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Indian Journal of Geo Marine Sciences Vol. 48 (12), December 2019, pp. 1864-1869

Genetic Evidence for two undescribed previously considered as sihama from Persian Gulf

Elaheh Fateh1, Amin Oujifard*1,2, Ahmad Shadi3, & Seyyed Javad Hoseini4 1Department of , Faculty of agriculture and Natural Resources, Persian Gulf University,Bushehr,7516913817, Iran 2Department of Fisheries, Faculty of Marine Sciences and Technology, Persian Gulf University, Bushehr, 7516913817, Iran 3Department of Marine Biotechnology, Faculty of Marine Sciences and Technology, Persian Gulf University, Bushehr, 7516913817, Iran 4Department of Molecular and Cell Biology, Faculty of Science, Persian Gulf University, Bushehr, 7516913817, Iran *[E-mail: [email protected]]

Received 26 April 2018; revised 04 June 2018

Traditionally from Sillaginidae considered as Sillago sihama in the North Persian Gulf. Genetic as well as morphological analyses were used to study species differentiation of Sillaginids in the North Persian Gulf. Meristic counts and geometric morphometrics were used to morphologically differentiate specimens. We sequenced a 620 bp fragment from the mitochondrial cytochrome c oxidase subunit I gene (COI) of Sillaginids collected from the north Persian Gulf and analysed in contrast to sequences from the GenBank. In spite of inconspicuous meristic features, no significant morphometric differences between samples was observed, however based on analysed sequences and phylogenetic relationships of the samples three species of Sillaginids were revealed. Except for Sillago sihama, no previous barcodes were available for other two species Sillago arabica and Sillago attenuata in the GenBank. The results of the present study revealed that at least three species of Sillaginids apparently identical are present in the North Persian Gulf, Therefore, precautionary consideration by environmental and fisheries managers is recommended.

[Keywords: Sillaginids; Mitochondrial DNA; Morphologically similar species; Persian Gulf]

Introduction specific segment of genome that embedded in every Morphology is the main tool used by biologists to cell5. Genetic markers can provide critical information categorize organisms; however, it has been proved regarding phylogenic relationships even in instances of that many inter and intra specific variations are morphological homogeneity, and when groups are 1,2 hardly identifiable based on external morphology . strongly diverged6-9 . Fish from family Sillaginidae are Most fish species have been identified based on commonly inhibiting shallow waters with sandy unique phenotypic characteristics; however in some substrate or estuarine areas of rivers10. This family cases species discrimination is difficult due to comprise 34 species in three genera worldwide11. morphological homogeneity of some sibling species. Sillaginids display a high degree of similarity in Differentiation of similar species based on phenotypic appearance and external morphology, having slender identification systems is more complicated in larval elongate fusiform-shaped bodies and long conical and juvenile stages. In such cases, monitoring and snouts12. Sillago considered a morphologically management of fish populations may be complicated. 13 Microgenomic identification systems, on the other difficult identifiable genus around the world . hand provide reliable discrimination of species based Traditionally fish from family Sillaginidae have been on a small fragment of the genome. Challenges in considered as S. sihama in the North Persian Gulf. Due identifying morphologically difficult taxa using to high similarity of morphologic characteristics of the phenotypic differences can be overcome by the use of Sillaginids in the present study, we employed molecular techniques, which have proved to be useful sequencing of COI gene to achieve basic genetic for discovering unexpected species diversity2-4. information on Sillaginids in the north Persian Gulf and Taxonomists overcome the limitations of phenotypic to determine the systematic relationships of what using DNA sequences (barcodes) in a known as S. sihama. FATEH et al.: GENETIC EVIDENCE FOR TWO UNDESCRIBED SPECIES AS SILLAGO SIHAMA 1865

Materials and Methods At first, some landmarks were specified on , Collection of sample and specimen photos were taken at given equal In total, over 100 specimens were collected from distance. The intended factors (morphometric two sites in the Persian Gulf, Iran: Bushehr province characters) were determined using Imagej Ver 1.48. and Hormozgan province (30°09'28.5"N 50°04'27.1"E In this research, allometric formula was used in to 27°09'49.8"N 56°18'03.7"E). Samples were preserved to modify the effect of the difference of specimens' in ice and carried back to the laboratory. Small measurements. fragments (2-3 gr) of the caudal fin of each sample b were preserved in 96% ethanol for further analysis. Madj = M(LS/L0) Overall shape of was used to sort samples for further analysis. where M is original measurement, Madj is the size adjusted measurement, L0 is the standard length of the Overall meristic and morphometric observation fish, Ls the overall mean of standard length for all fish Geometric morphometrics has proved to provide from all samples in each analysis, and b was high potential discrimination of populations and estimated for each character from the observed data as morphologically similar species. This method was the slope of the regression of logM on log L0 using all used to evaluate the potential of this method for fish in any group. The results derived from the differentiating diversity of studied fish. For this, allometric method were confirmed by testing 6 meristic characters including first spines, significance of the correlation between transformed second dorsal fin spines, second dorsal fin rays, anal variables and standard length. To visualize body fin rays, anal fin spines, scales and shape differences we generated thin-plate spline 16 morphometric characters including total length deformation grids using MorphoJ v. 1.06b. Thin-plate (T.L), postorbital length (P.O.L), first pre dorsal fin spline deformation grids are similar to D’Arcy length (F.Pr.D.F.L), second pre dorsal fin length Thompson's transformation grids12, where relative (Se.Pr.D.F.L), first posterior dorsal fin length shape differences are represented as a set of bent grid (F.Po.D.F.L), second posterior dorsal fin length lines superimposed over the landmark coordinates of (Se.Po.D.F.L), pre anal fin length (Pr.A.F.L), each specimen13. Morphometric characters in fishes posterior anal fin length (Po.A.F.L), pre pelvic fin and data analysis were compared using SPSS ver. 20 length (Pr.Pv.F.L), first dorsal fin length (F.D.F.L), and Excel 2010. second dorsal fin length (Se.D.F.L), anal fin length (A.F.L), head length ( H.L), pectoral fin length DNA Extraction DNA was extracted using the modified CTAB (Pt.F.L), body depth (B.D) and peduncle height (P.H) 14 were determined (Fig. 1). method . The DNA quality, concentration, and purity were assessed by spectrophotometry using a spectrophotometer and electrophoresis on agarose gel (1 %).

PCR Amplification Universal primers FISH F1(5´-ACCAAC CAC AAA GAC ATT GGC AC-3´) and FISH R1 (5´- ACT TCT GGG TGG CCA AAGAATCA-3´) were used for PCR amplification15. The reaction was performed in a final volume of 25 μl, containing 9 μl of Milli- Q water, 1μl of genomic DNA, 0.8 μl of dNTPs, 2.5 μl Fig. 1 — Specified landmarks on samples: 1- the beginning of the of PCR buffer (10x), 2 μl MgCl2, 1μl of forward and snout in the maxilla. 2- the front of the eye. 3- the beginning of the . 4- the beginning of the first dorsal fin. 5- the end reverse primers each, the Taq DNA polymerase 0.5μl. of the first dorsal f in. 6- the beginning of the second dorsal fin. The thermal cycling profile was as follows: 94°C 7- the end of the second dorsal fin. 8- the end of the peduncle. initial denaturing for 30s; 30 cycles of 94°C of 9- the end of the caudal fin. 10- the beginning of the pelvic fins. denaturing during 3 min, 53°C annealing for 30 s, and 11- the end of the pelvic fins. 12- the beginning of the pectoral fin. 13- the end of the pectoral fin. 14- the beginning of the anal 72°C extension for 45s; and a final extension at fin. 15- the end of the anal fin. 94°C for 10 min. The amplified product was 1866 INDIAN J. MAR. SCI., VOL. 48, NO. 12, DECEMBER 2019

electrophoresed through a 1% agarose gel. The purified subunit I (mtDNA COI) were obtained. The results of PCR product was sequenced on an ABI 3730 XL BLAST did not confirm previous identification of all automated sequencer. samples as S. sihama and only 7 specimens of 30 were genetically attributed to S. sihama, and no Phylogenetic Analysis barcodes were available for other samples in the Obtained sequences were aligned using Bio-Edit 16 GenBank. When the visualized ver.7.0 software (Applied program Clustal w) . using UPGMA (Fig. 2), the specimens divided into Sequence genetic divergences were calculated using 17 three different clusters. Some detailed morphology the Kimura 2 parameter (K2P) distance model . observations such as meristic counts in addition to Phylogenetic relationships were assessed using the morphology of swim bladder were used to Neighbor-Joining, the Maximum Likelihood and the differentiate samples with no registered COI Unweighted Pair Group Method with Arithmetic sequences (S. arabica and S. attenuata). Table 3 Mean methods from MEGA ver 5 software. shows some differences of the studied specimens. Furthermore, Bootstrap replication (500 numbers) Comparing the specimens on the basis of genetic was used to validate the tree. Dnasp ver. 5 was used to distance which is performed between the specimens in extract a genetic differentiation parameters set from this study and the other species of Sillaginidae family nucleotide sequences (haplotype number, haplotype suggested the genetic distance vary between 0 and diversity, nucleotide diversity and pair wise 0.27 (Table 4). difference). Some COI sequences from Sillaginidae species for comparison were obtained from GenBank Table 1 — COI sequences from GenBank used in the analysis. (Table 1). Species GenBank number Location Sillago sinica KC708229.1 South Korea Results Sillago sihama) JX260974.1 Morphologic observations Sillago parvisquamis HQ389249.1 The results of general morphometric observations Sillago maculata FJ223189.1 are given in Table 2. Based on the results of Sillago japonica HQ389252.1 China the geometric morphometrics, no significant Sillago ciliata JX887796.1 Australia differentiation was observed. Sillago chondropus JF494514.1 South Sillago bassensis HM131484.1 China and phylogeny Sillago analis JX875487.1 Australia Universal primers of Fish F1 and Fish R1 for COI Sillago aeolus HM131475.1 China gene and thermal profiles revealed clear, single-band Sillaginodes punctatus EF609465.1 Australia PCR products. After sequence alignment, a 620 bp Sillago vincenti KC774672.1 India fragment of mitochondrial DNA cytochrome oxidase Leucaspius delineatus KP794944.1 outgroup

Table 2 — General morphometric measurements of specimens Factor Max(mm) Min(mm) CV (X¯±SD) T.L 198.31 179.84 2.92 (187.79±5.49 ) P.O.L 31.66 16.68 15.17 (22.88±4.84 ) F.Pr.D.F.L 72.95 42.25 17.00 (57.79±9.82 ) F.Po.D.F.L 110.84 80.63 11.26 (95.69±10.78 ) Se.Pr.D.F.L 111.78 82.31 10.91 (97.07±10.60 ) Se.Po.D.F.L 160.97 146.80 2.68 (155.07±4.16 ) Pr.A.F.L 110.68 79.39 12.06 (95.03±11.46 ) Po.A.F.L 162.66 144.50 3.32 (154.00±5.11 ) Pr.Pv.F.L 67.72 39.90 19.43 (51.96±10.09 ) F.D.F.L 51.01 25.51 21.09 (38.18±8.05 ) Se.D.F.L 72.69 45.40 16.99 (57.59±9.78 ) A.F.L 73.87 42.65 15.40 (59.47±9.16 ) H.L 61.18 36.11 19.75 (47.82±9.44 ) Pt.F.L 36.86 17.07 19.00 (28.27±5.37 ) B.D 43.30 22.74 20.88 (30.1±6.29 ) P.H 16.28 8.91 16.96 (11.70±1.98 )

FATEH et al.: GENETIC EVIDENCE FOR TWO UNDESCRIBED SPECIES AS SILLAGO SIHAMA 1867

Fig. 2 — phylogenetic tree of the studied Sillaginids from the Persian Gulf and other Sillaginidae species by UPGMA. *Some of the sequences which were obtained during the present study

Table 3— Some discriminating morphologic traits of the studied species based on McKay (1992) Species Dorsal fin Anal fin Vertebra Swim bladder S. sihama XI+I,20-23 II,21-23 14+20=34 2 posterior extensions, 2 anterior extensions, 2 lateral extensions S. arabica XII-XIII+I,22-24 II,22-24 15-16+22-23=38-40 without anterior extensions, 1 posterior extension S. attenuata XII-XIII,19-21 II,18-20 15+22=37-39 anterior margin convex

The distance of S. sihama with S. arabica and S. Discussion attenuata were 0.23 and its value between S. arabica Overexploitation of major wild fish stocks of the and S. attenuata was 0.22. In addition, the intra world has resulted in increasing global demand for species genetic distance among specimens of S. development of the fish culture especially coastal attenuate and S. arabica were 0.063 and 0.02, , and hence some fish inhabiting the respectively. coastal area has been recommended as candidate for

1868 INDIAN J. MAR. SCI., VOL. 48, NO. 12, DECEMBER 2019

aquaculture. Fish from family Sillaginidae are Despite the fact that all three studies species exhibit important species in estuarine aquaculture18. Since the inconspicuous distinctive meristic characteristics Sillaginids display a high degree of similarity in (table2), description of management units based on coloration and external morphology, they are morphological features is complicated, and even considered as difficult species to identify and impossible in premature stages. Therefore, genetic classify18,19. Poor information is available on the differentiation of Sillago complex is essential for morphometrics and interrelationship between various management implications, as the Sillago genus is one body measurements in S. sihama especially in the of the difficult species to identify genetically and north Persian Gulf. morphologically. Therefore redescription and new Our study provides basic evidence for taxonomic species reports are common for this genus22,23,24,25. differentiation of Sillaginids of the north Persian Gulf. Based on the constructed phylogenetic tree, first The comparison of genetic distances between the cluster of specimens (S. sihama) separated into a specimens in the present study and the other species cluster along with other S. sihama samples from of Sillaginidae suggested the genetic distance vary GenBank. But other two populations were clustered between 0 and 0.27 (Table 4). Thorpe and Thrope et differently in the phylogenetic tree (Fig. 2) which al.,20 showed that the Nei21 genetic distance values represents two other different species in the North averaged 0.05 (range: 0.002-0.07) for con-specific Persian Gulf. In spite of the fact that in the previous populations and 0.30 (range: 0.03-0.61) for con- studies in the North Persian Gulf, Sillaginids were generic species. It can be concluded from the results assigned to one species: S. sihama, Shadi26 proposed of the present study that genetic distances between the possibility of existence of more than one species three clusters were in the range of species of the same of Sillaginids in the region based on 16s rDNA and gender. microsatellite analysis. Our results suggest the However, genetic identification of species was presence of at least three species of family complicated mostly by barcoding efficiency. As the Sillaginidae in the North Persian Gulf, thus providing results showed that due to lack of previously new basis for conservation and developmental goals. submitted sequences, two species S. arabica and S. Therefore, precautionary consideration by attenuate were not completely attributed to any of environmental and fisheries managers is Sillaginidae species on the basis of data accessible recommended for decision making related to from GenBank. This is one of the limitations of DNA Sillaginids of north Persian Gulf. barcoding, make difficulty when no listed sequence is In conclusion, geometric morphometrics did not available for the species. discriminate species of the present study, however The morphometric analysis of samples showed no based on genetic data revealed from COI gene discriminating feature in the present study (Table1). sequences, three species of Sillaginids were

FATEH et al.: GENETIC EVIDENCE FOR TWO UNDESCRIBED SPECIES AS SILLAGO SIHAMA 1869

recognized. Our results showed that the specimens 12 Thompson, D.W., On growth and form. On growth and under this examination which have been considered form. (1942). 13 Adams, D.C., Rohlf, F.J., & Slice ,D.E. , Geometric traditionally as S. sihama, actually are a complex of morphometrics: ten of progress following the Sillaginidae species, with three different species: S. ‘revolution.’ Ital. J. Zool. 71(2004)5–16. sihama, S. arabica, and S. attenuate which are not 14 Doyle, J., & Doyle ,J.L. ,Genomic plant DNA preparation easily identifiable. from fresh tissue-CTAB method. Phytochem Bull. 19(1987)11–15. 15 Ward, R.D., Zemlak ,T.S., Innes, B.H., Last ,P.R., & Hebert, Acknowledgement P.D.N., DNA barcoding Australia’s fish species. Philos. TR The authors are grateful to Dr. Dara Bagheri for his Soc. B, 360(2005), 1847–1857 assistance in geometric morphometric analysis and 16 Hall, T.A., BioEdit: a user-friendly biological sequence Mr. Hamid Ziarati for fish sampling. alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41(1999)95–98. 17 Kimura, M., A simple method for estimating evolutionary References rates of base substitutions through comparative studies of 1 Mayr, E.,The species concept: semantics versus semantics. nucleotide sequences. J. Mol. Evol. 16(1980)111–120. (N. Y). 3:(1949)371–372. 18 McKay, R.J. ,FAO species catalogue. v. 14: Sillaginid fishes 2 Bickford, D., Lohman ,D.J., Sodhi ,N.S., Ng ,P.K.L., of the world (Family Sillaginidae). An annotated and Meier ,R., Winker ,K., Ingram ,K.K., & Das ,I. ,Cryptic illustrated catalogue of the Sillago, Smelt or Indo-Pacific species as a window on diversity and conservation. Trends species known to date.(South Brisbane)1992 pp. 87. Ecol. Evol. 22(2007)148–155. 19 Mirzaei ,Mr., Valinassab ,T., Yasin ,Z., & shau hwai ,T. , 3 Taylor, D.J., Finston, T.L., & Hebert ,P.D.N., Biogeography Reproduction characteristics and length- relationship of a widespread freshwater : pseudocongruence of the whiting (Sillago sihama) in the south and cryptic endemism in the North American Daphnia laevis of Iran (Persian Gulf and Oman Sea). Ann. Biol. Res. complex. Evolution (N. Y). 52(1998)1648–1670. 4:4(2013) 269–278. 4 Pfenninger, M., & Schwenk, K., Cryptic species are 20 Thorpe, J.P., & Solé-Cava, A.M., The use of allozyme homogeneously distributed among taxa and biogeographical electrophoresis in invertebrate systematics. Zool. Scr. regions. BMC Evol. Biol. 7(2007)121. 23(1994)3–18. 5 Hebert, P.D.N., Cywinska, A., & Ball ,S.L. ,Biological identifications through DNA barcodes. Proc. R. Soc. London 21 Nei, M.,Genetic distance between populations. Am. Nat. B Biol. Sci. 270(2003)313–321. 106(1972)283–292. 6 Boissin, E., Féral, J.P., & Chenuil, A., Defining 22 Golani ,D., Fricke ,R., & Tikochinski ,Y. ,Rehabilitation of reproductively isolated units in a cryptic and syntopic species Sillago erythraea Cuvier, and redescription of Sillago sihama complex using mitochondrial and nuclear markers: the (Forsskål)(Teleostei: Sillaginidae) from the . brooding brittle star, Amphipholis squamata (Ophiuroidea). Stuttgarter Beiträge zur Naturkd. A. 4(2011)4:465–471. Mol. Ecol. 17(2008)1732–1744. 23 Kaga, T., & Ho, H.-C., Redescription of Sillago 7 Forsman, Z.H., Barshis ,D.J., Hunter, C.L., & Toonen ,R.J. , (Parasillago) indica McKay, Dutt & Sujatha, 1985 Shape-shifting corals: molecular markers show morphology (: Sillaginidae), with a reassignment to the is evolutionarily plastic in Porites. BMC Evol. Biol. subgenus Sillago. Zootaxa. 3513(2012)3513:61–67. 9(2009)45. 24 Kaga, T., & Heemstra, E., First record of a rare , 8 Souter, P., Hidden in a key model species of Sillago caudicula (Perciformes: Sillaginidae), from coral. Mar. Biol. 157(2010)875–885. Madagascar. Mar. Biol. Res. 9(2013)316–320. 9 Piggott, M.P., Chao, N.L., & Beheregaray ,L.B. ,Three fishes 25 Tikochinski,Y., Shainin,I., Hyams,Y., Motro, U. & Golani, in one: cryptic species in an Amazonian floodplain forest D., Genetic evidence for an undescribed species previously specialist. Biol. J. Linn. Soc. 102(2011)391–403. considered as Sillago sihama from the northern Red Sea. 10 Nadkarni, V.B., Structure of the kidney of marine fishes Mar. Biol. Res. 9(2013)309–315. in relation to their . Recent Adv. Zool. India. 26 Shadi,A., Zolgharnein, H., Salari-aliabadi, M., Nabavi, M., & (1963)157–170. Ronagh, M., Genetic characterization of Sillago sp. from 11 Kaga, T., Phylogenetic systematics of the family Sillaginidae Hormozgan coastal waters using microsatellite markers. (: order Perciformes). Zootaxa. 3642(2013)1–105. J. Mar. Sci. Technol. 13(2014)1–10.