Fluorescent in situ hybridization andcharacterization of the SalI family of satelliterepeats in the Haliotis L. species (abalone) of theNortheast Pacific

Norma Karina Hernandez-Ibarra, Andrew R. Leitch, Pedro Cruz, and Ana M. Ibarra

Abstract: The SalI satellite repeat previously identified in Haliotis L. (abalone) was thought to be present in H. rufescensand absent in H. fulgens. However, we show here that SalI is also found in H. fulgens and is not useful for screeninghybrid individuals bred in aquaculture or occurring naturally in the wild. SalI is a family of predominantly subtelomerictandemly repeated sequences, and sequenced clones revealed clustering to species and little intraspecific variation. Analy-sis of SalI sequence divergence between Haliotis species of the Northeast Pacific revealed that evolutionary distancescorrelate well with bathymetric and latitudinal species distributions. Analysis of the structure of the tandem repeatsrevealed two regions of high sequence conservation that may contain conserved transcription factor binding sites, asurprise for an apparently ‘‘non-coding’’ tandem repeat. We speculate that these regions might be involved in hetero-chromatin silencing, perhaps mediated via transcriptional activity and RNA interference. The repeats show substantialdifferences in their chromosomal distributions, even between individuals of the same species, indicating a dynamicorganization of repeats, perhaps mediated via sequence homogenization.

Key words: satellite repeat, subtelomeric repeats, FISH, mollusc, evolution, cytogenetics.

Resume : L’ADN satellite SalI identifie anterieurement chez le Haliotis L. (ormeau nordique) etait presume present chezle H. rufescens et absent chez le H. fulgens. Les auteurs montrent ici que le satellite SalI est egalement present chez leH. fulgens et n’est donc pas utile pour identifier des hybrides produits en aquaculture ou survenant naturellement. Lesatellite SalI est une sequence repetee principalement subtelomerique et les clones sequences suggerent un groupement surla base des especes et peu de variation intraspecifique. L’analyse de la divergence nucleotidique entre les especes de l’Ha-liotis du nord-est du Pacifique a revele que les distances evolutives sont bien correlees avec la repartition latitudinale etbathymetrique des especes. L’analyse de la structure des repetitions en tandem revele deux regions de forte conservationdes sequences qui pourraient contenir des sites conserves de liaison de facteurs de transcription, une observation surpre-nante au sein d’une region apparemment « non-codante ». Les auteurs speculent que ces regions sont peut-etre impliqueesdans l’inactivation de l’heterochromatine obtenue via l’activite transcriptionnelle et l’interference a l’ARN. Les repetitionsaffichent des differences substantielles dans leur distribution chromosomique, meme entre individus de la meme espece cequi indique une organisation dynamique des repetitions, peut-etre via l’homogeneisation des sequences.

Mots-cles : satellite repete, repetitions subtelomeriques, FISH, mollusque, evolution, cytogenetique.

[Traduit par la Redaction]

Introduction

Abalones are molluscs of the family Haliotidae, belongingto the order Archaeogasteropoda. There is a single genus,Haliotis, that includes about 56 species. Taxonomy in thisfamily is controversial, as species assignments and genussubdivisions are largely derived from phenotypic character-istics of the shell (Geiger and Poppe 2000). In the NortheastPacific there are 7 recognized species that live more or less

sympatrically: Haliotis rufescens, H. fulgens, H. corrugata,H. kamtschatkana, H. sorenseni, H. cracherodii, and H. wa-lallensis (Leighton 2000). These species are closely relatedto two species around Japan, H. discus and H. gigantea,and together form a phylogenetic group that differs fromIndo-Pacific, Australian, South African, and Mediterraneanabalones (Lee and Vacquier 1995; Coleman and Vacquier2002). The centre of origin of Haliotis and the ancestralcharacters of the genus remain unclear. Nevertheless,

Received 13 December 2007. Accepted 3 April 2008. Published on the NRC Research Press Web site at genome.nrc.ca on 12 July 2008.

Corresponding Editor: W. Traut.

N.K. Hernandez-Ibarra, P. Cruz, and A.M. Ibarra.1 Aquaculture Genetics Lab, Centro de Investigaciones Biologicas del NoroesteS.C., Mar Bermejo 195, Col. Playa Palo de Sta. Rita, La Paz B.C.S, 23090 Mexico.A.R. Leitch. School of Biological and Chemical Sciences, Queen Mary University of London, Mile End Road, London E1 4NS, UnitedKingdom.

1Corresponding author (e-mail: aibarra@cibnor.mx).

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chromosome number is consistent with geographic location,i.e., North Pacific species, 2n = 36; Mediterranean species,2n = 28; and Indo-Pacific species, 2n = 32 (Nakamura1986).

Many abalone species live sympatrically (Lindberg 1992),and there is a high possibility that hybrids can form in thewild. Indeed, based on phenotypic characters of the shelland epipodium, hybrids have been reported between wildnortheastern Pacific species (Owen et al. 1971). Unfortu-nately no genetic proof has verified this hypothesis. Morerecently, allozyme analysis has shown that adult hybrids ar-tificially produced by mating H. rufescens and H. fulgenscan exist under culture conditions (Ibarra et al. 2005). Kar-yotype analysis of the northeastern Pacific species H. rufes-cens and H. fulgens (Hernandez-Ibarra et al. 2004) indicatesthat even if hybrids are formed, reduced fertility and (or)gamete viability is expected and indeed has been observedin laboratory-induced hybrids (Ibarra et al. 2005). Neverthe-less, there may be sufficient fertility to facilitate some geneflow between species in the wild.

This paper aims to better characterize Haliotis geneticallyas part of an ongoing research program to evaluate hybridsfor commercial production. It also aims to search for geneticor cytogenetic markers that could be used to assist in thescreening of artificial and natural hybrids, including at dif-ferent life-cycle stages. One such marker is reported to bethe SalI repeat isolated from genomic DNA using the SalIrestriction enzyme. This repeat was thought to be present inall northeastern Pacific species except H. fulgens andH. cracherodii (Muchmore et al. 1998). It is that repeatwhich is analyzed here using PCR, sequence analysis, andFISH, but we find the repeat is also in H. fulgens and is ofno use for the screening of hybrids. This work did revealthat SalI is highly structured and dynamic, with evidencefor unit amplification and reduction at individual loci bothwithin and between species.

Materials and methods

Animal material and genomic DNA extractionDNA was extracted from muscle tissue preserved in 70%

ethanol, using a phenol–chloroform extraction protocol de-scribed by Sweijd et al. (1998). Tissue was sampled fromadult individuals of each abalone species, H. rufescens andH. fulgens, and used as templates in independent PCR am-plifications. The samples were from the same stock of indi-viduals utilized by Ibarra et al. (2005) and Cruz et al.(2005).

For chromosome preparations, abalone spawners fromeach species were obtained from a commercial farm andinduced to spawn. Twenty-four hours after spawning andfertilization, hatched trochophore larvae were sampled.

Primers designed for amplification of SalI satellite repeatfragment

The SalI satellite was amplified using two newly designedsets of primers. The first primer set, labeled rufSalI (for-ward: rufSalI-F, 5’-GACTCCTCAGGTCATTTGGG-3’; re-verse: rufSalI-R, 5’-GCCAAGTCTGAGACTCCTACC-3’),was designed using the satellite DNA sequence reported byMuchmore et al. (1998) for H. rufescens (GenBank acces-

sion number AF031155). Primers rufSalI-F and rufSalI-R lo-cate to positions 4–23 and 268–288, respectively, in thatreported sequence.

A second set of primers, labeled fulSalI (forward: fulSalI-F, 5’-CATTGGAGTGCCAGGTGTTA-3’; reverse: fulSalI-R,5’-GATCACGTTTTGACCGTCCT-3’), was designed from anew satellite DNA repeat sequence obtained after amplify-ing H. fulgens DNA (GenBank accession numberDQ474063) with the rufSalI set of primers. Primer fulSalI-Flocates to the downstream positions 198–217 and fulSalI-Rto positions 113–132 in the sequence obtained from H. ful-gens (with rufSalI primers) in this work (Fig. 1).

PCR amplification of SalI satellite repeat fragmentFor both rufSalI and fulSalI sets of primers, reactions

were performed separately for each species in a 25 mL reac-tion volume containing 4 ng/mL of template DNA from aspecific individual, 1� PCR buffer, 1 mmol/L of MgCl2,0.2 mmol/L of each dNTP, 0.5 mmol/L of each primer, and1.5 units of Platinum Taq DNA polymerase (Invitrogen,Carlsbad, California, USA). PCR was performed using aniCycler thermal cycler (Bio-Rad, Hercules, California,USA) under the following conditions: 5 min denaturation at94 8C, 30 cycles of 30 s at 94 8C, 30 s at 56 8C for the ruf-SalI primer set and 59 8C for the fulSalI primer set, and1 min at 72 8C, and a final extension of 10 min at 72 8C.PCR amplification products were subjected to 1% (w/v)agarose gel electrophoresis and stained with ethidium bro-mide.

Cloning and sequencing of SalI satellite repeat fragmentin H. rufescens and H. fulgens

The PCR amplification products obtained after using bothprimer sets with each species’ DNA were excised from theagarose gels using the QIAquick PCR Purification Kit(QIAGEN, Germany). For each species a band of ~300 bpand one of ~600 bp were obtained using the rufSalI primerset, and a band of ~200 bp and one of ~500 bp wereobtained with the fulSalI primer set. Purified DNA was li-gated into a plasmid vector (pCR4-TOPO, Invitrogen) andinserted into competent E. coli cells using the InvitrogenTOPO TA Cloning Kit for Sequencing. Transformed bacte-ria were cultured in a Petri dish with enriched LB agarmedium (Difco, Sparks, Maryland, USA) for 24 h at 37 8C.For the ~300 bp cloned fragments, 15 colonies were ran-domly selected from H. rufescens DNA and 10 coloniesfrom H. fulgens DNA. For the ~200 bp, ~500 bp, and ~600bp cloned fragments, 5 colonies from each species wererandomly selected. Colonies were diluted in 50 mL of Milli-Q water separately. Cells were heated at 100 8C for 5 minand centrifuged (16 000g) before the supernatant was col-lected to be used as template for PCR amplification usingthe cloning kit M13 primers. The reaction mixture (25 mL)contained 2 mL of template DNA, 1� PCR buffer,0.8 mmol/L of MgCl2, 0.2 mmol/L of each dNTP,0.8 mmol/L of M13F and M13R primers, and 1.25 units ofPlatinum Taq DNA polymerase (Invitrogen). PCR was per-formed under the following conditions: 5 min denaturationat 95 8C, 30 cycles of 1 min at 95 8C, 1 min at 55 8C, and1 min at 72 8C, and a final extension of 10 min at 72 8C.

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The PCR products obtained were sequenced (Macrogen Se-quencing Services, Korea).

Sequences and phylogenetic analysesPairwise sequence alignments and multiple alignments

were carried out with the program BioEdit (Hall 1999). Se-quence identity searches were performed in GenBank usingBLASTN (Altschul et al. 1997). The SalI satellite repeatfragment was analyzed in 6 abalone species of the NortheastPacific including H. fulgens (the 290 bp sequence investi-gated in this work) and H. corrugata, H. kamtschatkana,H. rufescens, H. sorenseni, and H. walallensis (the 290/291bp sequences published by Muchmore et al. 1998). ForH. fulgens the 290 bp repeat fragment was completed usingsequences obtained with both primers sets.

PAUP version 4.0b was used to conduct phylogenetic anal-yses (Swofford 2001), and gaps were treated as missing data.Using the DELTRAN character optimization to illustratebranch lengths, heuristic searches were performed using treebisection-reconnection (TBR) branch swapping and 100 rep-licates of random stepwise addition, holding 10 trees at eachreplicate. To assess branch support, 10 000 bootstrap repli-cates (Felsenstein 1985) with fast stepwise addition were per-formed with equal weight using TBR branch swapping.

The CLOVER program (Frith et al. 2004) and sequencesof Drosophila melanogaster available at the MotifViz Webpage (Fu et al. 2004) were used to search for possible tran-scription factor binding sites. The JASPAR matrices data-base (Sandelin et al. 2004) was screened against the SalIsatellite sequences of 6 abalone species. Motifs with anoverall score above 5.0, and present in all species, were con-sidered as putative recognition sites for transcription regula-tors. Motifs were considered conserved across the 6 speciesif they showed only a single base substitution in any onespecies.

Chromosome preparationsChromosome preparations followed the method of Miyaki

et al. (1997) for Asian abalones (Nordotis (Haliotis) discusand N. (H.) gigantea). Sampled trochophore larvae weretreated with 0.1% (w/v) colchicine (Sigma, St. Louis, Mis-souri, USA) in seawater for 2 h. A hypotonic shock was ap-plied for 10 min using 1% (w/v) sodium citrate in distilledwater. Larvae were then fixed in a 3:1 methanol–acetic acidsolution (v/v), pipetted into 60% acetic acid (v/v) to obtain acellular suspension, and spread by dropping onto a heated(45 8C) glass slide. Preparations were air-dried for 1 h.

Fluorescence in situ hybridization (FISH) of SalI satelliteAll PCR products obtained after amplification of H. rufes-

cens and H. fulgens DNA templates using the rufSalI primerset were labeled with biotin-16-dUTP separately (Roche,Mannheim, Germany). FISH was carried out as describedby Lim et al. (2004). Chromosome slides were pretreatedwith 100 mg/mL of RNase A (QIAGEN) and 0.25 mg/mLof pepsin for 3 min at 37 8C, and then denatured with 70%(v/v) formamide in 0.6� SSC (0.03 mol/L sodium citrateand 0.3 mol/L sodium chloride) at 71.5 8C for 2 min. La-beled probes were denatured at 75 8C for 13 min in the hy-bridization mix containing 50% (v/v) formamide, 10% (w/v)dextran sulphate, and 0.1% (w/v) sodium dodecyl sulphate in2� SSC. Post-hybridization washes were done in 20% (v/v)formamide in 0.1� SSC at 42 8C, with an estimated hybrid-ization stringency of 80%–85%. Hybridization sites weredetected with 5 mg/mL Cy3-conjugated streptavidin (Amer-sham Biosciences, Buckinghamshire, UK). Chromosomeswere counterstained with 2 mg/mL DAPI (4’,6-diamidino-2-phenylindole) and mounted in Vectashield medium (VectorLaboratories, Burlingame, California, USA). Slides wereexamined using an Olympus BX51 epifluorescence micro-scope (Olympus, Melville, New York, USA) and imagesdigitized with a CoolSNAP-Pro camera (Media Cybernetics,

Fig. 1. Locations of primers used for PCR amplification of the SalI repeat, and the fragments amplified, in Haliotis fulgens and H. rufescens.

Fig. 2. Fragments amplified by PCR using primers rufSalI-F andrufSalI-R: (a) H. rufescens, (b) H. fulgens.

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Silver Spring, Maryland, USA). Images were transferred toan image analysis program (Image-Pro Plus 4.5, Media Cy-bernetics) for analyses. Further editions of the images (ex-tracting chromosomes from metaphases to be pasted andaligned in the karyotypes, and adjusting contrast and bright-ness uniformly) were done in Adobe Photoshop 8.0.1(Adobe Systems Inc.).

Results

SalI repeat sequences in H. rufescens and H. fulgensPrimers against the SalI satellite repeat (rufSalI) amplified

predominantly a ~300 bp fragment in both species (Fig. 2).Other fragments (~600 and ~900 bp) at lower abundancewere also amplified; the ~900 bp fragment band was veryfaint in ethidium bromide stained agarose gels (not visiblein Fig. 2). The fulSalI primer set amplified a predominantfragment of ~200 bp in both species and less abundantproducts of ~500 and ~800 bp (data not shown), asexpected if the repeated sequence (cf. Fig. 1) were intandem array.

Consensus sequences were obtained for H. rufescens (15

clones) and H. fulgens (10 clones) after sequencing the~300 bp fragments amplified with the rufSalI primer set(Fig. S12). The sequence lengths were 285 bp for bothH. fulgens (Hful-rufSalI-285) and H. rufescens (Hruf-ruf-SalI-285). These sequences were aligned with publishedSalI satellite repeats from other abalone species (Muchmoreet al. 1998) and sequence identities were calculated(Table S12).

Sequence analysis of the ~200 bp fragments amplified byPCR with fulSalI primers from H. fulgens and H. rufescensrevealed a 225 bp sequence in H. fulgens (Hful-fulSalI-225)that contains the SalI restriction enzyme site at the 91–96 bpposition (equivalent to positions 1–6 in the published se-quence for H. rufescens (AF031155)) (Fig. S22). There was87% similarity between the consensus sequences amplifiedfrom the two abalone species, the same as was found forthe 285 bp fragments amplified with the rufSalI primer set.

The less abundant fragments amplified with rufSalI andfulSalI primers (~600 and ~500 bp, respectively) werecloned and sequenced. They contained contiguous repeatsof the SalI 285 bp fragments, as expected of a tandemly re-peated satellite sequence (data not shown).

Fig. 3. Alignment of consensus sequences of the SalI satellite repeat of 6 northeastern Pacific abalone species. The first conserved region(positions 70 to 170) and second conserved region (from position 248/249 of one repeat to position 18 of the next contiguous repeat) areshaded. Dots are identities and dashes are insertion/deletions. K = G, T; M = A, C; R = A, G; S = C, G; W = A, T.

2 Supplementary data for this article are available on the journal Web site (http://genome.nrc.ca) or may be purchased from the Depositoryof Unpublished Data, Document Delivery, CISTI, National Research Council Canada, Building M-55, 1200 Montreal Road, Ottawa, ONK1A 0R6, Canada. DUD 3770. For more information on obtaining material refer to http://cisti-icist.nrc-cnrc.gc.ca/cms/unpub_e.html.

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SalI sequence analysis of 6 northeastern Pacific abalonespecies

The aligned sequences of SalI repeat for 6 abalone speciesare shown in Fig. 3. For H. fulgens, the sequence Hful-ruf-SalI-285 (DQ474063) was used after manually adding thefirst 3 bp and the last 2 bp to the sequence, which are notamplified with the rufSalI primer set. The sequence addedwas determined from the Hful-fulSalI-225 clones at equiva-lent positions (positions 89–91, first 3 bp; positions 90–93,last 2 bp) (Fig. S2).

We conducted a phylogenetic analysis of 19 SalI satelliteclones from H. rufescens and 15 clones from H. fulgens andcombined this with GenBank sequence data from H. corru-gata, H. sorenseni, H. kamtschatkana, and H. walallensis.We observed more intraspecific variability in SalI sequencesin H. rufescens than in H. fulgens, but both formed well-supported clades (96% and 93% bootstrap support, respec-tively) (Fig. 4). Haliotis rufescens was sister to H. fulgensand H. corrugata (Fig. 4) and in unrooted trees they formeda clade that was sister to H. sorenseni, H. kamtschatkana,and H. walallensis with 100% bootstrap support, the latterthree species being used in Fig. 4 as the outgroup. Pairwiseevolutionary distances between consensus sequences of the 6species (Table S22), obtained in MEGA4 (Jukes and Cantor1969; Tamura et al. 2007), revealed that the highest evolu-tionary distance (0.205 ± 0.034) is seen between H. corru-gata and H. walallensis.

A comparative analysis of the consensus SalI repeat in the6 species revealed that the A+T content ranged from 49.83%to 53.79%. There is a G+C-rich region (70.6% to 73.5%) atpositions 79–112 in all species, and an A+T-rich region(62.5% to 72.3%) at positions 218–265 in all species. Thereare two long conserved regions found in all species: the firstregion is 101 bp in length, from positions 70 to 170, and thesecond region is a 61 bp sequence between two contiguousrepeats, from position 248/249 of one repeat to position 18in the next (shaded areas in Fig. 3).

Transcription factor binding sites analysis in CLOVER(Frith et al. 2004) indicated, with a significant score (P <0.002, log-likelihood ratio), putative recognition sites fortranscription regulators within the two conserved regionsin SalI. Motifs similar to D. melanogaster nuclear recep-tors RORalfa-1 (retinoid-related orphan receptor) and CF1-USP (chorion factor 1/ultraspiracle) are present in both thepositive (+) and negative (–) strands of the first conservedregion (positions 70–170), but RORalfa-1 is the only motifalso found in the second conserved region (+ strand). Athird conserved motif, for the zinc finger protein Staf(selenocysteine tRNA gene transcription activating factor)in D. melanogaster, is present within the high G+C contentregion (positions 79–112). In addition, the first conservedregion of SalI carries a motif similar to D. melanogasterARNT (aryl hydrocarbon receptor nuclear translocator).Most motifs are conserved in all species, with the excep-tion of CF1-USP (+ strand) in H. walallensis and CF1-USP (– strand) and RORalfa-1 (– strand) in H. fulgens. Inthese cases the exclusion is due to a single base substitu-tion (Fig. 3).

Some regulatory proteins, such as transcription factors,frequently require secondary folding of the DNA for binding(Spiro et al. 1993; Gohler et al. 2002). Because the SalI

repeat has several small inverted repeats, we used the DNA-fold program (Zuker 2003) to search for putative secondarystructures. We focused our effort at the two long conservedregions in SalI because they carry the putative transcriptionfactor binding sites. The program predicts that single-stranded DNA at these sites can form stem-loop structures(Fig. 5).

FISH with labeled SalI probesTwo probes were prepared by PCR, one derived from

H. rufescens (Hruf-rufSalI) and the second from H. fulgens(Hful-rufSalI). These were hybridized using FISH to H. ru-fescens and H. fulgens. Both probes gave a similar labelingpattern in each of the two species. Strong hybridization sig-nals of the SalI satellite were located to subtelomeric re-gions of the chromosomes and occasionally at interstitial

Fig. 4. One of the most parsimonious trees (10 000 bootstrap repli-cations) for the cloned SalI satellite repeats (H. fulgens and H. ru-fescens) and consensus sequences from four additional northeasternPacific abalone species taken from GenBank (H. corrugata, H. sor-enseni, H. kamtschatkana, and H. walallensis). Branch lengths(DELTRAN optimization) and bootstrap values (in parentheses) areshown above and below branches, respectively. Haliotis sorenseni,H. kamtschatkana, and H. walallensis were defined as the outgroup.

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positions (Figs. S3, S42). Signals were predominantly lo-cated on short arms of chromosomes. In a few metaphases,some chromosomes had signals on both arms but at low sig-nal intensity.

From 5 metaphases of each species (FISH with Hruf-ruf-SalI probe) we constructed karyotypes according to 3 crite-ria: chromosome length, centromere index, and presence andsize of signals. Because of the similarity in chromosomemorphology and the variation in the satellite repeat evenwithin a species, we cannot with confidence determinewhether the groupings are natural. Two of the metaphasesfrom each species are shown, representing the range ofobserved SalI intraspecific polymorphism. Karyotypesshown for H. rufescens (Fig. 6) differ in the occurrence ofprominent signals on pair 12, and one chromosome of pair16 lacks signal in karyotype b. Similarly, karyotypes ofH. fulgens (Fig. 7) differ in the presence of signals on pairs2 and 16, and signal is lacking on one homologue of pair 14in karyotype b and is present on only one homologue of pair17 in karyotype a. Table 1 lists the intraspecific and inter-specific polymorphisms observed in the distribution of SalIrepeat of the 5 karyotypes analyzed. The occurrence ofinterspecific differences cannot be established because ofthe substantial intraspecific variation.

DiscussionMuchmore et al. (1998) described the occurrence of SalI

satellite in 5 of 7 northeastern Pacific abalone species(H. rufescens, H. corrugata, H. walallensis, H. sorenseni,and H. kamschatkana) following restriction enzyme analysis.They concluded the sequence was absent in H. fulgens andH. cracherodii. Here we show by PCR amplification, se-quencing, and FISH that the sequence is present in H. ful-

gens. The strong hybridization signals to H. rufescens andH. fulgens chromosomes were obtained with probes thatshare 87% or more sequence identity in the 285 bp targetrepeat. Future work might show that the SalI satellite ispresent in H. cracherodii as well. Given the presence ofSalI in both H. fulgens and H. rufescens, the probe is notuseful for screening hybrid abalone individuals.

Nature of the SalI satellite repeatThe SalI satellite DNA in H. rufescens and H. fulgens is

organized as tandem repeats of 290 bp monomers, as evi-denced by the ~600 and ~900 bp PCR-amplified fragmentsand by sequencing of the ~600 bp fragments, which showedtwo consecutive repeats. In addition, the second set of pri-mers (fulSalI) amplifies a 225 bp fragment generated fromtwo consecutive repeats. The sequenced repeat monomersfrom both species, and those from the database, were simi-lar, indicating that they belong to the same family of SalIsatellite DNA.

FISH analyses of the SalI satellite in two abalone species,H. rufescens and H. fulgens, revealed that the sequence islocated predominantly at subtelomeric positions, but thedistribution of signals differs between chromosomes withina species. The telomere itself in most eukaryotes, includingabalones (Gallardo-Escarate et al. 2005), consists of a shortrepeat motif, (TTAGGG)n, or its variants (Fajkus et al.2005; Traut et al. 2007).

Divergence of the SalI satellite repeat family throughsequence homogenization

The existence of the SalI satellite repeat family in 6 ana-lyzed northeastern Pacific abalone species (this work, andMuchmore et al. 1998) indicates the repeat existed in the

Fig. 5. Secondary structures formed within the conserved regions of the SalI repeat. Alternative stem-loops formed for (a, b) the first con-served region (positions: bp 70 to 170) and (c) the second conserved region (positions: bp 248/249 of one repeat to bp 18 of the secondcontiguous repeat). The red arrow indicates the end of the first repeat and the beginning of the second repeat. Coloured lines outline theregulatory motifs: green, Staf; dark blue, CF1-USP (+); light blue, CF1-USP (–); red, RORalfa-1 (+); orange, RORalfa-1 (–); yellow,ARNT.

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common ancestor of these species. Satellite repeats and tan-demly repeated genes (e.g., ribosomal DNA) can change innature through the amplification and contraction in copynumber of particular units in the tandem array. The homog-enization processes are thought to be mediated via gene con-version (e.g., unequal recombination, perhaps between sister

chromatids) (Eickbush and Eickbush 2007). Overall, wefound high homogeneity within species and divergence be-tween species, both indicators of homogenization processesacting on SalI satellite repeats. The consequence of homog-enization is that mutational change in one repeat of thetandem array can amplify across the entire array (Dover

Fig. 7. (a, b) Two metaphases and associated karyotypes of H. fulgens after FISH with the Hruf-rufSalI probe. Red colour, probe detectedwith Cy3; blue colour, chromosomes counterstained with DAPI.

Fig. 6. (a, b) Two metaphases and associated karyotypes of H. rufescens after FISH with the Hruf-rufSalI probe. Red colour, probe detectedwith Cy3; blue colour, chromosomes counterstained with DAPI.

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1986). In addition, the apparent mobility of the tandemarrays between chromosome termini, as seen by the intra-specific variability after FISH, is evidence of how dynamicthe evolution of tandem arrays can be. These data show thatthe sequences can amplify and contract at individual chro-mosomes rapidly enough that substantial differences in se-quence distribution can occur even within a species.

The phylogenetic tree constructed for the SalI satelliteclones of H. fulgens and H. rufescens and the consensus se-quences of H. corrugata, H. kamtschatkana, H. walallensis,and H. sorenseni (Fig. 4) is congruent with that of Much-more et al. (1998), which lacks H. fulgens. Both trees differmaterially from a tree based on sequence divergence of the

open reading frame of lysin (Lee and Vacquier 1995). Inthat study H. rufescens, H. kamtschatkana, H. sorenseni,and H. walallensis are grouped together, whereas the SalIsatellite DNA tree reveals that H. rufescens is not groupedwith these species. Further work with other DNA markers isrequired to clarify species relationships.

In silico analysis of conserved regions of the SalI satelliterepeat

Two highly conserved regions exist in the SalI repeat.Conserved regions intermixed with variable regions havebeen found for other satellite sequences, suggesting that theconserved regions are under selective pressure (Ugarkovic2005). We investigated whether the conserved SalI domainsmight potentially bind transcription factors. The programCLOVER predicts that several transcription factor bindingsites included in the JASPAR matrices database (Sandelinet al. 2004) do occur in SalI. Furthermore, the programDNAfold (Zuker 2003) predicts that the DNA could poten-tially form stem-loop structures. Such structures are also po-tential recognition sites for transcription factors. These dataindicate that domains of the SalI satellite DNA are undersome type of transcription regulation. Transcription of othersatellite DNA sequences has been demonstrated in severalorganisms, and transcripts may have a regulatory role (for areview see Ugarkovic 2005).

In yeast, unequal recombination in tandemly arrangedrDNA units facilitates array expansion and sequence homog-enization. This is achieved through the transcriptional activ-ity of a bidirectional promoter in the intergenic spacer,which acts to displace cohesins and cause sister chromatidsto recombine asymmetrically (Kobayashi and Ganley 2005).In Arabidopsis thaliana, aberrant transcriptional activity ofrDNA results in the production of short interfering RNAs,which are targeted back to the rDNA units and cause theunits to become methylated and heterochromatinized (Ponteset al. 2006). Thus transcription of repetitive DNA can resultin both homogenization and heterochromatinization. Perhapsthe transcription factor binding sites in SalI play a similarrole, with transcription of the units acting to both hetero-chromatinize the sequences and facilitate their homo-genization.

Relationships between species, SalI, and ecologicalvariables

The rapid evolution of subtelomeric SalI tandem repeatsmay have had an influence on speciation in abalone, sincesatellite repeats can establish differences between chromo-somes and prevent their proper pairing at meiosis (Lim etal. 2006). Pairing at meiosis can commence at subtelomereregions, and divergence of subtelomeric repeats could leadto reduced homologue pairing and the establishment or rein-forcement of reproductive isolation barriers.

The evolutionary distances between the SalI satellite re-

Fig. 8. Distribution of abalone species along the North AmericanPacific coast. (a) H. sorenseni, (b) H. walallensis, (c) H. kamtschat-kana, (d) H. rufescens, (e) H. corrugata, (f) H. fulgens. Speciesfrom a to f are distributed from deep to shallow waters. Modifiedfrom Leighton (2000).

Table 1. Distribution of SalI satellite signals in five karyotypes of each species, H. rufescens and H. fulgens.

SpeciesNo. of chromosomeswith signal

No. of chromosome pairs withsignal on both homologues

No. of chromosomeswith signal on both arms

No. of chromosomes withsignal only on long arms

H. rufescens 14–17 6–8 0–3 0–1H. fulgens 10–17 4–8 0 0

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peats from the different abalone species correlated with theextent of overlap in their ecological variables (temperaturetolerance, bathymetric range), probably reflecting speciationthrough ecological partitioning. All species overlap in somepart of their latitudinal distribution (Fig. 8), but their bathy-metric ranges are more varied (Table 2), presumably reflect-ing each species’ temperature tolerance (Leighton 2000). Forexample, H. corrugata and H. fulgens form a clade (Fig. 4)and are most abundant in the southern part of the range.They live in temperate waters and have overlapping bathy-metric ranges. Likewise, H. kamtschatkana and H. walallen-sis are closely similar species and have a northerndistribution (up to Canada) and overlapping bathymetricranges. The highest evolutionary distance (0.205) obtainedwith the consensus sequences was between H. walallensisand H. corrugata. These species share the smallest latitudi-nal range and have minimal bathymetric overlap. Thus geo-graphic and ecological partitioning of the species iscorrelated with the degree of SalI divergence. Whetherhybrid zones exist where species ranges overlap, as pre-dicted by morphology and by laboratory experiments, re-mains to be verified genetically. Recent work using lysin-SSCP markers has found one H. fulgens and H. corrugatahybrid in canned abalone of unknown origin (P. Cruz,unpublished results). Potentially, therefore, gene flow maybe occurring between these broadly sympatric species.

AcknowledgementsThis research was funded by grant CONACYT 38860-B

to A.M. Ibarra. We are indebted to Jose Luis Ramırez forhis technical support during the gathering of animal samplesand to Elizabeth McCarthy for help with the phylogeneticanalysis. The first author was supported by a Ph.D. fellow-ship from CONACYT (166332).

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