Determination of the phylogenetic relationships among Pacific salmonids by using short interspersed elements ( SINEs ) as temporal landmarks of evolution ( retroposon / PCR / phylogeny / orthologous locus / salmon )

Several subfamilies of the salmonid Hpa I short interspersed element (SINE) family were isolated from salmonid genomes and were sequenced. For each genomic locus that represented the subfamily, amplification by PCR of the orthologous loci in the 12 fish aflowed us to determine the order of branching of the Pacific salmonid species. The deduced phylogeny suggests three evolutionary lines, namely, a line of chum salmon, pink salmon, and kokanee; a line ofcoho salmon and chinook salmon; and a line ofsteelhead trout. Our data also support a change in the phylogenetic ament of steelhead trout from Salmo to Oncorhynchus. We present here an extensive phylogenetic tree constructed from an analysis of differential insertion of SINEs, and we propose that SINE insertion analysis is one of the best available methods for clarifying the order of divergence of closely related species. A retroposon is defined as a nucleotide sequence, present initially as a cellular RNA transcript, that has been reincorporated into the genome, presumably via a cDNA intermediate. Retroposons constitute roughly 10% of the human genome and are similarly abundant in other mammalian genomes (1, 2). As a result, the remarkable fluidity of eukaryotic genomes reflects the contributions of retroposition (2) as well as mechanisms operating at the DNA level such as mutation and recombination (1-4). Retroposons can be unique to one species, a few species, a genus, or in some cases a family. Retroposition is therefore a specialized form of gene duplication, which is believed to be of major importance in the creation ofgenetic diversity during evolution (5). Nonviral retroposons are classified into three main groups: processed retropseudogenes, LINEs (long interspersed elements), and SINEs (short interspersed elements) (6). Except for the rodent type 1 and human Alu families (7, 8), all of the SINE families examined to date have been shown to be derived from tRNAs (9-14). In contrast toDNA transposable elements, which are often capable of being excised precisely, SINEs appear to be inserted irreversibly and should therefore provide an ideal evolutionary and phylogenetic marker (4). The Pacific salmon and trout (Oncorhynchus) are a group of closely related species with complex life histories and an interesting global distribution (reviewed in ref. 15). Previously, in an attempt to elucidate a possible role of SINEs in the genomic organization and speciation of salmonids, we characterized three families of tRNA-derived SINEs in salmonid genomes (16, 17). The salmon Sma I family is restricted to the genomes of chum salmon and pink salmon. The charr Fok I family is present only in species that belong to the genus Salvelinus. The third family, the salmonid Hpa I family, is present in all species in the family Salmonidae but The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. not in other species (16). These results suggest that these SINEs were amplified specifically within certain salmonid lineages during evolution. Our data prompted us to attempt to construct a phylogenetic tree for the salmonid species by using SINE insertions as irreversible events that would serve as informative markers of evolution. In this report, we present a characterization of the four subfamilies of the Hpa I family.t These subfamilies were amplified in the four different ancestral species within the genus Oncorhynchus. Such characterization provides a highly reliable order of branching of the various species of Oncorhynchus. MATERIALS AND METHODS Experiments were performed by using standard techniques (18-21). The fish species examined in this study and their geographic sources are listed in Table 1. The family Salmonidae consists mainly of four genera: Oncorhynchus, Salmo, Salvelinus, and Hucho. The genus Oncorhynchus includes eight species, of which six species were analyzed in this study. Genomic DNAs from chum salmon (0. keta), kokanee (0. nerka adonis), and coho salmon (0. kisutch) were used to construct three genomic libraries. Each genomic library was screened for phage clones that contained the salmonid Hpa I family and their sequences were determined by the chaintermination method (19). When a unit of the family appeared to be integrated into a unique region of the genome, we synthesized 5' and 3' 20-meric primers that flanked the unit. Then PCR was performed (20), using the DNAs from the 12 listed species as templates. Each locus was named after the number of the clone and the name of the species from which it was isolated. For example, when aphage clone was isolated from the genomic library of kokanee and the number of the clone was 345, the locus was named Hpa(ON)-345 (whereON stands for 0. nerka). The orthologous loci of chum salmon (0. keta), pink salmon (0. gorbuscha), coho salmon (0. kisutch), and steelhead trout (0. mykiss), which could be detected by PCR, were named Hpa(OK)-345, Hpa(OG)-345, Hpa(OKi)-345, and Hpa(OMy)-345, respectively. To confirm the presence or absence of a SINE unit, Southern hybridization experiments were performed (21) and several sequences of products of PCR at the orthologous loci were determined. To distinguish different loci from one another, different numbering systems were adopted for the different genomic libraries. Abbreviation: SINE, short interspersed element. *To whom reprint requests should be sent at the present address: Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 227, Japan. tThe sequences reported in this paper have been deposited in the GenBank data base (accession nos. D16238-D16246).