The objectives of this study were barcoding and taxonomic analysis of the five tilapiine species (Oreochromis aureus, O. niloticus, O. mossam- bicus, Sarotherodon galilaeus, and Tilapia zillii), two tilapia hybrid strains (Florida red tilapia and Philippine red tilapia), and two endemic wild cichlids (Tristramella simonis and Astatotilapia flaviijosephi) available in Israel, as well as O. urolepis hornorum. Cytochrome oxidase subunit I (COI) 619 bp sequence traces of 104 individuals were assembled, aligned, and compared (GenBank project GI 209553463). The DNA sequences of two hybrid strains were identical to those of O. hornorum and O. aureus. Absence of intra-specific variability was detected in the commercially used species, O. aureus, S. galilaeus, O. mossambicus, and O. urolepis horno- rum. Two DNA sequence variants were detected in O. niloticus originating from Ghana and Egypt. In contrast, 2-3 variants were detected in the DNA of each of the non-commercial species. Amino-acid sequences were identical in all “true tilapias” and different from the sequences in the endemic cichlids. As a whole, the protein phylogenetic tree fitted the expected conventional taxonomy as opposed to the respective DNA-based tree. Sequences FJ348047-FJ348150 were submitted to GenBank via the BOLD database (identical to FISH001-08 - FISH104-08 in this database)

Barroso, Renata M.

Cohen-Zinder, Miri

Hulata, Gideon

Ron, Micha

Seroussi, Eyal

Shirak, Andrey

eVols at University of Hawaii at Manoa

The Open Access Israeli Journal of Aquaculture – BamidgehAs from January 2010 The Israeli Journal of Aquaculture - Bamidgeh (IJA) will be published  exclusively  as  an  on-line  Open  Access  (OA) quarterly  accessible  by  all AquacultureHub  (http://www.aquaculturehub.org)  members  and  registered  individuals and institutions.  Please visit our website (http://siamb.org.il) for free registration form, further information and instructions.  This transformation from a subscription printed version to an on-line OA journal, aims at supporting the concept that scientific peer-reviewed publications should be made available to all, including those with limited resources. The OA IJA does not enforce author or subscription fees and will endeavor to obtain alternative sources of income to support this policy for as long as possible.Editor-in-ChiefDan Mires Editorial BoardSheenan Harpaz Agricultural Research OrganizationBeit Dagan, IsraelZvi Yaron Dept. of ZoologyTel Aviv UniversityTel Aviv, IsraelAngelo Colorni National Center for Mariculture, IOLREilat, IsraelRina Chakrabarti Aqua Research LabDept. of ZoologyUniversity of DelhiIngrid Lupatsch Swansea UniversitySingleton Park, Swansea, UKJaap van Rijn The Hebrew University Faculty of AgricultureIsraelSpencer Malecha Dept. of Human Nutrition, Food and Animal SciencesUniversity of HawaiiDaniel Golani The Hebrew University of JerusalemJerusalem, IsraelEmilio Tibaldi Udine UniversityUdine, ItalyCopy Editor Ellen Rosenberg Published under auspices ofThe Society of Israeli Aquaculture and Marine Biotechnology (SIAMB), University of Hawaii at Manoa Library andUniversity of Hawaii Aquaculture Program in association withAquacultureHub http://www.aquaculturehub.org              ISSN 0792 - 156X Israeli Journal of Aquaculture - BAMIGDEH.PUBLISHER: Israeli Journal of Aquaculture - BAMIGDEH -Kibbutz Ein Hamifratz, Mobile Post 25210, ISRAELPhone: + 972 52 3965809http://siamb.org.il The Israeli Journal of Aquaculture – Bamidgeh  61(2), 2009, 83-88. 83Full article available to e-journal subscribers only athttp://www.siamb.org.ilDNA Barcoding of Israeli Indigenous and Introduced CichlidsAndrey Shirak, Miri Cohen-Zinder, Renata M. Barroso, Eyal Seroussi, Micha Ron and Gideon Hulata*Institute of Animal Science, Agricultural Research Organization (ARO), Volcani Center, Bet Dagan 50250, Israel(Received 7.12.06, Accepted 1.1.09)Key words: barcoding, taxonomic analysis, cichlids, cytochrome oxidase subunit I, variability, commercial species, conventional taxonomy* Corresponding author. E-mail: vlaqua@volcani.agri.gov.ilAbstractThe objectives of this study were barcoding and taxonomic analysis of the five tilapiine species (Oreochromis aureus, O. niloticus, O. mossam-bicus, Sarotherodon galilaeus, and Tilapia zillii), two tilapia hybrid strains (Florida red tilapia and Philippine red tilapia), and two endemic wild cichlids (Tristramella simonis and Astatotilapia flaviijosephi) available in Israel, aswell as O. urolepis hornorum. Cytochrome oxidase subunit I (COI) 619 bp sequence traces of 104 individuals were assembled, aligned, and compared (GenBank project GI 209553463). The DNA sequences of two hybrid strains were identical to those of O. hornorum and O. aureus. Absence of intra-specific variability was detected in the commercially usedspecies, O. aureus, S. galilaeus, O. mossambicus, and O. urolepis horno-rum. Two DNA sequence variants were detected in O. niloticus originating from Ghana and Egypt. In contrast, 2-3 variants were detected in the DNA of each of the non-commercial species. Amino-acid sequences were identical in all “true tilapias” and different from the sequences in the endemic cichlids. As a whole, the protein phylogenetic tree fitted the expected conventionaltaxonomy as opposed to the respective DNA-based tree. Sequences FJ348047-FJ348150 were submitted to GenBank via the BOLD database (identical to FISH001-08 - FISH104-08 in this database).Shirak et al.84 DNA barcoding of cichlids 85IntroductionDNA sequence analysis has been used for over 30 years to assist in species identification, butdifferent sequences have been used for different taxonomic groups (Ward et al., 2005). The con-served sequence of the 5’ region of the mitochondrial gene cytochrome oxidase subunit I (COI or cox1) was proposed as a platform for the universal DNA barcoding of life (Hebert et al., 2003). Of the estimated 670,000 extant species in the world, more than 49,000 have already been for-mally characterized with DNA barcodes and are registered in the Barcode of Life Data System (BOLD, www.barcodinglife.org). Under that system, the Fish Barcode of Life initiative (FISH-BOL, www.fishbol.org) focuses on fish species, comprising half the global vertebrate species. Currently,barcodes of more than 5,600 out of 30,000 fish species are included in the database.Tilapiine species (family Cichlidae) inhabit the fresh and brackish water of Africa, the Middle East, coastal India, and Central and South America. “True tilapias”, a group of approximately 50 species of perch-like fishes, are endemic only to Africa and the Middle East. The group consistsof three genera Oreochromis, Sarotherodon, and Tilapia (Trewavas, 1982). The objective of this study was to DNA barcode the cichlids available in Israel and compare them with O. urolepis hornorum. Materials and MethodsThe cichlid species were sampled from native waters (Tilapia zillii, Tristramella simonis, and As-tatotilapia flaviijosephi), as well as from aquaculture and experimental facilities. Fin samples werecollected from 104 individuals of six tilapiine species, two tilapia hybrid strains and two endemic wild cichlids (Table 1). The species O. urolepis hornorum is not available in Israel; thus, preserved fin samples from Brazil were used in this study. Florida red tilapia and Philippine red tilapia arecommercially cultured hybrids.DNA extraction was performed using the salting out procedure (Ma et al., 1996). DNA samples were diluted 1:10-1:50 and 3 µl of samples were used as a template for PCR amplification of theCOI sequence. COI-3 primer cocktail and M13 primers were used for amplification and sequenc-ing, respectively, following Ivanova et al. (2007). PCR products were run on 1.5% agarose gel. Relevant band sizes were excised from the gel, purified with DNA Montage Gel Extraction Kit(Millipore, Bedford, MA, USA), and sequenced. Sequence trace files were assembled using theGAP4 program (Staden et al., 1999). Amino-acid sequences were aligned with ClustalW using the default parameters and the GONNET weight matrix (http://clustalw.genome.jp/). Phylogenetic trees were constructed using MEGA (version 4.0.2) using the default models recommended by FISH-BOL bioinformatic tool for similar analyses. Results and DiscussionAll 104 examined samples were successfully amplified and sequenced. Sequence traces of COI619 bp region were assembled, aligned, and compared. Sequence alignment may be generated using NCBI PopSet module (www.ncbi.nlm.nih.gov/sites/entrez?db=PopSet&cmd=search&term=209553463). The COI sequences of the Florida and Philippine red tilapia hybrids were identical to those of O. hornorum and O. aureus, respectively, in agreement with documentation of the history of these strains (Watanabe et al., 1989, Galman and Avtalion, 1983, respectively).Sequence comparison showed absence of intra-specific variability in the four commerciallycultured species O. aureus, S. galilaeus, O. mossambicus, and O. urolepis hornorum. Two Shirak et al.84 DNA barcoding of cichlids 85Table 1. Israeli indigenous and introduced cichlid species and stocks barcoded in this project.ARO = Agricultural Research Organization; BIU = Bar-Ilan University; CHESF = Fish Aquaculture Station, BrazilSpeciesSarotherodon  S. galilaeusOreochromis  O. mossambicus  O. urolepis hornorum  O. aureus  O. aureus  O. aureus  O. aureus  O. aureus  O. niloticus  O. niloticus  O. niloticus  O. niloticus  Florida red tilapia hybrid  Philippine red tilapia hybridTilapia  T. zillii  T. zillii  T. zilliiOther cichlids  Tristramella simonis  Astatotilapia flaviijosephiTotalOrigin of speciesIsraelEast AfricaNorth-East AfricaEgypt, Lower NileIsraelIsraelIsraelIsraelEgyptEgypt, Lake Manzala     (red mutant)EgyptGhanaFlorida-IsraelIsraelIsraelIsraelIsraelOrigin of stockSea of GalileeNatal, South AfricaBrazilUniv. of Stirling, UKMehadrin ReservoirEin Feshkha Ein Feshkha Bet Shean ValleyUniv. of Swansea,     WalesUniv. of Stirling, UKCanadaVolta RiverJamaicaPhilippinesYarkon streamSea of Galilee-Jordan-Dead Sea     rift valleyJordan-Dead Sea     rift valleyOrigin of sampleARO, BIUAROCHESF AROAROAROBIUNir DavidAROAROAROARO, BIUSde Eliyahu BIUBIUAROGan ShmuelSea of GalileeSea of GalileeSample size109657134343246375436104Shirak et al.86 DNA barcoding of cichlids 87sequence variants (FJ348104 and FJ348106) were detected in O. niloticus originating from Ghana, while only one variant (FJ348115) was detected in all other stocks originating from Egypt. In contrast to commercial species, 2-3 variants were detected in each of the non-commercial species T. zillii, Tr. simonis, and A. flaviijosephi. Analysis of intra-specific variation in T. zilliipopulations revealed two variants that differ by a single nucleotide substitution. Both variants were found in the Yarkon River (FJ348145 and FJ348147), but only one in the Sea of Galilee (FJ348140) and Gan Shmuel near Hadera stream (FJ348135). Phylogenetic trees based on protein and DNA sequences are presented in Figs. 1a and 1b, respectively. Conventional taxonomy (Gittleman, 1981) predicted grouping the examined species into three groups: (1) A. flaviijosephi, (2) Tr. simonis, and (3) all other species that are the membersof “true tilapias” and their sub-groups i.e. Oreochromis, Sarotherodon, and Tilapia. The protein phylogenetic tree (Fig. 1a) fits the expected conventional taxonomy; “true tilapias” are grouped inthe same branch due to identical amino-acid sequences. However, the DNA-based tree (Fig. 1b) does not fit this taxonomy. In general, the same phylogenetic tree was obtained whether the thirdcodon nucleotide or the whole nucleotide sequence was compared. This tree has three major branches: (1) T. zillii; (2) A. flaviijosephi; and (3) all other species including Tr. simonis. The lattergroup is sub-grouped into two major branches: (1) three species from the Sea of Galilee and (2) three African Oreochromis species. Thus, two major findings were unexpected: first - O. aureusclusters with S. galilaeus and Tr. simonis, according to the common geography, but not with the other members of the Oreochromis genus; and second - T. zillii is distant from other “true tilapias”, even more than A. flaviijosephi.In accordance with Russo et al. (1996), we conclude that the protein sequence rather than the DNA sequence represents the more likely phylogenetic tree. However, the protein sequence has low phylogenetic resolution for evolutionary-young species such as the Tilapiini, in which case a DNA-based tree might be advantageous. The convergence of the DNA sequences of the three taxonomically distant species from the Sea of Galilee (O. aureus, S. galilaeus, and Tr. simonis) into a single branch (Fig. 1b) is rather surprising and should be further studied.AcknowledgementsWe thank Dr. Daniel Golani and Dr. Alexandre W.S. Hilsdorf for their help in collecting some of the fin samples used in this study. Contribution from the Agricultural Research Organization, Instituteof Animal Science, Bet Dagan, Israel, No. 538/2008.Shirak et al.86 DNA barcoding of cichlids 87Fig. 1. Phylogenetic trees of endemic and introduced cichlid species. Distances were calculated using either (a) amino-acids translation of the 619 bp region of COI mitochondrial gene and the Dayhoff Matrix model, or (b) the third nucleotide in codons present in the 619 bp region of COI mitochondrial gene and the Kimura 2-parameter model. The trees were generated by MEGA4 using the neighbor-joining method and bootstrap (500 replicates) analysis. Numbers at tree junctions indicate the percentage of trees that correspond to the consensus bootstrap tree. The scale on the X-axis represents the distance in number of (a) amino-acid or (b) nucleotide substitutions per site. v = variant.S. galilaeusO. niloticus v1O. niloticus v2T. zillii v2O. mossambicusT. zillii v1O. aureusPhilippine red hybridO. hornorumFlorida red hybridTr. simonis v2Tr. simonis v1A. flaviijosephi v3A. flaviijosephi v1A. flaviijosephi v20.009 0.008 0.007 0.006 0.005 0.004 0.003 0.002 0.001 0.000548967aO. aureusPhilippine red hybridS. galilaeusTr. simonis v1Tr. simonis v2O. niloticus v1O. niloticus v2O. mossambicusO. hornorumFlorida red hybridA. flaviijosephi v3A. flaviijosephi v1A. flaviijosephi v2T. zillii v1T. zillii v20.20 0.15 0.10 0.05 0.00b681006385829947100100100100Shirak et al.88ReferencesGalman O.R. and Avtalion R.R., 1983. A preliminary investigation of the characteristics of red tilapias from the Philippines and Taiwan. pp. 291-301. In: L. Fishelson, Z. Yaron (Comps.). Proc. Int. Symp. Tilapia in Aquac., May 8-13, Nazareth, Israel. Tel Aviv Univ., Ramat Aviv, Israel.Gittleman J.L., 1981. The phylogeny of parental care in fishes. Anim. Behav., 29:936-941.Hebert P.D.N., Cywinska A., Ball S.L. and J.R. de Waard, 2003. Biological identificationsthrough DNA barcodes. Proc. Royal Soc. B: Biol. Sci., 270:313-321.Ivanova N.V., Zemlak T.S., Hanner R.H. and P.D.N. Hebert, 2007. Universal primer cocktails for fish DNA barcoding. Mol. Ecol. Notes, 7:544-548.Ma R.Z., Beever J.E., Da Y., Green C.A., Russ I., Park C., Heyen D.W., Everts R.E., Fisher S.R., Overton K.M., Teale A.J., Kemp S.J., Hines H.C., Gurin G. and H.A. Lewin, 1996. A male linkage map of the cattle (Bos taurus) genome. J. Hered., 87:261-271.Russo C., Takezaki N. and Nei M., 1996. Efficiencies of different genes and different tree-building methods in recovering a known vertebrate phylogeny. Mol. Biol. Evol., 13:525-536.Staden R., Beal K.F. and J.K. Bonfield, 1999. The Staden package, 1998. Methods Mol. Biol.,132:115-130.Trewavas E., 1982. Generic groupings of Tilapiini used in aquaculture. Aquaculture, 27:79-81. Ward R.D., Zemlak T.S., Innes B.H., Last P.R. and P.D.N. Hebert, 2005. DNA barcoding Austral-ia’s fish species. Phil. Trans. Royal Soc. London, Ser. B, Biol. Sci., 360:1847-1857.Watanabe W.O., Ernst D.H., Olla B.L. and R.I. Wicklund, 1989. Aquaculture of red tilapia (Oreochromis sp.) in marine environments - state of the art. AQUACOP IFREMER Actes Colloq., 9:487-498.

DNA Barcoding of Israeli Indigenous and Introduced Cichlids

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DNA Barcoding of Israeli Indigenous and Introduced Cichlids

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