five irregular large dark brown cross bars, large orange-brown spots present between these cross bars. First bar originated from third dorsal spine to pelvic fin base; second from eighth to tenth dorsal spine to in front of anus; third brown band from sixth dorsal ray to second anal spine; fourth from ninth to thirteenth dorsal ray to second anal soft ray base and fifth on caudal peduncle. Orange-brown spots present on head maxilla and lower jaw. Dorsal, anal and pelvic brownish with dark brown spots, edges black. Pectoral fin dusky with dark brown spots at base, edges black in colour. Caudal fin brown with dark brown spots on twothird of caudal, edges black. Epinephelus angularis (Valenciennes, 1828) - Angular perch: Dorsal 16-17; Pectoral 17; Two rows of teeth on upper jaw and on lower jaw; Caudal fin emarginated; Pyloric caeca Colour: Body dorsally light grey and colourless ventrally with brown spots having green tinge. Orange spots present on head, maxilla and operculum. Dorsal pale in colour with two rows of spots, Pectoral transparent with bright orange spots. Pelvic colourless at base, with orange spots towards its margin; anal fin with orange spots. Dorsal, pectoral, pelvic and anal fins edges black in colour. Caudal with orange spots and white margin. Epinephelus bleekeri (Vaillant, 1877) - Dusky tail grouper: Dorsal 16-18; Pectoral 17-18; Three rows of teeth on upper jaw and two rows of teeth on lower jaw; Caudal fin truncate; Pyloric caeca Colour: Head and body greyish brown in colour with golden yellow spots. On head spots present on snout, interorbital space, upper jaw, lower jaw, and operculum. From chin to pelvic origin creamy colour with no spots. Dorsal fin grey in colour with golden yellow spots, soft dorsal edges are white in colour. Pectoral fin dusky with no spots, behind pectoral fin base golden yellow spots present. Pelvic fin with light grey in colour with golden yellow spots, edges black. Anal fin pale grey in colour with black edges, spots absent. Caudal fin with golden yellow spots on upper basal half, lower half dusky without any spots. Epinephelus chlorostigma (Valenciennes, 1828) - Brownspotted grouper: Dorsal 16-17; Pectoral 17; Three rows of teeth on upper jaw and two rows of teeth on lower jaw; Seven or eight serrae at preopercular angle; Pyloric caeca Colour: Head and body pale brown with small, irregular, close set dark brown spots. The ground colour between spots forming a pale network. Ventral surface of the body from chin to anal base pale in colour with no spots. Orange irregular spots with brown margin present on maxilla and

4 ISWARYA DEEPTI et al.: TAXONOMY AND PHYLOGENETIC RELATIONSHIP OF SEVEN GROUPER SPECIES 2257 lower lips. Dorsal with closely set spots, the ground colour forms white colour network, rare edges white in colour. Pectoral pale in colour with orange spots at base, brown spots confined to rays, edges white. Anal with orange spots at base and towards margin brown coloured closely set spots and pelvic with small irregular, closely set dark brown spots, the edges of fins are darker. Caudal fin with closely set irregular dark brown spots and margins are white in colour. Epinephelus longispinis (Kner, 1864) Longspine grouper: Dorsal 16-17; Pectoral 17-18; Three rows of teeth on upper and lower jaws; Pyloric caeca Colour: Body pinkish brown to dull brown in head region, becoming gradually paler posteriorly. Dark round spots on head, operculum; posterior spots on body appear oval; pectoral pale brown with dark brown spots. Dorsal fin pinkish brown with dark brown spots, soft dorsal edge white in colour. Pelvic, anal and caudal fins dark brown with no spots. Caudal and anal fin rear edge white in colour. PCA results of Group I: Epinephelus coioides, E. malabaricus and E. tauvina Factor loadings for principal components of the above three species given in the Table 3. The variance explained by the first two components was 73.92% and characters for which the factor loadings are above 0.95 are considered. The first factor is mainly defined by measurements of size (Standard length (SL), Body depth (BD), Head length (HL)), by the position of fins (Predorsal, Prepelvic, and Preanal), size of fins (dorsal base, anal base, dorsal spine height, second dorsal height, pectoral length, pelvic length) and by preorbital distance. The second component was mainly correlated with meristic character of count of gill raker on upper and lower arms. Tukey test for all the above characters was carried out that revealed significant difference (P<0.01) in 13 out of 25 morphometric characters and in two out of five meristic characters, thus serving as an aid in differentiating these three species Table 4. PCA results of Group II: Epinephelus angularis, E. bleekeri, E. chlorostigma and E. longispinis The factor loadings of the first two principal components given in Table 6. The variance explained by these components was and characters for which the factor loadings are above 0.95 were considered. The first factor was mainly defined by measurements of size (Standard length, Body depth, head length and head depth), by the position of fins (prepectoral, prepelvic, preanal distance), size of fins (dorsal base, anal base, dorsal height, second dorsal height, pectoral length, pelvic length, anal ray), by preorbital and postorbital. Results of Tukey test (p<0.05) for these four species given in Table 7 that revealed significant difference in 16 out of 26 morphometric characters. In the eleven enzyme systems studied, twenty six consistent, reproducible, scorable loci were detected. In the present study three loci detected for allozymes AAT*, ALD*, G6PDH* and GSN*, two loci detected for the remaining allozymes. A total of 71 alleles were detected and their frequencies given in Table 8. The number of polymorphic loci (P 0.95) exhibited by species of this genus Epinephelus are in the range two to five. The results yielded species specific diagnostic markers in five species E. coioides, E. malabaricus, E. tauvina, E. bleekeri and E. angularis. The results yielded species specific diagnostic markers in five species, E. coioides, E. malabaricus, E. tauvina, E. angularis and E. bleekeri. Diagnostic locus where no alleles shared with any other species 17 for E. tauvina observed at G6PDH-3*. Among the seven species number of diagnostic alleles are in the range four to seven (Table 9). Parameters of genetic variation are given in Table 10. The mean number of alleles per locus was found to be high in E. coioides and E.tauvina. The observed heterozygosity varied from to Probability test was performed to assess the conformity of allele frequencies to that expected under Hardy Weinberg expectations. A significant deviation from HW expectation after sequential Bonferroni adjustment (p<0.005) of probability levels was observed at loci AAT- 2*, ADH-3*, EST-2*, GDH-1*, GSN-1*, MDH* and SOD-1*. Estimates of genetic identity and genetic distance between pairs of seven species given in Table 11. The overall relationships were apparent from UPGMA derived dendrogram (Fig 8) that revealed two main branches where Epinephelus coioides, E. malabaricus and E. tauvina formed one cluster and E. angularis, E. bleekeri, E. chlorostigma and E. longispinis formed another cluster.

5 2258 INDIAN J. MAR. SCI., VOL. 43 No.12 DECEMBER 2014 Table 1 Frequency distribution of meristic character of seven species of groupers of genus Epinephelus in pooled samples collected in the catches of Visakhapatnam Dorsal rays n E. coioides E. malabaricus E. tauvina E. angularis E. bleekeri E. chlorostigma E. longispinis Pectoral rays n E. coioides E. malabaricus E. tauvina E. angularis E. bleekeri E. chlorostigma E. longispinis Caudal rays n E. coioides E. malabaricus E. tauvina E. angularis E. bleekeri E. chlorostigma E. longispinis Gill rakers, upper arm n E. coioides E. malabaricus E. tauvina E. angularis E. bleekeri E. chlorostigma E. longispinis Gill rakers, lower arm n E. coioides E. malabaricus E. tauvina E. angularis E. bleekeri E. chlorostigma E. longispinis Gill rakers, total n E. coioides E. malabaricus E. tauvina E. angularis E. bleekeri E. chlorostigma E. longispinis X X X X X X SD SD SD SD SD SD SE SE SE SE SE SE

6 ISWARYA DEEPTI et al.: TAXONOMY AND PHYLOGENETIC RELATIONSHIP OF SEVEN GROUPER SPECIES 2259 Table 2 Comparison of morphometric data of the three species of genus Epinephelus, represented in the catches of Visakhapatnam Min-Max X ±SD Min-Max X ±SD Min-Max X ±SD As percentage of standard length: Total length ± ± ±0.44 Body depth* ± ± ±0.38 Head length* ± ± ±2.38 Predorsal distance* ± ± ±0.88 Prepectoral distance ± ± ±1.97 Prepelvic distance* ± ± ±1.09 Preanal distance ± ± ±1.00 Dorsal base* ± ± ±2.69 Anal base* ± ± ±1.57 Dorsal 3 spine height* ± ± ±0.55 Soft dorsal height* ± ± ±0.36 Pectoral length* ± ± ±0.29 Pelvic length* ± ± ±1.67 Anal third spine height ± ± ±0.54 Soft anal height ± ± ±0.89 As percentage of head length Head depth* ± ± ±7.83 Head width* ± ± ±0.55 Preorbital ± ± ±1.33 Postorbital* ± ± ±5.20 Upper jaw ± ± ±4.70 Lower jaw ± ± ±2.41 Snout length ± ± ±5.26 Eye diameter ± ± ±1.97 Interorbital width ± ± ±2.16 Maxillary width ± ± ±1.82 *Significant characters obtain from PCA (factor loading 0.95) Species name Standard length E. coioides, n= 136 E. malabaricus, n=36 E. tauvina, n= mm mm mm

7 2260 INDIAN J. MAR. SCI., VOL. 43 No.12 DECEMBER 2014 Table 3 Factor loadings for principal components of three species of genus Epinephelus E. coioides, E. malabaricus and E. tauvina off Visakhapatnam. Characters Component 1 Component 2 Standard length Total length Predorsal distance Prepectoral distance Prepelvic distance Preanal distance Dorsal base Anal base Dorsal spine height Second dorsal height Pectoral length Pelvic length Anal height Anal length Body depth Head length Head depth Head width Eye diameter Preorbital Postorbital Interorbital width Snout length Maxillary width Lower jaw length Dorsal rays Pectoral rays Caudal rays Upper arm of gill raker Lower arm of gill raker Table 4 Results of Tukey test among the three species Epinephelus coioides, E. malabaricus and E. tauvina off Visakhapatnam. Characters F value p Predorsal distance P<0.01 Pre Pelvic distance P<0.01 Dorsal base P<0.01 Anal base P<0.01 Dorsal spine height P<0.01 Soft dorsal height P<0.01 Pectoral length P<0.01 Pelvic length P<0.01 Body depth P<0.01 Head length P<0.01 Head depth P<0.01 Head width P<0.01 Postorbital length P<0.01 Discussion UPGMA dendrogram constructed based on allozyme electrophoretic studies revealed that in cluster I, E. tauvina radiated forming a separate branch from E. coioides and E. malabaricus showing that these two species are closely related. Based on colour description and biometric data E. tauvina has overlapping meristic and morphometric characters and colour pattern with E. coioides and E. malabaricus. However they can be distinguished by the following characters. - Lateral line scale series in E. tauvina is while in E. coioides and in E. malabaricus. - The shape of the preopercle is rounded while sub angular in E. coioides and E. malabaricus. - Absence of bony plates on lateral side of gill rakers while bony plates present in E. coioides and E. malabaricus. E. malabaricus differs from E. coioides by the following characters - preanal distance in SL vs in SL in E. coioides - body depth in SL vs in SL in E. coioides - post orbital distance in HL vs in HL in E. coioides - number of gill rakers in upperarm 9-10 vs 6-10 in E. coioides So PCA has been carried out for meristic and morphometric data of these closely related species to identify the significant variables. E. coioides and E. malabaricus, two closely related and similar species in appearance 18. The two species occupy the same habitats along continental and large islands shore of the Indo- Pacific. They show great adaptability to different water salinity. During the present study both the species are encountered in the catches during the same period. Based on RAPD analysis also it was stated that the genetic relationship was very close among E. coioides, E. malabaricus and E. tauvina 4. Allozyme electrophoretic studies in E. coioides revealed 21 loci from 17 enzyme systems, out of which ten loci were found to be

8 ISWARYA DEEPTI et al.: TAXONOMY AND PHYLOGENETIC RELATIONSHIP OF SEVEN GROUPER SPECIES 2261 E. longispinis, n= mm Min - Max As percentage of standard length: Total length ± ± ± ±3.02 Body depth* ± ± ± ±4.27 Head length* ± ± ± ±3.30 Predorsal distance ± ± ± ±2.12 Prepectoral distance* ± ± ± ±1.70 Prepelvic distance* ± ± ± ±1.70 Preanal distance* ± ± ± ±2.63 Dorsal base* ± ± ± ±2.51 Anal base* ± ± ± ±1.39 Dorsal 3spine height* ± ± ± ±1.31 Soft dorsal height* ± ± ± ±2.01 Pectoral length* ± ± ± ±2.55 Pelvic length* ± ± ± ±1.61 Anal 3 spine height ± ± ± ±0.49 Soft anal height* ± ± ± ±2.25 As percentage of head length: Head depth* ± ± ± ±3.97 Head width ± ± ± ±3.37 Preorbital* ± ± ± ±1.74 Postorbital* ± ± ± ±3.93 Upper jaw ± ± ± ±1.96 Lower jaw ± ± ± ±3.07 Snout length ± ± ± ±4.00 Eye diameter ± ± ± ±2.12 Interorbital width ± ± ± ±1.22 Maxillary width ± ± ± ±1.75 *Significant characters obtain from PCA (factor lading 0.95) X ± SD Table 5 Comparison of the morphometric data of the four species of genus Epinephelus off Visakhapatnam Species name E. angularis, n= 52 E. bleekeri, n= 41 E. chlorostigma, n= 6 Standard length mm mm mm Min - Max X ± SD Min - Max X ± SD Min - Max X ± SD

9 2262 INDIAN J. MAR. SCI., VOL. 43 No.12 DECEMBER 2014 Table 6 Factor loadings for principal components of four species Epinephelus angularis, E. bleekeri, E. chlorostigma E. longispinis off Visakhapatnam Chacters Component1 Component2 Standard length Total length Predorsal distance Prepectoral distance Prepelvic distance Preanal distance Dorsal base Anal base Dorsal spine height Second dorsal height Pectoral length Pelvic length Anal height Anal length Body depth Head length Head depth Head width Eye diameter Preorbital Postorbital Interorbital width Snout length Maxillary width Lower jaw length Dorsal rays Pectoral rays Caudal rays Upper arm of gill raker Lowerarm of gill raker Table 7 Results of Tukey test for morphometric characters among four species: Epinephelus angularis, E. bleekeri, E. chlorostigma E. longispinis off Visakhapatnam. Characters F value P Prepectoral distance P<0.01 Prepelvic distance P<0.01 Preanal distance Dorsal base Anal base P<0.01 Dorsal spine height P<0.01 Second dorsal height P<0.01 Pectoral length P<0.01 Pelvic length Anal length P<0.01 Body depth P<0.01 Head length P<0.01 Head depth P<0.01 Preorbital P<0.01 Postorbital P<0.01 polymorphic and the mean heterozygosities given as ± This value is correlating with the present study. Genetic structure of E. tauvina was studied employing eight allozymes where 16 loci were observed, only ADH was found to be polymorphic 20. Cluster II revealed two main branches where E. bleekeri and E. chlorostigma formed one cluster and E. angularis and E. longispinis formed one cluster showing that they are closely related species. E. chlorostigma is closely related to E. bleekeri but can be distinguished by following characters: - Pre anal distance in E. bleekeri vs in E. chlorostigma - Pre orbital vs in E. chlorostigma - Interorbital convex in E. bleekeri vs slightly concave in E. chlorostigma - Central gill raker almost double the length of gill filament in E. bleekeri vs gill raker slightly longer than gill filament in E. chlorostigma - Maxillary scaly with 12 rows of scales in E. bleekeri vs rows of scales in E. chlorostigma According to Groupers and Wrasses Specialist group, this is Near Threatened (NT) species in IUCN Red List 21. Description of the present specimens of E. angularis is in good agreed with the earlier authors 22,23. Following earlier author 24,25,26,27,2829 E. angularis 22 was usually considered as synonym of E. areolatus. Based on following characters E.angularis 22 is stated as a valid species Caudal fin shape emarginated vs truncate in E. areolatus. - More number of closely set spots present on the body in E. chlorostigma. E. longispinis is being recorded for the first time from Visakhapatnam towards north of Chennai. E. longispinis was first recorded and described from Madras 31, India and the species is subsequently been recorded from several parts of

10 ISWARYA DEEPTI et al.: TAXONOMY AND PHYLOGENETIC RELATIONSHIP OF SEVEN GROUPER SPECIES 2263 Table 8 Allele frequencies at twenty six loci in the seven grouper species of genus Epinephelus off Visakhapatnam Locus Allele E.coioides (38) E.malabaricus(36) E.tauvina (3) E.bleekeri(26) E.angularis(30) E.chlorostigma(4) E. longispinis(10) AAT-1* 100(a) (b) (c) AAT-2* 100(a) (b) AAT-3* 100(a) (b) ACP-1* 96(a) (b) (c) ACP-2* 100(a) (b) ALD-1* 98(a) (b) (c) (d) ALD-2* 98(a) (b) (c) ALD-3* 98(a) (b) EST-1* 96(a) (b) (c) EST-2* 96(a) (b) (c) GDH-1* 98(a) (b) GDH-2* 98(a) (b) G6PDH-1* 100(a) (b) (c)

11 G6PDH-2* 100(a) (b) (c) G6PDH-3* 100(a) (b) GSN-1* 98(a) (b) (c) GSN-2* 100(a) (b) GSN-3* LDH-1* 100(a) (b) (c) LDH-2* 100(a) (b) (c) MDH-1* 100(a) (b) (c) MDH-2* 100(a) (b) PGM-1* 98(a) (b) (c) (d) PGM-2* 100(a) (b) SOD-1* 100(a) (b) (c) (d) SOD-2* 96(a) (b) (c) (d) INDIAN J. MAR. SCI., VOL. 43 No.12 DECEMBER 2014

12 ISWARYA DEEPTI et al.: TAXONOMY AND PHYLOGENETIC RELATIONSHIP OF SEVEN GROUPER SPECIES 2265 Table 9 Alleles present at each locus, given as letters in alphabetic order according to their anodal mobility a representing the fastest migrating. Diagnostic alleles of species are underlined. Locus E.coioides E.malabaricus E.tauvina E.bleekeri E.angularis E.chlorostigma E.longispinis AAT-1* ab abc ab ab ab ab ab AAT-2* ab ab ab ab AAT-3* ab ab ab ACP-1* bc bc ac bc - abc - ACP-2* - - ab ab ab ab ab ALD-1* abc abcd bc ab bc ab ab ALD-2* ab bc ALD-3* - ab ab EST-1* ac ac abc bc bc - - EST-2* bc bc ab bc bc bc abc GDH-1* ab ab ab ab ab ab ab GDH-2* ab - ab ab ab ab ab G6PDH-1* abc abc bc ab ab ab - G6PDH-2* ab bc bc ab ab ac ab G6PDH-3* - - Ab GSN-1* ab ab ab bc bc bc ab GSN-2* ab ab GSN-3* ab ab ab LDH-1* - - ab ab abc ab - LDH-2* abc abc ac ac ac MDH-1* ab ab abc ab ac bc ac MDH-2* ab ab - - PGM-1* bcd bcd ac ab - - ab PGM-2* ab - ab SOD-1* ab ab ab abcd ab ab abd SOD-2* abcd ac cd - - -

13 2266 INDIAN J. MAR. SCI., VOL. 43 No.12 DECEMBER 2014 Table 10 Parameters of genetic variation at each allozyme locus in the seven grouper species of genus Epinephelus Parameter/ E.coioides E.malabaricus E.tauvina E.bleekeri E.angularis E.chlorostigma E.longispinis Species Sample size No. of loci screened No. of polymorphic loci Mean heterozygosity 0.065± ± ± ± ± ± ±0.04 Table 11 Estimates of genetic identity (above diagonal) and genetic distance (below diagonal) (Nei, 1978) between pairs of seven grouper species of genus Epinephelus off Visakhapatnam Species E.coioides E.malabaricus E.tauvina E.bleekeri E.angularis E.chlorostigma E.longispinis E.coioides **** E.malabaricus **** E.tauvina **** E.bleekeri **** E.angularis **** E.chlorostigma **** E.longispinis ****

14 ISWARYA DEEPTI et al.: TAXONOMY AND PHYLOGENETIC RELATIONSHIP OF SEVEN GROUPER SPECIES Head covered with cycloid scales in E. angularis vs ctenoid scales in E. longispinis - Two rows of teeth present on both jaws in E. angularis vs three rows of teeth on both jaws as in E. longispinis - Inter orbital profile slightly convex in E. angularis vs flat in E. longispinis - Caudal fin emarginated in E. angularis vs truncate in E. longispinis Based on PCA result, significantly different characters derived from PCA for these two species are Prepectoral, Head length, Preorbital and Postorbital (Table 4). Thus to solve taxonomic ambiguities among closely related species and for proper identification allozyme electrophoresis has been very useful as it is quite faster, easier and cheaper method and moreover even though there are several overlapping morphological characters closely related species could be clearly identified and segregated without any confusion. the Indo-West Pacific but surprisingly not reported later any time till now from Chennai (Madras) i.e. east coast of India. This is the second report of E. longispinis from east coast of India. This species closely related to E. angularis and can be distinguished by following characters - Pectoral 17 in E. angularis vs in E. longispinis - Gill rakers in E. angularis vs in E. longispinis - Maxillary scaly with rows of scales in E. angularis vs 14 rows in E. longispinis - Central gill rakers longer than gill filament in E. angularis vs central gill raker smaller than gill filament in E. longispinis Acknowledgements Financial help from Ministry of Earth Science, New Delhi is gratefully acknowledged. Authors thank Head of the Department of Marine Living Resources, Andhra University for providing facilities and Visakhapatnam Regional Centre of Fishery Survey of India (FSI), for providing some samples. First author is thankful to Department of Science and Technology (DST) New Delhi for the award of DST-Women Scientist (A) Fellowship. References 1. Rangaswamy, V.S., Marichamy, R., Rajapackiam, S. & Sundararajan, D., Collection and transportation of groupers for farming. In: The Fourth Indian Fishes Forum, Proceedings 24-25, November Kochi pp James, C. M., Al-Thobaiti, B.M., Rasem, B.M. & Carlos, M.H., Potential of grouper hybrid (Epinephelus fuscogutatus x E. polyphekadion) for aquaculture. Aquacult. Eur. Mag., 24(2) (1999), Craig, M.T. & Hastings, P.A., A molecular phylogeny of the subfamily Epinephelinae (Serranidae) with a revised classification of the Epinephelini. Ichthyol. Res., 54, (2007), Govindaraju G.S. & Jayasankar, P., Taxonomic relationship among seven species of Groupers (Genus Ephinephelus: Family Serranidae) as revealed by RAPD fingerprinting. Mar. Biotechnol., 6, (2004),

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