Karyotype and chromosomal location of 18S–28S and 5S ribosomal DNA in

the scallops Pecten maximus and Mimachlamys varia (Bivalvia: Pectinidae)

Ana Insua*, Marıa Jose Lopez-Pinon, Ruth Freire & Josefina MendezDepartamento de Biologıa Celular y Molecular, Universidade da Coruna, A Zapateira s/n, 15071, A Coruna,Spain; *Author for correspondence (Phone: +34-981167000 ext. 2055; Fax: +34-981167065;E-mail: insuax@udc.es)

Received 23 February 2005 Accepted 17 May 2005

Key words: FISH, karyotype, Mimachlamys varia, Pecten maximus, ribosomal DNA, scallops

Abstract

This work describes the karyotype and chromosomal location of the ribosomal DNA (rDNA) of Pectenmaximus and Mimachlamys varia, two commercial scallop species from Europe. According to the chro-mosome centromeric index values found, the karyotype of P. maximus is composed of 1 metacentric, 2metacentric–submetacentric, 1 telocentric–subtelocentric and 15 telocentric pairs, and that of M. varia of 4metacentric, 2 subtelocentric–submetacentric, 9 subtelocentric, 3 subtelocentric–telocentric and 1 telocen-tric–subtelocentric pairs. In P. maximus, 18S-28S rDNA was located by FISH on a metacen-tric–submetacentric pair, and in M. varia on a subtelocentric–submetacentric pair using both silver stainingand FISH. PCR amplification of the 5S rDNA unit yielded a single product of about 460 bp (P. maximus)and 450 bp (M. varia), that used as probe revealed a 5S rDNA site on a telocentric pair in P. maximus and asubtelocentric pair in M. varia. Two-color FISH or sequential silver staining of 5S rDNA-FISH-meta-phases corroborated that the two gene families are located on different chromosomes in both species. Acomparative analysis of the data allowed the inference of karyotypic relationships within scallops.

Introduction

The family Pectinidae includes some 400 livingspecies (Brand, 1991) out of the 8,000 (Boss, 1982;Morton, 1996) to 20,000 (Kilias, 1982) speciesdesignated to the class Bivalvia. They are com-monly called scallops and occur in all the seas ofthe world as important members of numerousbenthic communities (Brand, 1991). They oftendisplay attractive shells, used as ornamental mo-tifs, and many species have a long history of beinggastronomic delicacies. In the European coasttwenty-four species have been found (Wagner,1991), five of which are exploited commercially.

Cytogenetic studies, aimed at assessing scalloprelationships, require the analysis of many familymembers, independently of their commercial sig-nificance. However, chromosome analysis ofcommercial species is needed to improve produc-

tion by genetic procedures such as chromosomemanipulation, selection strategies or genetic engi-neering.

Thus far, 16 scallops have been investigatedcytogenetically (Wang & Guo, 2004a) and the datareported concern mostly the description of thechromosome number and the conventionalkaryotype. Nevertheless, studies carried out in thelast few years often include the chromosomallocation of the nucleolus organizer regions(NORs) which contain multiple copies of the genesencoding the 18S, 5.8S and 28S ribosomal RNA(18S–28S rDNA). Differential silver staining ofNORs (Ag-NORs) and/or 18S–28S rDNA map-ping by fluorescent in situ hybridization (FISH)were carried out in Aequipecten opercularis (Insua,Lopez-Pinon & Mendez, 1998), Nodipecten nodo-sus (Pauls & Affonso, 2000), Argopecten purpura-tus (Gajardo, Parraguez & Colihueque, 2002),

Genetica (2006) 126: 291–301 � Springer 2006DOI 10.1007/s10709-005-7408-7

Chlamys farreri, Argopecten irradians irradians(Wang & Guo, 2004a) and Hinnites distortus(Lopez-Pinon, Insua & Mendez, 2005). The chro-mosomal location of Ag-NORs or 18S–28S rDNAsites in these species has led to the identification ofsome chromosome pairs and provided usefulcharacters for scallop comparative studies.

Other repetitive sequences related to the 18S–28S rDNA from a functional point of view are thegenes encoding the 5S rRNA (5S rDNA). Thesewere already mapped in A. opercularis (Insua,Lopez-Pinon & Mendez, 1998), C. farreri,A. i. irradians (Wang & Guo, 2004a) and H. dis-tortus (Lopez-Pinon, Insua & Mendez, 2005) by insitu hybridization. Their location allowed toidentify chromosome pairs or regions other thanthose carrying the 18S–28S rDNA.

This work focuses on the great scallop Pectenmaximus and the black scallop Mimachlamys var-ia, two commercial species of the European coast.P. maximus is the basis of important commercialfisheries in several countries and its aquacultureproduction is increasing. M. varia makes a smallcontribution to the scallop production at presentdue to a reduction in catches but efforts are beingmade to develop its culture (Louro et al., 2003).The karyotype and chromosomal location of boththe 18S–28S rDNA and the 5S rDNA in the twospecies are described and the data obtained arecompared to those available for other scallops inorder to infer karyotypic relationships.

Materials and methods

Sample collection and chromosome preparation

Samples of P. maximus and M. varia (1–2 cm)were collected from a wild population in Rıa deArousa (northwest Spain) and fed in the labora-tory with the microalgae Isochrysis galbana for10 days before performing chromosome spreads.

Metaphase chromosomes were obtained fromgill cells. The animals were maintained in 0.005%colchicine in seawater for 7–9 h. The gills wereexcised, placed in 50 and 25% seawater solutionfor 30 min each and fixed by three incubations of20 min each in a freshly prepared mixture ofabsolute ethanol and acetic acid (3:1). Gill cellswere dissociated in 50% acetic acid and thesuspension obtained was dropped onto slides

heated at 42�C (Thiriot-Quievreux & Ayraud,1982).

Karyotype and silver staining

Metaphases stained with Giemsa (4%, pH 6.8) for10 min were photographed, the chromosomes often (P. maximus) and nine (M. varia) cells werepaired according to their morphology and thenshort and long arms measured using the Leica Q-Win 2.2 program (Leica Imaging Systems Ltd).For each chromosome pair, mean and standarddeviation of relative length (100�absolute length/total length of the haploid complement) and cen-tromeric index (100�length of short arm/totalchromosome length) were calculated. Nomencla-ture for centromere position follows that of Levan,Fredga & Sandberg (1964) but when the 95%confidence limits of the centromeric index meancovered two chromosome categories a binary ter-minology was adopted.

Silver staining was carried out according to thetechnique of Howell & Black (1980) modified byGold & Ellison (1982) on Giemsa-stained slidesand subsequently discolored with alcohol.

Probe construction and labelling

To locate the 18S–28S rDNA, a recombinantplasmid containing 18S, 5.8S, 28S genes plus in-tergenic spacers of Drosophila melanogaster wasused as probe. Plasmid DNA was purified usingthe alkaline lysis method (Sambrook, Fritsch &Maniatis, 1989) and labeled with digoxigenin-11-dUTP employing a nick translation kit (RocheDiagnostics).

For the 5S rDNA a specific probe was gener-ated by PCR. Genomic DNA was extracted from a20 mg piece of ethanol-preserved adductor muscleaccording to Winnepenninckx, Backeljau & DeWachter (1993) after two 15 min washes in phos-phate buffered saline (PBS) and sterile deionizedwater. The 5S rDNA repeat unit (gene plus non-transcribed spacer) was PCR amplified first using amixture containing 10 ng template DNA/ll,0.25 mM of each dNTP, 1 lM of each primer(5¢-AGCCCGGTTAGTACTTGG-3¢ and 5¢-CGACGTTGCTTAACTTCG-3¢), buffer (10 mMTris–HCl, 1.5 mM MgCl2 and 50 mM KCl), and0.025 U Taq polymerase/ll. Thirty standard PCRamplification cycles were performed including an

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annealing temperature of 54�C for P. maximus and60�C forM. varia, and PCR products evaluated byelectrophoresis in 2% agarose gels. Labeling wasachieved by PCR according to the proceduredescribed but using in the reaction mixture (1)35 lM digoxigenin-11-dUTP, 160 lM dTTP,100 lM dATP, 100 lM dCTP and 100 lM dGTPor (2) 50 lM biotin-16-dUTP, 150 lM dTTP,200 lM dATP, 200 lM dCTP and 200 lM dGTP.

Fluorescent in situ hybridization (FISH)

Chromosome spreads were pre-treated accordingto Insua, Lopez-Pinon and Mendez, (1998). Probeswere denatured by heat at 75�C for 10 min in thehybridization solution (50% formamide, 10%dextran sulphate, 2�SSC, 250 ng/ll salmon spermDNA, 0.125% sodium dodecyl sulphate and 4 ng/ll of the labelled probe). The chromosomal DNAwas denatured at 83�C for 2.5 min in 70% form-amide, 2�SSC and 50 mM sodium phosphatesolution, the slides were then immersed into cold70% and absolute ethanol for 5 min and air-dried.Hybridization solution containing the denaturedprobe was dropped onto the denatured spreads, theslides were covered and sealed, and kept overnightin a humid chamber at 37�C. After hybridization,the slides were washed twice (7 min each) with 50%(18S-28S rDNA FISH) or 20% (5S rDNA FISH)formamide in 2�SSC at 42�C, three times (5 mineach) with 0.1�SSC at 60�C (18S–28S rDNAFISH) or 2�SSC at 42�C (5S rDNA FISH), andonce (5 min) in 2�SSC at room temperature.Detection of the digoxigenin-labelled probes inone-color FISH was carried out after treating theslides for 30 min in TNB (0.1 Tris–HCl, 0.15 MNaCl, 0.5% Roche blocking reagent) and threeconsecutive incubations (30 min each) at 37�C withTNB containing one of the following antibodies:mouse anti-digoxigenin, rabbit anti-mouse-FITC(fluorescein isothiocyanate), or goat anti-rabbit-FITC. Three slide washes (5 min each) with TNT(0.1 M Tris–HCl, 0.15 M NaCl, 0.05% tween-20,pH 7.5) were inserted in between the antibodyincubations. Dehydrated and air-dried spreadswere counterstained with 50 ng/ml propidium io-dide in antifade and viewed under a photomicro-scope equipped with appropriated filters. In thecase of two-color FISH, the procedure was identi-cal to that described for 5S rDNA FISH, exceptthat the hybridization solution contained 4 ng/ll of

each of the labelled probes (18S–28S rDNA withdigoxigenin and 5S rDNA with biotin). Thedetection of hybridization sites was performedusing Digoxigenin-Rhodamine and Biotin-FITCdetection kits (Oncor, Inc) and the slides werecounterstained with 20 ng/ml DAPI in antifade.

Once the FISH slides were examined, they werewashed three times (5 min each) in 2�SSC, brieflywashed in PBS, dehydrated through an ethanolseries and Giemsa or silver stained according tothe procedure previously described.

Results

The diploid chromosome number, counted onGiemsa-stained metaphases, was 2n=38 in 71 and72% of the P. maximus and M. varia cells exam-ined, respectively. Table 1 indicates the relativelength, centromeric index and classification of thechromosome pairs in each species. The P. maximuskaryotype consisted of 1 metacentric, 2 metacen-tric–submetacentric, 1 telocentric–subtelocentricand 15 telocentric pairs. The chromosome relativelength ranged from 8.02 to 3.45%, with very smallsize differences between successive pairs (less than0.7%). A representative karyotype is shown inFigure 1(a). TheM. varia karyotype was composedof 4 metacentric, 2 subtelocentric–submetacentric,9 subtelocentric, 3 subtelocentric–telocentric and 1telocentric–subtelocentric pairs. The chromosomerelative length ranged from 7.32 to 3.41%, withalso minimal differences in size between successivepairs (less than 0.64%). Figure 1(b) shows a rep-resentative karyotype.

FISH using 18S–28S rDNA plasmid probe in P.maximusmetaphases revealed strong hybridizationsignals on the telomere of an arm of a chromosomepair (Figure 2(a)). Sequential Giemsa staining ofFISH metaphases and subsequent chromosomepairing showed that the 18S–28S rDNA bear-ing chromosomes correspond to the metacen-tric–submetacentric pair 1. A set of primers,annealing on the 5S rRNA gene and designed in theopposite orientation to amplify the whole 5SrDNA repeat unit, yielded a main product of about460 bp (Figure 3). FISH, using the 5S rDNA PCRproduct as probe, showed hybridization signals atinterstitial positions on the long arm of a telocen-tric pair (Figure 2(b)). Simultaneous detection of18S–28S and 5S rDNA by two-color FISH

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corroborated that the two ribosomal gene familiesare located on different chromosome pairs (Fig-ure 2(c)).

In M. varia, the location of the 18S–28S rDNAwas first examined by silver staining of NORs inGiemsa-stained metaphases and subsequently

Table 1. Chromosome morphology of the scallops studied

Chromosome pair Relative length Centromeric index Classification

Mean SD Mean SD

P. maximus

1 4.99 0.49 41.43 5.29 m–sm

2 4.29 0.35 39.06 4.86 m–sm

3 3.45 0.50 42.05 3.10 m

4 8.02 0.55 5.59 2.62 t

5 7.32 0.42 5.24 2.03 t

6 6.83 0.34 5.05 2.58 t

7 6.47 0.40 5.40 1.80 t

8 6.11 0.28 5.40 2.01 t

9 5.82 0.20 6.93 2.51 t

10 5.56 0.19 6.10 2.03 t

11 5.35 0.17 8.40 5.66 t–st

12 5.11 0.22 6.59 3.05 t

13 4.96 0.21 5.53 2.20 t

14 4.78 0.24 5.48 1.16 t

15 4.66 0.23 6.17 2.69 t

16 4.46 0.23 6.43 2.59 t

17 4.24 0.22 5.41 3.11 t

18 3.91 0.23 6.11 1.98 t

19 3.67 0.25 6.51 2.17 t

M. varia

1 6.64 0.57 42.41 2.94 m

2 5.91 0.41 43.81 3.38 m

3 5.51 0.44 43.81 2.36 m

4 5.15 0.46 41.52 3.15 m

5 7.32 0.33 12.1 3.22 t–st

6 6.68 0.54 22.39 3.28 st–sm

7 6.35 0.4 12.9 2.47 st–t

8 5.8 0.38 21.02 4.6 st

9 5.55 0.37 24.25 3.86 st–sm

10 5.3 0.54 12.67 1.95 st–t

11 5.29 0.41 16.23 3.98 st

12 5.07 0.47 17.4 3.66 st

13 4.95 0.42 18.39 4.02 st

14 4.78 0.25 15.85 3.49 st

15 4.29 0.2 15.28 4.61 st–t

16 4.23 0.43 17.39 4.59 st

17 4.03 0.43 16.54 4.01 st

18 3.76 0.33 17.75 4.21 st

19 3.41 0.35 17.25 2.84 st

m: metacentric; sm: submetacentric; st: subtelocentric; t: telocentric.

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discolored. The number of Ag-NORs per cellvaried from 1 to 2 and they were identified asterminally or subterminally on the short arm of achromosome pair, which according to the Giemsa-karyotype corresponds to the subtelocentric–sub-metacentric pair 9 (Figure 1(b)). FISH revealedtwo hybridization signals, often of unequal size,spread along the short arm of the bearing chro-mosome pair (Figure 2(d)). PCR amplification ofthe 5S rDNA repeat unit generated a singleproduct of about 450 bp (Figure 3). The FISHcarried out with this PCR product yieldedhybridization signals on the short arm of a subt-elocentric pair. Sequential silver staining of 5SrDNA FISH metaphases and chromosome pairingrevealed that in M. varia the two ribosomal gene

families are also located on different chromosomepairs (Figures 2(e–f)), the 5S rDNA bearingchromosome pair being shorter than that bearing18S–28S rDNA.

Discussion

The specimens of P. maximus and M. variaexamined in this work displayed a diploid chro-mosome number of 2n=38. This corresponds tothe number previously reported by Beaumont &Gruffydd (1974) for both species after the analysisof eggs and early embryos obtained from speci-mens collected at the North Irish Sea, and also tothat reported for M. varia from the Adriatic

Figure 1. Karyotypes of P. maximus (a) and M. varia (b) after conventional Giemsa staining. In the box, the Ag-NOR bearing

pair after Giemsa and silver staining.

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Italian coast (Rasotto, Altieri & Colombera, 1981)and from the Atlantic French coast (Baron, Diter& Bodoy, 1989). 2n=38 is the most commonchromosome number in the family Pectinidae, asthe data compiled in Table 2 show, and also in theclass Bivalvia (Insua, 1993; Thiriot-Quievreux,2002). However, when the scallop chromosome

number is considered, taking into account thesubfamilies recognized by Waller (1991; 1993), allthe Pectininae (which include P. maximus) display2n=38, while the Chlamydinae (which include M.varia) display a variable chromosome number,from 2n=26 to 2n=38 (Table 2). If 2n=38 is as-sumed to be the ancestral chromosomal number,

Figure 2. Metaphases of the scallops studied, showing the location of the rDNA. FISH with 18S–28S rDNA (a), 5S rDNA (b) and

both probes (c) in P. maximus. FISH with 18S-28S rDNA (d) and 5S rDNA FISH (e) followed by silver staining (f) in M. varia.

Arrows indicate the hybridization sites (white) and an Ag-NOR (black).

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the available data indicate that the chromosomeevolution in Chlamydinae entailed a reduction inthe number of chromosomes.

The karyotype reported here for P. maximusdiffers slightly from that reported in 1974 byBeaumont & Gruffydd (Table 2). The differencescould be due to some type of rearrangement of thechromosome structure, which could be elucidatedby the application of longitudinal banding tech-niques. However, it seems likely that they resultfrom the application of different methods for thechromosome preparations and/or the analysis ofchromosomes differing in the condensation state.The chromosomes analyzed by Beaumont &Gruffydd (1974) were from embryos and usuallythey are less condensed than those obtained fromgill tissue (personal observation); moreover, theirmaterial was squashed by pressure, a step not in-cluded in the procedure used here. Compared tothe Pectininae species (Table 2), P. maximus andP. albicans display the closest karyotypes: the twoscallops show similar chromosome morphologywith bi-armed chromosomes (i.e. metacentric orsubmetacentric chromosomes) among the shortones, suggesting that few structural rearrange-ments have taken place between them. Differenceswith respect to the other subfamily species includethe number of telocentric chromosomes, thesebeing scarcer than in P. maximus (Table 2).

The karyotype of M. varia was determinedhere for the first time using chromosome lengthand centromeric index. Differences were also ob-served with respect to the karyotype described byBaron, Diter & Bodoy (1989), but the absence ofcentromeric index values in their work precludesan accurate comparison. The karyotype of M.varia studied here was characterized by theoccurrence of a higher number of mono-armed(subtelocentric or telocentric) than bi-armed(metacentric or submetacentric) chromosomes, as

occurs in P. maximus and most of the scallopsexamined, but the mono-armed chromosomesshowed larger short arms (centromeric index val-ues between 12.1 and 24.25) than those inP. maximus (centromeric index values between5.05 and 8.40). Taking into consideration theChlamydinae species with 2n=38 (Table 2),M. varia displays the same proportion of mono-and bi-armed chromosomes as did H. distortus(Lopez-Pinon, Insua & Mendez, 2005), and thedifferences between the two scallops lie mainly inthe tendency of H. distortus chromosomes to havethe centromere towards more submedian-subter-minal positions. Other species with 2n=38,C. farreri and Patinopecten yessoensis (Komaru &Wada, 1985), display a higher number of bi-armedthan of mono-armed chromosomes, which sug-gests that H. distortus is the closest Chlamydinaespecies to M. varia.

Both scallops studied have the 18S-28S rDNAin terminal positions, but P. maximus has it on anarm of a metacentric–submetacentric pair and inM. varia it is spread along the short arm of asubtelocentric–submetacentric pair. Althoughchromosomal location of the 18S-28S rDNA wasinvestigated in few Pectinidae species, availabledata indicate the occurrence of considerable vari-ation. 18S-28S rDNA was found at the telomere ofthe long arm of a telocentric pair in A. opercularis(Insua, Lopez-Pinon & Mendez, 1998), of theshort arm of a submetacentric–subtelocentric pairin C. farreri and two subtelocentric pairs in A. i.irradians (Wang & Guo, 2004a), and at the cen-tromere level of two subtelocentric pairs in H.distortus (Lopez-Pinon, Insua & Mendez, 2005).On the other hand, the Ag-NORs reported for A.purpuratus are terminal on the short arm of twopairs and in the pericentromeric region of the longarm of one pair (Gajardo, Parraguez & Colihue-que, 2002); and in N. nodosus the number of Ag-NORs varied from two to four (Pauls & Affonso,2000). Since silver staining only detects the trans-criptionally active NORs at the preceding inter-phase (Miller et al., 1976a, b; Howell, 1977), thedata provided may be imprecise in determiningthe number of chromosomes involved and also theextension of the chromosomal region bearing the18S–28S rDNA. Thus, data coming from in situhybridization are preferable for more accuratecomparisons. Despite the variation observed, allthe Chlamydinae scallops, except A. opercularis,

Figure 3. PCR product of the 5S rDNA repeat unit of three

individuals of P. maximus and M. varia. First lane on the

right, DNA molecular marker (100 bp ladder).

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display 18S–28S rDNA at the telomere of shortarms or near the centromere on chromosomesmostly mono-armed. This can be due to the factthat this is a frequent chromosome type in theirkaryotypes but it can also suggests that the speciesshare an ancestral 18S–28S rDNA site undergoingfew changes (if any), such as reduction/expansionprocesses by unequal crossing-over and/or peri-centric inversions. It is worth noting that in the

phylogenetic trees derived from the internal tran-scribed spacer sequences of the 18S–28S rDNAH. distortus and M. varia group in a clade separatefrom that grouping P. maximus and A. opercularis(Insua et al., 2003). Cytogenetic evidence relatingthese two latter species was not found in this workbut it will be interesting to test in the future thedegree of correspondence between the scalloprelationships revealed by 18S–28S rDNA cytoge-

Table 2. Chromosome number and karyotype of scallop species grouped according to the subfamilies recognized by Waller (1991,

1993)

Subfamily/Species n 2n Karyotype References

Camptonectinaea – – – –

Chlamydinae

Chlamys opercularis 13 26 1

14 2

=Aequipecten opercularis 26 2 m, 4 m–sm, 7 t 3

Argopecten purpuratus 32 2 m, 7 m/sm, 3 st, 4 t 4

32 5 st, 11 t 5

A. irradians irradians 16 32 6

32 5 st, 11 t 7

C. varia 19 38 1

19 2

38 5 m, 10 sm, 4 t 8

= Mimachlamys varia 38 4 m, 2 st–sm, 9 st, 3 st–t, 1 t–st This study

C. distorta 19 38 1

=Hinnites distortus 38 1 sm, 3 sm–m,1 st–sm, 14 st 9

C. farreri farreri 19 38 3 m, 1 sm, 6 sm–st, 7 st, 2 st–t 10

C. farreri 38 3 m, 4 sm, 7 sm–st, 4 st, 1 st–t 7

C. glabra 14 2

C. islandica 19 1

C. nobilis 16 32 3 m, 13 t 10

Patinopecten yessoensis 19 38 2 m, 1 m–sm, 4 sm, 6 sm–st, 3 st, 3t 10

Pectininae

Pecten albicans 19 38 3 m–sm, 4 st, 12 t 11

38 1 m, 1m–sm, 3 sm–st, 14 t 10

P. jacobaeus 19 2

P. maximus 19 38 2 m, 2 sm, 1st, 14 t 1

38 1 m, 2 m–sm, 1 t–st, 15 t This study

Euvola ziczac 19 38 5 m, 6 sm, 7 st, 1 t 12

Nodipecten nodosus 19 38 4 m, 5 sm, 7 st, 3 t 12

19 38 6 m, 6 sm, 7 st 13

Placopecten magellanicus 19 38 1

19 38 5 sm, 10 st, 4 t 14

1: Beaumont & Gruffydd (1974); 2: Rasotto, Altieri & Colombera (1981); 3: Insua, Lopez-Pinon & Mendez (1998); 4: Von Brand,

Bellolio & Lohrmann (1990); 5: Gajardo, Parraguez & Colihueque (2002); 6: Wada (1978); 7: Wang & Guo (2004a); 8: Baron, Diter &

Bodoy (1989); 9: Lopez-Pinon, Insua & Mendez (2005); 10: Komaru & Wada (1985); 11: Ieyama, (1975); 12: Basoa et al. (2000); 13:

Pauls, Pacheco & Cabeza (1996); 14: Xiang, Desrosiers & Dube (1993). aData not available.

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netic and molecular data. It is known that 18S–28SrDNA shows a pattern of concerted evolution andthat the number of repeats, the number of chro-mosomes and the chromosomal location of thesites involved are among the factors influencingthis pattern (Linares, Bowen & Dover, 1994; Li,1997).

The variation observed in the distribution ofthe 18S–28S rDNA in scallops is similar to thatreported in other bivalve families more extensivelystudied, such as Mytilidae and Ostreidae. Themussels studied display from one to four 18S–28SrDNA sites per haploid complement, often locatedterminally on an arm of submetacentric or subt-elocentric chromosomes but also on metacentricchromosomes and other chromosomal positions(e.g. Insua & Mendez, 1998, Torreiro et al., 1999;Gonzalez-Tizon et al., 2000; Vitturi et al., 2000).Oysters show one to three 18S–28S rDNA sites,mostly at the telomere of an arm of metacentric orsubmetacentric chromosomes (e.g. Leitao et al.,1999; 2002; Wang & Guo, 2004b).

The whole 5S rDNA repeat unit was success-fully PCR amplified both in P. maximus andM. varia using primers annealing in a contiguousstretch of the coding region, revealing that at leastin part the 5S rDNA is arranged in tandem repeats.Moreover, little variation occurs in the repeat unitsize for the scallops studied: in P. maximus being�460 bp, as in A. opercularis (Insua, Lopez-Pinon& Mendez, 1998), and in M. varia �450 as inH. distortus (Lopez-Pinon, Insua &Mendez, 2005).Nevertheless, the same is not reflected for chro-mosomal location. While in P. maximus the 5SrDNA is at interstitial position on the long arm ofa telocentric pair, in M. varia it is on the short armof a subtelocentric pair. In turn, in A. opercularis itwas identified at two sites (near the centromere andthe telomere) on an arm of a metacentric pair(Insua, Lopez-Pinon & Mendez, 1998) and inH. distortus at the pericentromeric region of thelong arm of one subtelocentric pair (Lopez-Pinon,Insua & Mendez, 2005). Chlamys farreri and A. i.irradians have 5S rDNA interstitially on the longarm of a telocentric and submetacentric–subtelo-centric pair, respectively (Wang & Guo, 2004a).Unlike with the 18S–28S rDNA, 5S rDNA tendedto be located outside of the telomere and on thelong arm of the bearing chromosome. This sug-gests that in M. varia a pericentric inversioninvolving the 5S rDNA site could be occurring,

reinforcing the importance of such structuralchanges during the scallop evolution.

The scallop 5S rDNA distribution differsclearly from that of the three bivalves investigatedso far. Two mussels, Mytilus galloprovincialis andM. edulis, display four 5S rDNA sites outside thetelomere on three metacentric pairs (Insua et al.,2001) and the cockle Cerastoderma edule has fivepairs carrying these sequences terminally (Insua,Freire & Mendez, 1999).

Two approaches, rarely applied to bivalvechromosomes, such as two-color FISH in the caseof P. maximus and silver staining after 5S rDNAFISH in that of M. varia, allowed us to corrobo-rate that 18S–28S and 5S rDNA map on differentchromosome pairs. This is in line with that re-ported for the other scallops examined and theother three bivalve species, except C. farreri, wherea single chromosome pair carries both gene fami-lies at different sites (Wang & Guo, 2004a).Although sinteny or even a close ligation ofthe18S–28S and 5S rDNA is not a rare phenom-enon in animals (e.g. Drouin 1999; Colomba et al.,2002; Vitturi et al., 2002), the most frequent situ-ation is that the two types of rDNA map on dif-ferent chromosomes (e.g. Wimber & Steffensen,1970; Matsuda et al., 1994; Kost et al., 1995;Martins & Galetti, 2001). This feature is useful forthe identification of chromosome markers in spe-cies scarcely characterized cytogenetically, as is thecase of scallops.

In conclusion, P. maximus and M. varia displaya chromosome number (2n=38) that is commonamong scallops and bivalve species. Their karyo-types are characterized by the occurrence of ahigher number of mono-armed than bi-armedchromosomes, as in most of the scallops examined,however, the closest karyotypes were found amongmembers of their respective subfamilies (P. albi-cans and H. distortus, respectively). Although bothspecies have one chromosome pair with 18S–28SrDNA in terminal position, as observed in someother scallops and bivalves, the morphology ofsuch pair differs andM. varia could share one 18S–28S rDNA ancestral site with other Chlamydinaespecies. The chromosomal location of the 5SrDNA in P. maximus follows the scallop tendencyof being outside of the telomere on the long arm ofthe bearing pair but this is not the case of M. variawhere a pericentric inversion could account for itsposition on a short arm.

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Acknowledgments

We thank Dr. Guillermo Roman for supplyingthe samples and Ms. Rosa Garcıa Dıaz for hertechnical assistance in the laboratory. This workwas funded by Xunta de Galicia through projectXUGA10302B97.

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