The University of Maine DigitalCommons@UMaine Marine Sciences Faculty Scholarship School of Marine Sciences 3-1994 The Utility of SATA Satellite DNA Sequences for Inferring Phylogenetic Relationships an1ong the Three Major Genera of Tilapiine Cichlid Fishes Jens P. C. Franck Irv Kornfield Jonathan M. Wright Follow this and additional works at: https://digitalcommons.library.umaine.edu/sms_facpub Part of the Oceanography and Atmospheric Sciences and Meteorology Commons This Article is brought to you for free and open access by DigitalCommons@UMaine. It has been accepted for inclusion in Marine Sciences Faculty Scholarship by an authorized administrator of DigitalCommons@UMaine. For more information, please contact [email protected]. MOLECULAR PHYLOGENETICS AND EVOLUTION Vol. 3, No. 1, March, pp. 10-16, 1994 The Utility of SATA Satellite DNA Sequences for Inferring Phylogenetic Relationships an1ong the Three Major Genera of Tilapiine C:ichlid Fishes 1 2 JENs P. C. FRANCK,*· IRv KoRNFIELD.t AND JoNATHAN M. WRIGHT*· *Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada, 83H 417; and tDepartment of Zoology and Center for Marine Studies, University of Maine, 5757 Murray Hall, Orono, Maine 04469-5757 Received June 21, 1993; revised November 3, 1993 blage are the tilapiine and haplochromine fishes (Re­ The SATA satellite DNA family of sequences, com­ gan, 1920). Taxonomic classification of tilapias has posed of three size variants of approximately 237, 230, relied primarily upon morphological characters (Stias­ and 209 hp, is conserved in the genomes of tilapiine and sny, 1991) and reproductive behavior (Trewavas, 1982, haplochromine cichlid fishes. In the present study we 1983). Trewavas identified three major tilapiine gen­ examined the utility of the SATA sequences for infer­ era as maternal mouthbrooders (Oreochromis), bipa­ ring phylogenetic relationships among the three major rental and paternal mouthbrooders (Sarotherodon), genera of tilapiine fishes, Oreochromis, Sarotherodon, and substrate spawners (Tilapia). and Tilapia. Hybridization of the monomer SATA re­ Two models have been proposed for the evolution of peat to genomic DNA of representative cichlid species brooding behaviors in tilapias. Peter and Berns (1982) established conservation of the sequence in the African argued that the mouthbrooding strains periodically di­ tilapiine and haplochromine lineages and its absence verged from the substrate spawning lineage, in which from other cichlid lineages. Bootstrapped DNA parsi­ the most ancient lineage is represented by the mater­ mony and neighbor-joining analyses of derived consen­ nal mouthbrooders while the more recently diverged sus sequences revealed two distinct clades, one con­ taining the mouthbrooding genera Oreochromis and species are represented by the biparental mouthbrood­ Sarotherodon, and the other containing the substrate ers. Trewavas (1982, 1983), however, proposed that the spawning genus Tilapia. These results are consistent mouthbrooding lineage originated from a Tilapia-like with recent independent studies using mitochondrial substrate spawner which subsequently split into the DNA and establish the utility of the SATA satellite DNA maternal and biparental mouthbrooding lines. Allo­ family for phylogenetic reconstruction. Concerted evo­ zyme analyses of the major tilapiine genera suggested lution of the SATA sequences was also demonstrated a clos·~ relationship of the mouthbrooding Oreochromis within the tilapiine tribe. @ 1994 Academic Press, Inc. and Sarotherodon genera as distinct from the substrate spawning Tilapia genus (Kronfield et al., 1979; McAn­ drew and Majumdar, 1984). Mitochondrial DNA INTRODUCTION (mtDNA) restriction analysis resolved the trichotomy among· these three tilapiine genera, and supported Cichlids are the predominant percoid fishes of the Trewavas' hypothesis (Seyoum, 1989; S. Seyoum and tropical regions of the world. Members of the cichlid I. Kornfield, unpublished). Restriction endonuclease family are broadly distributed in the Old World (Af­ analysis of mtDNAs has been used to clarify the taxon­ rica) and New World (Neotropical; Central and South omy of the Oreochromis subspecies complex (Kornfield, America), with a few additional taxa in the Indian sub­ 1991; Seyoum and Kornfield, 1992). In addition, two continent (Fryer and Iles, 1972). The startling explo­ recent mtDNA studies employed sequence information sive radiation of the cichlids, especially in the rift lakes to elucidate the relationships of the haplochromine of eastern Africa, has resulted in taxa with a wide di­ cichlids of Africa (Meyer et al., 1990; Sturmbauer and versity of ecology, morphology, and behavior (Stiassny, Meyer, 1992). 1991). Two major tribes of the African cichlid assem- Sat•:illite DNAs have been extensively studied in many organisms including invertebrates (Miklos, 1982, 1985), amphibians (Hummel et al., 1984), mam­ 1 Present address: Department of Organismal Biology and Anat­ mals (Singer, 1982; Amason et al., 1984), and plants omy, University of Chicago, 1025 East 57th Street, Chicago, Illinois 60637 (Flavell et al., 1983). Satellite DNAs in tilapiine fishes 2 To whom correspondence should be addressed. were first observed as intensely staining bands of ap- 10 1055-7903194 $6.00 Copyright© 1994 by Academic Press, Inc. All rights of reproduction in any form reserved. TILAPIINE PHYLOGENY 11 proximately 200 hp in EcoRi- and Haelll-digested ge­ Cloning Methodology and Sequencing nomic DNA of Oreochromis mossambicus x Oreoch­ The cloning methodology for the tilapiine satellite romis hornorum hybrids by agarose gel electrophoresis monomer repeats has been previously described (Wright, 1989). The consensus sequence is 237 hp long (Franck et al., 1992). The Protomelas similis SATA se­ and the satellite constitutes 7% of the haploid genome. quence was cloned from a Hinfl digest of genomic Subsequently, the homologous sequences were cloned DNA. The approximate 230-bp intensely staining band and characterized from representatives of Oreoch­ from the digest was eluted from the gel using a silica romis, Sarotherodon, and Tilapia (Franck et al., 1992). bead matrix (Geneclean II, Bio. 101). The eluted DNA The SATA satellite DNA family includes three major was incubated with Mung Bean nuclease, and the size variants of approximately 237 hp (type I), 230 hp blunt end was ligated into the Smal site of M13mpl8. (type II), and 209 hp (type Ill). Phage lysates were immobilized on a nylon membrane Recent studies have demonstrated the utility of sat­ (Hybond-N, Amersham) with a slot blot apparatus and ellite DNA sequences for inferring phylogenetic rela­ hybridized to the radiolabeled SATA monomer repeat, tionships. The resolving power of satellite DNA se­ Opll-5, from Oreochromis placidus. Five of the posi­ quences in systematic studies ranges from the tively hybridizing clones were sequenced and used to identification of conspecific populations of the Sheeps­ construct a consensus sequence. Single-stranded re­ head Minnow (Turner et al., 1991) to interfamilial rela­ combinant M13 templates were sequenced by the tionships of cetaceans (Amason et al., 1992; Gretars­ chain-terminating method (Sanger et al., 1977) using dottir and Amason, 1992). In the present study we a-35S-dATP (1000 Ci/mmol) with T7 DNA polymerase investigated the potential utility of the SAT A satellite (Pharmacia). DNA family for inferring phylogenetic relationships among the major genera of the tilapiine cichlid fishes. Radiolabeling Techniques and Hybridization The results supported the close relationship of the Conditions mouthbrooding genera Oreochromis and Sarotherodon in a clade distinct from the substrate spawning Tilapia The Opll-5 monomer repeat was labeled either by genus consistent with the monophyletic model of random priming (Feinberg and Vogelstein, 1983) or mouthbrooding evolution proposed by Trewavas (1982, by Klenow filling of the recessed 5' ends with [a- 32P]dATP (300 Ci/mmol). DNA was routinely labeled 1983). Additional analyses established the concerted 8 evolution of the SAT A sequences. to a specific activity of 10 cpm/µg. Nylon membranes were incubated for at least 2 h in a prehybridization mixture (50% formamide; 5 x SSPE; 1 x Denhardt's solution; 100 µg/ml yeast tRNA; 0.1 % SDS) (1 x SSPE MATERIALS AND METHODS = 0.015 M NaCl, 10 mmol sodium dihydrogenortho­ phosphate, 1 mmol EDT A) at 42°C. Radiolabeled probe Sources of Fish Specimens and Genomic DNA was added to a final concentration of 106 cpm/ml and All tilapiine specimens with the exception of 0. ni­ hybridization was allowed to proceed for 24 to 48 h. loticus, which was maintained at Dalhousie Univer­ Membranes were washed under low stringency condi­ sity, were obtained from Dr. Brendan McAndrew at tions (0.2 x SSC, 0.1% SDS) at room temperature the Institute of Aquaculture, University of Stirling, ( ~20°C). Membranes were exposed to Kodak XAR5 X­ Scotland. All other cichlid specimens were purchased ray film. To estimate the copy number of the repetitive from a local tropical fish store. Total genomic DNA DNAs, digitized images of the slot blot autoradio­ was extracted either from the caudal peduncle tissue graphs were quantified using the program ScanAna­ or from blood samples collected by caudal vein punc­ lysis Densitometry for the Macintosh (Release 2.20; ture of anesthetized fish. The extraction of DNA from Burcham, 1987). blood has been described previously (Wright, 1989). For DNA extraction from tissue, approximately