Slow Rates of Evolution and Sequence Homogenization in an Ancient Satellite DNA Family of Sturgeons

Satellite DNA sequences are noncoding, short, tandemly repeated sequences located mainly at centromeres and telomeres (Charlesworth, Sniegowski, and Stephan 1994 ). The main concerns of evolutionary patterns of satellite DNA sequences are their rapid change rate and their concerted mode of evolution. When a satellite DNA family is shared by several related species, this enables one to find specific diagnostic sites for use in phylogenetic studies (Arnason, Grettarsdottir, and Widegren 1992 ; Gretarsdottir and Arnason 1992 ). Here, we analyze an exceptional ancient satellite DNA family of sturgeons which does not fit the evolutionary features of most satellite DNAs. Specifically, we found that the evolution of this satellite DNA family is very slow, as reflected by low mutation and homogenization rates. Notwithstanding, our data offer some information on the tracing of mechanisms accounting for the formation and origin of this satellite DNA family and on the phylogeny of the sturgeons. This satellite DNA family, the HindIII family, was previously found within the genome of Acipenser naccarii (Garrido-Ramos et al. 1997 ). Now, we extend this analysis in an attempt to determine the presence of this satellite DNA and its evolutionary pattern at the molecular level in the genomes of six species of Acipenser (the Adriatic sturgeon A. naccarii, the Siberian sturgeon Acipenser baerii, the Russian sturgeon Acipenser gueldenstaedtii, the sterlet Acipenser ruthenus, the white sturgeon Acipenser transmontanus and the Atlantic sturgeon Acipenser sturio) and one species of Huso (the great sturgeon Huso huso). Three individuals from each species were analyzed. By conventional cloning or by PCR amplification (primers 5′-CGAACCTGTAAGCTT-3′ and 5′-TGATCTTCAGAACTACC-3′, designed from conserved parts of the HindIII satellite DNA) and posterior cloning (Garrido-Ramos et al. 1995, 1999 ), we obtained a total of 52 monomeric units of the HindIII satellite DNA: 11 from A. naccarii, 9 from A. baerii, 12 from A. gueldenstaedtii, 9 from A. transmontanus, 4 from A. ruthenus, and 7 from H. huso. The EMBL accession numbers for these sequences are AJ286564–AJ286615 and Z49941. Acipenser sturio proved not to have the HindIII satellite DNA sequences. This extreme has also been confirmed by in situ hybridization techniques (unpublished data). According to biogeographic and molecular data on phylogenetic analyses of the sturgeon species (Birstein and deSalle 1998 ), our results indicate that we have analyzed an ancient satellite DNA more than 80 Myr old. As discussed below, since that time, the HindIII sequence pattern change has been very slow. This allows us to infer major abrupt changes that could have occurred in the process of formation and spreading of the HindIII satellite DNA. In all of the species, the length of the satellite monomers varied between 169 and 172 bp. However, in all of the species, the current monomer length appeared to be the consequence of several duplication events from smaller subunits (fig. 1A ). Analyzing the characteristics of this satellite DNA family, we propose the following events to explain the apparition of the current repeat unit of the HindIII satellite DNA family (fig. 1B ): (1) several partial duplications of the sequences AAATTAT and GGACC led to the amplification of a initial monomer of about 50 bp, generating a monomer of about 85 bp in length; (2) one entire duplication event led to the amplification of the monomer length from 85 bp to 170 bp; after that, (3) sequence divergence occurred between the two parts of the new monomer (these three events must have occurred early in the evolution of HindIII sequences, as they are shared by all species analyzed); finally, (4) a new event of partial duplication occurred, now of the sequence CTTTT in positions 1–5 and 11–15. This event must be recent (about 15 MYA according to Birstein and deSalle [1998] ). This duplication could lead to the appearance of two HindIII subfamilies (the CTTTT subfamily and the AGCTC subfamily) in A. naccarii, A. baerii, and A. gueldenstaedtii, which currently coexist in the process of homogenization. The appearance of the CTTTT subfamily after the AGCTC subfamily is supported by the fact that the duplication event is observed only in the first part of the sequence and only in three species (apparently closely related), while it is absent in the other species, such as A. transmontanus, which has the sequence AGCTC (or variants) at positions 11–15. Although monomers having the CTTTT sequence in this region are constant (presumably because of their recent origin), the alternative subfamily is highly variable, with monomers having AGCTC, AGTTC, or AGAAC. This polymorphism is ancient, and it is shared by all the species having HindIII sequences, except A. naccarii, in which the AGCTC variant has apparently been fixed or almost fixed. The latter variability for A. naccarii, A. baerii, and A. gueldenstaedtii is the most significant detected in the HindIII satellite DNA of these species. Apart from the variable region, the intraspecific variabilities (nucleotide diversity; Nei 1987 ) for these three species were 2.27%, 8.17%, and 4.27%, respectively. Aside from the variable region between positions 11 and 15, intraspecific sequence variabilities in A. transmontanus, A. ruthenus, and H. huso were 6.02%, 5.71%, and 10.65%, respectively. The 52 monomeric units sequenced were analyzed using the MEGA package (Kumar, Tamura, and Nei 1993 ). Genetic distances within and between species were calculated according to the Jukes-Cantor method. UPGMA and neighbor-joining trees showed similar topologies (fig. 2 ). Two main branches are visible: one leading to sequences of A. naccarii, A. baerii, A. gueldenstaedtii, and A. transmontanus, and another leading to sequences of A. ruthenus and H. huso, although some sequences of A. baerii are intermixed in this second branch. Two main features concerning the clustering are evident. First, the sequences appear intermixed without reflecting taxonomic affinity: this reflects the fact that many repeat variants are shared by different species and that genetic distances between monomers of different species are similar to or even shorter than genetic distances between monomers within species. Actually, by performing an analysis of molecular variance (AMOVA) (Excoffier, Smouse, and Quattro 1992 ) within and between species using ARLEQUIN software (Schneider et al. 1996 ), we found that 70% of the total genetic diversity could be ascribed to intraspecific variability, and the remaining 30% represented the amount of differentiation between species. Furthermore, within the first main branch, two groups of sequences corresponding to the two sequence subfamilies (the CTTTT and the AGCTC subfamilies, or variants) could be identified. In general, satellite DNAs follow concerted evolution, with very few exceptions: the best known case is that of a satellite DNA in tsetse flies (Trick and Dover 1984 ), in which absence of concerted evolution was attributed to low rates of sequence homogenization. However, in this case, both the intraspecific and the interspecific sequence divergences were high. In contrast, the HindIII satellite DNA of sturgeons is characterized by low intraspecific variability and also by similar or even lower interspecific sequence divergence. Thus, the explanation for HindIII sequences may be related to the fact that not only the rate of homogenization, but also the rate of mutation is low. With respect to the rate of mutation, slow sequence evolution appears to be the norm for sturgeon DNA, either nuclear or mitochondrial (Kedrova, Vladychenskaia, and Antonov 1980 ; Birstein and deSalle 1998 ; Ludwig and Kirschbaum 1998 ; Tagliavini et al. 1999a ), and for fish DNA in general (Martin, Naylor, and Palumbi 1992 ; Rico, Rico, and Hewitt 1996 ), but also for aquatic mammals and cold-blooded animals. To explain the unusually high conservation of sequences flanking microsatellites in whales, Scholötter, Amos, and Tautz (1991) suggested that aquatic environments are less mutagenic than terrestrial ones. Martin, Naylor, and Palumbi (1992) and Rico, Rico, and Hewitt (1996) suggested that the observations that metabolic rates in poikilotherms are lower and that rates of DNA damage are proportional to specific metabolic rates (Schmidt-Nielsen, 1986 ; Shigenaga, Gimeno, and Ames 1989 ) could explain the lower rates of nucleotide substitution for mitochondrial DNA found in sharks and for microsatellite flanking sequences found in turtles and fish. The homogenization rates should be interpreted in terms of parameters influencing homogenization in the chromosome pool, such as generation time and effective population size (Ohta and Dover 1983, 1984 ). Differences in homogenization rates between multigene families could be also related to functional requirements (Gojorobi and Nei 1984 ) and the structure of the repeat units (Zimmer et al. 1980 ). However, in the case of the HindIII sequences of sturgeons, we could explain the low homogenization rate among single species in terms of the primary rates of the homogenization process. That is, it is possible that the exchange between nonhomologous chromosomes having HindIII sequences is limited. The HindIII sequences are restricted to the centromeres of a small percentage of chromosomes in these species (unpublished data). However, these chromosomes are very heterogeneous in shape and size, and this could hamper interchromosomal exchange and homogenization (Jantsch et al. 1990 ). A fish family of more recent origin than sturgeons, the Sparidae (50–35 MYA; Tortonese 1975 ), shows a satellite DNA family which is localized in centromeres of all chromosomes in all species and follows concerted evolution (Garrido-Ramos et al. 1995, 1999 ). However, one difference between sparids and sturgeons is that the karyotypes of sparids are rather symmetrical, and there is thus no evident physical limitation to interchromosomal exchange. Actually, sparid species in which karyotypes are less symmetrical (for example, Diplodus bellotii) show higher intraspecific variability (Garrido-Ramos et al. 1999 ). However, we cannot disregard the possibility that events of interspecific hybridizations between sturgeon species followed by polyploidization (i.e., reticular evolution; Vasil'ev 1999 ) could preclude interspecific sequence divergence for HindIII satellite DNA. Our data offer some clues concerning the controversial phylogenetic relationships of the species of the Acipenserinae subfamily of Acipenseridae. The HindIII satellite DNA is shared by six of the seven species analyzed here (A. naccarii, A. gueldenstaedtii, A. baerii, A. transmontanus, A. ruthenus, and Huso huso). If we assume that the sharing of a satellite DNA family is a strong signal of cladistic relationships, we find that H. huso is a species belonging to the Acipenser group, supporting the view of Birstein and deSalle (1998) and the doubts (Artyukhin 1995 ) about the systematic position of the apparently artificial genus Huso. Furthermore, we demonstrated that the HindIII sequences are absent from the genome of A. sturio. These data support the hypothesis that A. sturio differs from the other species and most likely had an independent evolution (Birstein and deSalle 1998 ). We found that the HindIII sequences were identical between A. gueldenstaedtii and A. naccarii and almost identical to those of A. baerii (although a significant proportion of its sequence variants are shared with species such as A. ruthenus and H. huso). Presumably, the first three species are closely related (Tagliavini et al. 1999a ; Artyukhin 1995 ) and, together with A. transmontanus, must represent a group of species separated from the other group made up of A. ruthenus and H. huso (Tagliavini et al. 1999a ). It is striking that H. huso and A. ruthenus are diploid species and the rest of species are tetraploid species (Fontana 1994 ; Fontana et al. 1998 ; Tagliavini et al. 1999b ). Pierre Capy, Reviewing Editor Keywords: sturgeons satellite DNA concerted evolution slow mutation rates phylogeny Address for correspondence and reprints: Roberto de la Herrán, Departamento de Genética, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain. E-mail: [email protected]. Fig. 1.—A, Sequence comparison between the two distinguishable parts of the HindIII monomers. Arrows indicate short tandem direct repeats within each part. See text for details. The sequence depicted in this figure is a consensus sequence obtained from the comparison of all 52 monomeric units analyzed. All of the species had similar HindIII sequences and the same subrepeat organization as that shown in this figure. B, Scheme indicating the possible origin and formation of HindIII sequences from an initial monomer of about 50 bp to a current monomer of 170 bp throughout several partial duplication events and one whole duplication event. See text for details. Fig. 2.—Neighbor-joining tree of the 52 monomeric HindIII sequences analyzed in this study (pHAn = clones of Acipenser naccarii; pGU = clones of Acipenser gueldenstaedtii; pBa = clones of Acipenser baerii; pTR = clones of Acipenser transmontanus; pRu = clones of Acipenser ruthenus; pHH = clones of Huso huso). Numbers are bootstrapping indices for the levels of support for individual nodes (5,000 replicates) This work was supported by grants of the FEDER funds (1FD97-2289) and of Plan Andaluz de Investigación (Group CVI0200). This work was also supported by the Fourth Triennial Plan “Aquaculture in Seas and Lagoons” of the Italian Ministry of Agriculture, Food Resources and Forestry (4C14t), and by research grants from the Italian Ministry of University and Research. We are grateful for the valuable comments and suggestions on the manuscript given by two anonymous referees.