J. Appl. Ichthyol. 18 (2002), 290–297 Received: July 13, 2001 Ó 2002 Blackwell Verlag, Berlin Accepted: May 29, 2002 ISSN 0175–8659 Evidence of multiple alleles of the nuclear 18S ribosomal RNA gene in sturgeon (Family: Acipenseridae) By J. Krieger1 and P. A. Fuerst1,2 1Department of Molecular Genetics and 2Department of Evolution, Ecology and Organismal Biology, The Ohio State University, Columbus, OH, USA Summary The number of copies of the rRNA gene array is species Nuclear 18S ribosomal RNA (rRNA)gene sequences were specific; there may be hundreds or thousands of copies of the determined during a phylogenetic study of the evolutionary transcription unit in a particular species (Appels et al. 1980), relationships of the North American sturgeon and paddlefish. but there may also be as few as one copy, as in Tetrahymena In the course of this study, intra-individual variation of the 18S (Yao and Gall 1977). In some species, copies of the rRNA gene rRNA gene was discovered in all nine species of sturgeon array are found on more than one pair of chromosomes. examined, while no variation was detected for this gene in the Because of the large number of copies found in most North American paddlefish (Polyodon spathula)or in other genomes, various rRNA gene repeats in a chromosome or non-acipenseriform fish species included in the study (bichirs, genome may at first be thought to evolve independently, even reedfish, gar, bowfin and lungfish). The discovery of multiple though selection acts upon rRNA to retain essential and sequence variants of the 18S rRNA gene in an individual is conserved functions in protein synthesis. Previous studies of unexpected; although most eukaryotes possess many copies of rDNA in several organisms have shown, however, that the the rDNA unit in their genomes, the copies are normally gene repeats in an array do not evolve independently (Arnheim thought to be homogenized within an individual as well as in a 1983). The 18S, 28S and 5.8S rRNA genes normally show high, species by concerted evolution. The effect of the variation on usually complete, homogeneity of sequence among repeats the determination of species level relationships within the within an organism and even within a species, although order and of the relationship of the order to other fish groups differences in gene sequences appear when species are com- is examined. The actual cause(s)for the presence of intra- pared. The process of homogenization of repetitive sequences individual variation of the 18S rRNA gene are as yet is termed concerted evolution (Arnheim et al. 1980), and may unknown, however, it is possible that the origin and mainten- be caused by unequal crossing over, replicative transposition, ance of multiple 18S rDNA sequence variants in sturgeon may gene amplification or gene conversion (Dover et al. 1982). The be related to the polyploid status of the sturgeon genome. homogenization of rRNA gene repeats within individuals and species by concerted evolution ordinarily allows the species to be represented by a single 18S rRNA gene sequence in Introduction molecular phylogenetic studies (Hillis and Davis 1988). Here we report the first observation of extensive intra- With the assumption that the 18S rRNA genes of acipen- individual variation of the DNA sequences of the 18S rRNA seriforms would show no unusual characteristics, we obtained gene in any vertebrate. As an extension of a molecular 18S rRNA gene sequences from the North American paddle- phylogenetic study of the North American sturgeon and fish and nine species of North American sturgeon for the paddlefish (Krieger et al. 2000), sequences of the nuclear 18S purpose of a phylogenetic study. A single homogenous rRNA gene were determined. The 18S rRNA gene has been sequence was obtained for the gene in paddlefish, as generally used widely for phylogenetic studies of many organisms (for expected, and as seen in non-acipenseriform fish studied in our example, Stock et al. 1991; Stock and Whitt 1992; Wada laboratory and by other researchers. The results, however, 1998). Even though the rRNA genes exist in multiple copies were far from expected when sturgeon were studied. Multiple within all vertebrate genomes, sequence homogeneity, or near sequence variants were unexpectedly detected for the 18S homogeneity, within species has been the rule and a single 18S rRNA genes of all nine species of sturgeon. These findings rRNA gene sequence is usually used to represent a species in represent the first discovery of extensive intra-individual molecular phylogenetic studies (Hillis and Davis 1988). The variation of the 18S rRNA gene in any vertebrate, and we conserved function and structure of the 18S rRNA molecule describe here the relevance of this discovery to molecular allow sequences to be aligned, even among divergent species. phylogenetic analyses and our assumptions about the evolu- However, the molecule also possesses phylogenetically inform- tion of repetitive DNA sequences. ative variable regions that are useful for determining relation- ships among species (Hillis and Dixon 1991). In eukaryotes, Materials and methods the ribosomal RNA transcription unit usually exists in Specimens tandemly repeated arrays, containing three rRNA genes, 18S, 5.8S and 28S RNA genes, with the genes separated by Nuclear 18S rRNA gene sequences were examined in one spacer sequences (ITSs)(Fig. 1).Nontranscribed spacers individual for each of 10 acipenseriform taxa: Polyodon (NTS)separate tandem copies of the entire transcribed unit. spathula, Scaphirhynchus albus, S. platorynchus, S. suttkusi, U.S. Copyright Clearance Centre Code Statement: 0175–8659/2002/1804-06–0290$15.00/0 www.blackwell.de/synergy Multiple alleles of the nuclear 18S rRNA gene 291 Fig. 1. Diagram of the rDNA array of eukaryotes. The above represents a single transcription unit of rDNA that is found in tandem repeats on various homologous and/or non-homologous chromosomes of an organism. (NTS ¼ non-transcribed spacer; ETS ¼ external transcribed spacer; ITS ¼ internal transcribed spacer.)The detail of the small subunit (18S)rRNA gene shows PCR primers (and their orientations)used for amplification and sequencing of the gene Acipenser brevirostrum, A. fulvescens, A. medirostris, A. oxyrin- amplification of sturgeon and paddlefish genes because the chus oxyrinchus, A. o. desotoi, and A. transmontanus. Sequences lower error rate of Elongaseâ reduced the possibility that were also determined for one individual each of Polypterus sequence variation was a result of DNA polymerase errors senegalus and P. delhezi (bichirs), Erpetoichthys calabaricus during amplification. Details of amplification, amplification (reedfish), Lepisosteus osseus (longnose gar), Amia calva (bow- primers and sequencing primers are provided in Krieger 2000. fin)and a member of the lungfish genus Protopterus. Tissue The total size of the PCR gene products analyzed was samples (fin snips, muscle, blood or barbels)were obtained from approximately 1800 base pairs (bp)in all fish species examined. researchers and fisheries in the US or collected from fish Cloning of the entire 18S rRNA gene PCR product amplified purchased at a local aquarium store as described in Table 1. with the Elongaseâ enzyme mix was carried out on lake sturgeon (Acipenser fulvescens), shortnose sturgeon (A. brev- irostrum)and North American paddlefish ( Polyodon spathula) DNA extraction, PCR amplification, cloning and DNA sequencing using the TOPOâ TA PCR XL cloning kit (Invitrogen, Detailed molecular protocols are given in Krieger, 2000. After Carlsbad, CA)(Krieger, 2000).Clones were assigned names tissue homogenization and digestion, DNA extraction was based on the species plus a number (for example, 18S rDNA carried out following the standard phenol-chloroform method clones from lake sturgeon, A. fulvescens, were labeled Af-1, (Maniatis et al., 1982). The Polymerase chain reaction (PCR) Af-2,…, while clones from shortnose sturgeon, A. breviros- amplification (Saiki et al., 1988)of nuclear 18S rRNA genes trum, clones were labeled Ab-1, etc.). from total DNA was carried out in 100 ll reactions, using Gene sequences from PCR products and clones were either Taq DNA polymerase or Elongaseâ enzyme mix determined (in most cases for both strands)by manual (Gibco-BRL, Gaithersburg, MD). Taq DNA polymerase was sequencing using the dsCycle Sequencing System Kit (Gibco- used for PCR amplifications from all the non-acipenseriform BRL, Gaithersburg, MD)with 32P end-labeled sequencing species and preliminary amplifications from the acipenseriform primers. Sequencing products were separated on 6% poly- species. Elongaseâ Enzyme Mix was used for subsequent acrylamide gels. Sequences were deposited in GenBank under Table 1 Species examined in this study Species name Tissue sample Origin of specimen Collector/Provider of sample Polyodon spathula Fin snip Hatchery fish–Parents originally Blind Pony Lake Conservation Area, Missouri from Osage River Basin Scaphirhynchus albus Fin snip Hatchery fish–Parents National Biological Services Midwest Science Center, from Mississippi Missouri and Missouri rivers a Scaphirhynchus platorynchus Fin snip Missouri River National Biological Services Midwest Science Center, Missouri Scaphirhynchus suttkusi Muscle Alabama River (UAIC# 1885.01)University of Alabama at Tuscaloosa Acipenser brevirostrum Barbel Hatchery fish–Parents from Bears Bluff National Fish Hatchery, South Carolina Savannah River Acipenser fulvescens Fin snip Wolf River Wisconsin Department of Natural Resources Acipenser medirostris Muscle Klamath