Proceedings of the 2006 WSEAS Int. Conf. on Cellular & Molecular Biology, Biophysics & Bioengineering, Athens, Greece, July 14-16, 2006 (pp154-159)
Phylogeny of the genus Artemia based on RFLP analysis of mtDNA in IRAN
MAHBOBEH HAJIROSTAMLOO 1, SOHRAB REZVANI GILKOLAEI 2, SEID MOHAMMAD REZA FATEMI 3, MAJEED SADEGHIZADEH 4, SHAHROKH KAZEMPOUR OSALOO 5 1Department of Biology, Islamic Azad University, Marand Branch, Marand 54165 161, IRAN 2Iranian Fisheries Research Organization, Tehran 14155 6116, IRAN 3Department of Marin Biology, Islamic Azad University, Science and Research Branch, Tehran 14515 775, IRAN 4Department of Genetics, Faculty of Basic Sciences, Tarbiat Modares University, Tehran 14115 175, IRAN 5Department of Plant Biology, Faculty of Basic Sciences, Tarbiat Modares University, Tehran 14115 175, IRAN Email: [email protected] Abstract: Phylogenetic relationships among the Iranian populations of Artemia belonging to three species of Artemia parthenogenetica, A. franciscana and A. urmiana were inferred by PCR RFLP analysis of a 1566 bp segment of the mitochondrial 16S rRNA gene. A total of 270 specimens from 9 Artemia populations inhabiting in the inland Iranian lakes (Urmia, Shoor, Inche Burun, Maharloo, Bakhtegan, Namak, Hoze Soltan, Mighan and Nough) was surveyed using 10 restriction enzymes. Twelve different haplotypes were detected in A. parthenogenetica populations, including four haplotypes in Shoor and Inche Burun, one in Bakhtegan, one in Namak and Hoze Soltan, three in Mighan and three in Maharloo. Four different haplotypes of A. franciscana were found in Nough population and nine different haplotypes of A. urmiana were found in Urmia population. Phylogenetic analysis showed these nine populations were clustered into three clades: 1 Shoor, Inche Burun, Hoze Soltan, Namak, Bakhtegan and Maharloo 2 Nough and Mighan and 3 Urmia. In this study referring to literature and basing on the oldness of A. urmiana, it selected as the outgroupe. Obtained results also show the ability of used technique for division of different populations of Artemia in Iran and parthenogenetic Artemia`s derivation from bisexual populations and high genetically resemblance with those of A. urmiana. Key-word: Artemia, mtDNA, phylogeny, PCR RFLP, rRNA , IRAN 1 Introduction [22]. In spite of its great interest and extensive use Iran is in south half of temporal region in northern in aquaculture, little is known about the systematic hemisphere and most parts of country have warm of the different Artemia strains and species [18]. climate. Water of some river, lakes and pools are Molecular DNA tools have been widely applied brine because they pass or settling in lands with in different organisms during the post decade in high salt content, most of this lakes and pools are studying genetic similarities and differences, habitats of Artemia, likes Urmia lake in West phylogenetic relationships and molecular evolution. Azerbaijan, Shoor and Inche Borun lakes in This is especially important in phyla where no Golestan, Hoze Soltan and Namak lakes in Qom, fossil records are available and/or there are no clear Maharloo and Bakhteghan lakes in Fars, Nough morphological traits to be used as markers of the pool in Kerman and Mighan pool in Markazi. different population [18]. Over the last decade, The brine shrimp Artemia (Family Artemidae, modern techniques and new molecular markers order Anostroca, class Branchiopoda, subphylum have been increasingly used in Artemia research [1, Crustacean and phylum Arthropoda) is a widely 24, 22, 2, 3, 10, 11, 20, 12]. Their application, in distributed throughout the world in inland salt combination with complementary information from lakes, coastal lagoons, and salterns. The genus of other techniques, makes them particularly useful in Artemia consists of a set of bisexual species providing better descriptions and interpretations of defined by the criteria of reproductive isolation and biological diversity [24, 3] a large number of parthenogenetic forms can be a Its high rate of evolution, maternal mode of great variety of ploidies grouped under the binomen inheritance, and lake of recombination has made A. partenogenetica, for taxonomic convenience mtDNA the most useful molecule in this kind of Proceedings of the 2006 WSEAS Int. Conf. on Cellular & Molecular Biology, Biophysics & Bioengineering, Athens, Greece, July 14-16, 2006 (pp154-159)
analysis [18]. It is small, compactly organized with 2.2 DNA extraction and PCR few intergenic spaces and with few exceptions its DNA extracted using the differential centrifugation gene content is strictly conserved in the animal and phenol chloroform method with some kingdom [14]. It is well established that its modification [12]. Extracted DNAs electrophoresed mutation rate is quite high, 5 10 times higher on %1 agarose gel containing 0.5 µg/ml ethidium compared with single copy nuclear DNA, probably bromide in TBE buffer (90 mM Tris, 90 mM boric be cause there are no DNA repair mechanisms in acid, 2 mM EDTA, PH=8) and visualized with gel the mitochondria [10]. Very preliminary attempts documentation system for their quality. based on mtDNA restriction fragment length One mtDNA segments amplified with PCR using polymorphism (RFLP) analysis, were ventilated in the following primers: the early 1990s for determining evolutionary Forward: 5′ CCG GTC TGA ACT CAG ATC – 3′ divergence in Artemia [6, 7]. Two ribosomal RNAs Reverse: 5′ CTA GGA TTA GAT ACC CTA – 3′ (12 S and 16 S) are fragments of mt DNA that These primers designed based on a part of the separated from each other by tRNA Val. In this Artemia franciscana 16sr RNAs sequence [25]. research effort we use 16srRNA for designing Each PCR reaction composed of 1 µl of extracted primers, because previous studies [11, 20, 12] DNA, 5 µl of 10X PCR buffer (500 mM kcl, 200 showed this gene is a highly favorable fragment for mM Tris Hcl), 0.5 µl of d NTP mix (10 mM each genetically similarities and differences, to estimate of dATP, dTTP,dCTP and dGTP , PH=7.5), 2 µl of the genetic relationship among different Artemia each of 2 primers to final concentration of 20 pM, population of Iran and modeling their phylogenetic 0.5 µl of Taq polymerase (5 unit per µl) , 2 µl pattern by PCR RFLP analysis of mt DNA. Mgcl (50 ml M) and deionized water added to a 2 final volume of 50 µl. The amplification conditions consisted of initial denaturation at 94oc for 4 min 2 Materials and Methods and 30 sec, 30 cycles performed at 94oc for 1 min and 15 sec, 54oc for 50 sec, 72oc for 1 min as 2.1 Artemia population denaturation, annealing and extension respectively. Adults Artemia individuals were collected from Finally 72oc for 4 min as final extension performed. night different population: Urmia, Shoor, Inche Electrophoresis of 10 µl of the amplified product burun, Namak, Hoze Soltan, Maharloo and performed for their quality on a %1 agarose gel. Bakhtegan lakes and Mighan and Nough flaques in The size of amplified fragments estimated with a Iran (Fig.1). Samples were fixed in ethanol (%95) DNA size marker (Lambda DNA Marker, 3) on a and transferred to Caspian Sea Ecological Research %6 polyacrylamid gel in TBE buffer and bands Institute for molecular study. Molecular visualized with silver staining [12]. information of A.franciscana as reference species calculated on its complete mtDNA sequence [25]. 2.3 RFLP analysis PCR products (4 7 µl) separately digested by 10 restriction endonucleases: AluI, EcoRI, Eco47I, HaeIII, HindIII, HinfI, MobI, MspI, RsaI, TaqI in a final volume 20 µl according to the manufacture suggested protocol, to display DNA polymorphism. Then the digested samples run on %6 polyacrylamide gel and staining. The size of restriction fragments estimated in comparison with a DNA size marker (Ladder 50 bp).
2.4 Phylogenetic analysis For each endonuclease, produced restricted patterns were identified and all aligned by size. Individuals were coded based on existence or non existence of each restricted fragment with specific number Fig1. Sampling sites of Iranian Artemia population: (1or0) and assigned a multi number code that 1) Urmia 2) Inche borun 3) Shoor 4) Hoze Soltan described their composite mt DNA genotypes. 5) Namak 6) Mighan 7) Nough 8) Maharloo 9) Phylogentic analysis was performed on multi Bakhtegan number codes using maximum parsimony method Proceedings of the 2006 WSEAS Int. Conf. on Cellular & Molecular Biology, Biophysics & Bioengineering, Athens, Greece, July 14-16, 2006 (pp154-159)
(MP) as implemented in the version 4.0b16 of %6 polyacrylamid gel, M= Marker PAUP [23]. The strict Consensus and Distance trees were obtained and there Bootstrap values, 3.2 Digestion with restriction endonuclease Length, Consistency Index (CI), Retention Index PCR products digested using 10 restriction (RI), Rescaled Consistency Index (RC) were enzymes. All enzymes restricted amplified DNA, calculated. With attend to condition that Maddison, produced fragments were clear and there totally etal. [16] were pointed about correct selection of sizes in comparison with DNA marker ladder 50bp Out group likes: Out group must be older than In were same with PCR Product. Nine of ten group and there must be no far distance between endonucleases (MspI, HinfI, HindIII, Eco47I, AluI, Out and In groups and with attention to previous MobI, HaeIII, RsaI, TaqI) were showed inter and studies that indicated to oldness of Artemia interapopulation polymorphism that indicated to Urmiana in compare with other genus [7] it was different cut site in nucleotides sequence of selected as Out group. Strict consensus tree 16SrRNA within and among Iranian Artemia adequately summarizes the available evidence exist population. Results of enzymatic digestive in different phylogenic tree offered with PAUP and indicated to existence of 25 haplotypes of Artemia distance tree that able to explain genetically in Iran: 9 in Urmia, 4 in Shoor and Inche Borun, 4 distance between clades and phylogenic modeling in Nough, 1 in Namak and Hoze Soltan, 3 in were obtained using the neighbor joining method Mighan, 1 in Bakhtegan and Maharloo and 3 in [21] in PAUP using the two parameter method of Maharloo. Kimura [15] with 0.005 change scale. To obtain confidence limits for various clades, a bootstrap 3.3 Phylogenetic Analysis analysis [9] was conducted. Bootstrap values were Maximum parsimony analysis of the 16srRNA data calculated from 1000 replicate analyses with a set set resulted many parsimonious topologies. The Maxtrees limit of 100 trees per replicate. strict consensus of these trees with accompanying bootstrap values is presented in fig .3, these trees have a length of 203 steps, a consistency index of 3 Results 0.718, a retention index of 0.980 and a rescaled consistency index of 0.703. The neighbor joining 3.1 DNA extraction and PCR distance tree (fig.4) rendered with few exception, a All extracted DNA had acceptable quality and similar topology for phylogenetic relationships quantity without any pollution and the size of PCR within and between Artemia populations. amplified mtDNA fragment in comparison with Phylogenetic analyses of the 16srRNA data set DNA marker (Lambada DNA Marker, 3) on %6 by the two methods demonstrated that all Artemia polyacrylamid gel was approximately 1570 (1566) populations and species sampled in Iran belong to bp. This size was the same for all samples and there monophyletic group. Both MP strict consensus and was no heteroplasmy in samples (Fig.2). N.J tree (figs.2 and 3) reveal that genus Artemia in Iran is composed of three large clades. One clade (hereafter called the clade "A") is weakly supported 1584 by 54 % bootstrap and contains Shoor, Inche Burun, Hoze Soltan, Namak and Maharloo samples (this clade is different in MP and N.J trees). The second clade (clade "B") contain Nough and Mighan samples, is a strongly supported clade by 100% bootstrap (this clade is same in Both MP and N.J trees). The third clade is clade "C", a moderately supported clade by 64% bootstrap, contain 9 different haplotype of Urmia sample (this clade is same in Both MP and N.J trees too). Clade A is a large assemblage that, in turn, comprises three core clades: A1, A2, A3. A1 contain different haplotypes of Inche Burun and Shoor with 93% bootstrap. A2 contain different haplotypes of M Maharloo, samples of Hoze Soltan and Namak with Fig.2 PCR amplified fragment electrophoresed on 68% bootstrap, there is a trichotomy among Proceedings of the 2006 WSEAS Int. Conf. on Cellular & Molecular Biology, Biophysics & Bioengineering, Athens, Greece, July 14-16, 2006 (pp154-159)
samples of Hoze Soltan, Namak and haplotype regions and its similar to those explain for highly number 2 of Maharloo. A3 contain haplotype geographic dispersal organism. number 4 of Maharloo and samples of Bakhtegan In addition, the result indicate to with 93% bootstrap. parthenogenetic Artemias derivation from bisexual Clade B comprises two core clades: B1, B2. B1 populations and high genetically resemblance with contains different haplotypes of Nough and A. urmiana, that similar to same studies. A.franciscana as reference (100% bootstrap). B2 The phylogenic analysis of Artemia population contains different haplotypes of Mighan (3 in Iran suggests that Iranian Artemia is grouped in 3 different haplotype with 100% bootstrap). main clades (with well and strongly bootstrap): Clade C Contains 9 different haplotypes of 1 Samples of Shoor, Inche burun, Hoze Soltan, Urmia populations with 64% bootstrap, most of Namak, Bakhtegan and Maharloo. these haplotypes have sister position to each other. 1 Samples of Nough and Mighan 3 Samples of Urmia There are some differences in first clade of MP and 4 Discussion N.J trees, the phylogenetic placement of haplotypes The brine shrimp Artemia is a highly favorable number 4 of Inche Burun and Namak are organism for evolutionary studies as it has a range unresolved. In MP tree they are placed in A1 (sub of key factor driving speciation not very often seen clade) and grouped by other haplotypes of their in other organism [10]. population but in N.J tree they are placed in A3 and Several authors have demonstrated the grouped by haplotypes number 4 of Maharloo and discriminatory power of RFLP analysis of Bakhtegen samples. There is also a trichotomy amplicons obtained from mitochondrial DNA for among samples of Hoze Soltan, Namak and the identification of animal species [17, 5, 19, 4, 8]. haplotype number 2 of Maharloo, it’s indicated to Using this approach are not subjected to sequencing hesitancy to phylogenic relation and derivation but are digested directly with restriction time of these samples, for resolving this problem endonucleases. needs that more amount of molecular characters or MtDNA can be considered as one, Artemia samples studied. hypervariable haploid locus, representing a small No genetic information was available so far for part of the total genome, and its evolutionary Iranian Artemia populations, therefore, this dynamics are quite distinct from those of the demonstrates the power of molecular markers for a nuclear genome [13]. simple and fast genetic identification of samples. As a first step towards getting a clearer picture of Our results reveal that PCR RFLP is a suitable phylogenic relation ship between 9 Artemia technique with enough sensitivity for recognition populations this study aimed at confronting and separation of different population of Artemia previous data on the population structure of Iranian and appoint their phylogenetic relationship. Artemia to that revealed by the analysis of RFLPs of mtDNA. In particular, focus was placed on mtDNA variability with in populations from References: different geographic locations in Iran as a way to [1] Abatzopolous, T.J., Triantaphyllidis, G.V., establish their levels of phylogenetic relationship. Beardmore, J.A. and Sorgeloos, P., Cyst Ten restriction endonucleases were used in this membrane composition as a discriminant study that nine enzymes were found to give character in the geneus Artemia (International polymorphism, this enzymes were performed 84 study on Artemia LV), Journal of Marine molecular character based on restricted fragment Biological Association of the United Kingdom, that they produced, 7 of this character were Vol.77, 1997, pp.265 268. symplesimorphic, its mean that they were common [2] Abatzopolous, T.J., Beardmore, J.A., Clegg, between all samples and did not have phylogenic J.S. and Sorgeloos, P., Artemia: basic and worth. 5 enzymes: Eco47I, HaeIII, HinfI, MspI and applied biology. Kluwer Academic TaqI revealed non symplesiomorphic which make Publishers, Dordrecht, 2002. them reliable markers for phylogenic purpose. [3] Abatzopolous, T.J., Kappas, I., Bossier, P., All samples are monophylic and have common Sorgeloos, P. and Beardmore, J.A., Genetic ancestor with A.urmiana. Existence of several characterization of Artemia tibetiana haplotypes indicated to high diversity of (Crustacea: Anostraca). Biological journal of mitochondrial genome of Artemia in studied the Linnean society, Vol.75, No.3, 2002, pp.333 344. Proceedings of the 2006 WSEAS Int. Conf. on Cellular & Molecular Biology, Biophysics & Bioengineering, Athens, Greece, July 14-16, 2006 (pp154-159) Strict 100 Nough NO.4 70 Inche Borun No.2 94 A. franciscana Shoor No.2 76 95 Nough NO.3 Inche Borun No.3 100 85 Nough NO.2 93 Shoor No.3 100 BB11 Nough NO.1 Inche Borun No.4 93 99 100 Mighan NO.2 Shoor No.4 B 100 Mighan NO.3 A1 B A1 Inche Borun No.1 94 Mighan NO.1 B2 87 Shoor No.1 B2 82 Urmia NO.6 Hoze Soltan 87 Urmia NO.7 Namak CC 94 Urmia NO.8 54 Maharloo NO.2 68 68 Urmia NO.9 Maharloo NO.3 85 Urmia NO.4 AA AA22 Maharloo NO.1 Urmia NO.5 93 Maharloo NO.4 Urmia NO.2 Bakhtegan 77 75 AA33 Urmia NO.3 96 Nough NO.4 Inche Borun No.2 97 A. franciscana 97 Shoor No.2 68 Nough NO.3 90 100 Inche Borun No.3 Nough NO.2 AA11 92 Shoor No.3 Nough NO.1 98 100 BB11 Inche Borun No.1 Mighan NO.2 65 80 AA22 Shoor No.1 B 100 Mighan NO.3 B Hoze Soltan Mighan NO.1 BB22 Maharloo NO.1 64 Urmia NO.6 95 Urmia NO.7 Namak 92 67 A Maharloo NO.2 92 Urmia NO.8 A 64 Urmia NO.9 Maharloo NO.3 Maharloo NO.4 68 58 Urmia NO.4 93 Urmia NO.5 Bakhtegan 64 Urmia NO.3 100 Inche Borun No.4 AA33 CC Urmia NO.2 Shoor No.4 Urmia NO.1 Urmia NO.1 (A.Urmiana) (A.Urmiana) 0.005 changes Fig.3 Strict consensus of most parsimonious trees resulting from Fig.4 Neighbor joining distance tree resulting from phylogenetic analysis phylogenetic analysis of 16srRNA gene of mtDNA for Artemia of 16srRNA gene of mtDNA for Artemia. Branch lengths are proportional (Length=203 steps, CI=0.718, RI=0.980). Numbers above branches are to distances estimated from kimura’ s (1980) two parameter method (note bootstrap values for 1000 replicate analyses (with a set maxtrees limit of scale bar). Numbers above branches are bootstrap values for 1000 replicate 100 trees per replicate); values < 50% are not indicted. Clades identified analyses; Values < 50% are not indicated. Clades identified by letters (A by letters (A C) are discussed in the text. C) are discussed in the text. Proceedings of the 2006 WSEAS Int. Conf. on Cellular & Molecular Biology, Biophysics & Bioengineering, Athens, Greece, July 14-16, 2006 (pp154-159)
[4] Behrens, M., Unthan, m., Brinkmann, Y., [17] Meyer, R., Hofelein, C., Luthy, J., Candrian, Buchholz, R., Latus, N., Identification of animal U., Polymerase chain reaction restriction species in heated and complex meat products fragment polymorphism analysis: a simple using specific PCR reactions, Fleischwirtschaft, method for species identification in food, Journ. Vol.4, 1999, pp.17 22. AOAC int., Vol.78, No.6, 1995, pp.1542 1551. [5] Borgo,R., Souty Grosset, C., Bouchon, D., [18] Perez, M.L., Valverde, J.R., Batuecas, B., Gomot, L., PCR RFLP analysis of Amat, F., Marco, R., Garesse, R., Speciation in mitochondrial DNA for identification of snail the Artemia genus: Mitochondrial DNA analysis meat species, Journal Food Science, Vol.61, of bisexual and parthenogenic brine shrimps, No.1, 1996, pp.1 4. Journal of molecular evolution, Vol.38, 1994, [6] Browne, R.A. and Hoopes, C.W., Genotype pp.156 168. diversity and selection in asexual brine shrimp [19] Ram, J.L.,Ram, M.L., Baidoun, F., (Artemia), Evolution, Vol.44, 1990, pp.1035 Authentication of canned tuna and bonito by 1051. sequence and restriction site analysis of [7] Browne, R.A. and Bowen, S.T., Taxonomy and polymerase chain reaction products of population genetics of Artemia, Artemia biology mitochondrial DNA, Journal Agriculture food (ed. by R.A. Browne, P. Sorgeloos and C.N.A chemistry, Vol.44, No.8, 1996, pp.2460 2467. Trotman), CRC Press. Boca Raton, FL, 1991. [20] Rezvani Gilkolaei, S., Molecular study of [8] Cocolin, L., Maifreni, M., Manzano, M., Comi, Artemia Urmiana, Iranian Fishery Research G., Cattaneo, P., Cantoni, C., A PCR RFLP for Organization, unpublished, 2004. identification of various animal meat, Industrie [21] Saitou, N. and Nei, M., The neighbor joining Alimentari, Vol.37. 1999, pp.657 661. method: A new method for reconstructing [9] Felsenstein, J., Confidence limits on evolutionary tress. Molec. Biol. Evol., Vol.4, phylogenies: An approach using the bootstrap, 1987, pp.406 425. Evolution, Vpl.39, 1985, pp.783 791. [22] Sun, Y., Cheng Zhong, Y., Qin Song, W., [10] Gajardo, G. ; Abatzopoolos, T.J. ; Kappas, I. Sheng Zhang, R. and Yang Chen, R., Detection and Beardmore, J.A. , Evolution and speciation, of genetic relationships among four Artemia Artemia: Basic and Applied Biology, Kluwer species using randomly amplified polymorphic academic publishers, 2002. DNA. International journal of Salt Lake [11] Gajardo, G. etal. , Species identification of research, Vol.8, 1999, pp.139 147. Chilean Artemia population based on [23] Swofoord, D.L., PAUP. Phylogenetic Analysis mitochondrial DNA RFLP analysis, Journal of Using Parsimony (and other methods). Version. Biogeography, Vol.31, 2004, pp.547 555. 4. Sinauer Associates, Sunderland, Massachuset, [12] Hajirostamloo, M., Study of interaspecies 2002.. diversity and population structure in Iranian [24] Triantaphyllidis, G.V., Criel, G.R.J., Artemia by molecular genetic technique, PhD Abatzopolous, T.J., Thomas, K.M., Peleman, J., thesis, Islamic Azad University Science and Beardmore, J.A. and Sorgeloos, P., International Research branch, Tehran, 2005. study of Artemia LVII. Morphological and [13] Hansen, M.M and Loeschcke, V., Genetic molecular characters suggest conspecifity of all differentiation among Danish brown trout bisexual European and North African Artemia population, as detected by RFLP analysis of populations. Marine biology, Vol.129, 1997, PCR amplified mitochondrial DNA segments, pp.477 487. Journal of fish Biology, Vol.48, 1996, pp.422 [25] Valverde, J.R., Batuecas, B., Moratilla, C., 436. Marco, R. and Garesse, R. The complete [14] Hoffman, R.J., Boore, J.L., Brown, W.M., A mitochondrial DNA sequence of the crustacean novel mitochondrial genome organization from Artemia Franciscana. Journal of molecular the blue mussel. Mytilus edulis, Vol.131, 1992, Evolution, No.39, 1994, pp.400 408. pp.397 412. [15] Kimura, M., A simple method for estimating evolutionary rates of base substation through comparative studies of nucleotide sequence. Journal of Molecular Evolution, Vol.16, 1980, pp.111 120 [16] Maddison, W.P., Gene trees in species tree. Syst. Biol., Vol.46, 1997, pp.523 536.