Research Article Evolutionary Dynamics of rDNA Clusters in …downloads.hindawi.com/journals/bmri/2014/754012.pdf · 2019-07-31 · Research Article Evolutionary Dynamics of rDNA

Research ArticleEvolutionary Dynamics of rDNA Clusters inChromosomes of Five Clam Species Belonging to the FamilyVeneridae (Mollusca Bivalvia)

Concepcioacuten Peacuterez-Garciacutea12 Ninoska S Hurtado1 Paloma Moraacuten1 and Juan J Pasantes1

1 Departamento de Bioquımica Xenetica e Inmunoloxıa Universidade de Vigo 36310 Vigo Spain2Department of Biogeochemistry and Ecotoxicology Laboratory of Ecotoxicology rue de lrsquoIle drsquoYeu BP 2110544311 Nantes Cedex 03 France

Correspondence should be addressed to Juan J Pasantes pasantesuvigoes

Received 20 February 2014 Revised 23 April 2014 Accepted 25 April 2014 Published 22 May 2014

Academic Editor Stephan Koblmuller

Copyright copy 2014 Concepcion Perez-Garcıa et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

The chromosomal changes accompanying bivalve evolution are an area about which few reports have been published To improveour understanding on chromosome evolution in Veneridae ribosomal RNA gene clusters were mapped by fluorescent in situhybridization (FISH) to chromosomes of five species of venerid clams (Venerupis corrugata Ruditapes philippinarum Ruditapesdecussatus Dosinia exoleta and Venus verrucosa) The results were anchored to the most comprehensive molecular phylogenetictree currently available for Veneridae While a single major rDNA cluster was found in each of the five species the number of 5SrDNA clusters showed high interspecies variation Major rDNA was either subterminal to the short arms or intercalary to the longarms of metacentric or submetacentric chromosomes whereas minor rDNA signals showed higher variability Major and minorrDNAs map to different chromosome pairs in all species but in R decussatus one of the minor rDNA gene clusters and the majorrDNA cluster were located in the same position on a single chromosome pair This interspersion of both sequences was confirmedby fiber FISH Telomeric signals appeared at both ends of every chromosome in all species FISH mapping data are discussed inrelation to the molecular phylogenetic trees currently available for Veneridae

1 Introduction

The nuclear genes that code for the ribosomal RNA ineukaryotes are organized into two different multigene fam-ilies Major rDNA repeats consist of a transcriptional unitwhich codes for the 18S 58S and 28S rRNAs separatedby two internal transcribed spacers (ITS1 and ITS2) and anintergenic spacer (IGS) Minor rDNA repeats consist of asequence which codes for the 5S rRNA and a nontranscribedspacer (NTS) Although in most eukaryotes in which theorganization of these genes is known the two types of rDNAsare located on different chromosome pairs an increasingnumber of studies have reported the existence of clusters ofboth major and minor rRNA genes mapping to the samechromosomal position in many species [1 2] Furthermorethe changes in both number and chromosomal position of

the rDNA clusters have helped to solve some uncertainties inthe phylogenies of some groups of organisms [3 4]

The family Veneridae (Bivalvia Heterodonta) includesmore than 800 living species distributed worldwide whichhave adapted to a wide range of marine environments [5 6]The phylogenetic relationships among the species of thisfamily have been investigated using both morphological andmolecular features [6ndash9] but in terms of characterizingtheir chromosomes current knowledge is limited to mitoticchromosome numbers and karyotypes of just a few species[10ndash13] a restriction endonuclease banding pattern inRuditapes decussatus [14] and the location of telomericrDNA andor core histone gene sequences in Mercenariamercenaria [15 16] Dosinia exoleta [13] R decussatus andR philippinarum [17] Polititapes (Venerupis) aureus and P(Tapes) rhomboides [18]

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 754012 9 pageshttpdxdoiorg1011552014754012

2 BioMed Research International

In order to advance the chromosomes characterizationof bivalve species and gain some insights on their evolutionwe hybridized major rDNA 5S rDNA and telomeric probesto mitotic chromosomes and surface spread synaptonemalcomplexes of five species of clams of different subfamiliesof the family Veneridae Venerupis corrugata Ruditapesphilippinarum and R decussatus (Tapetinae clade A1 in [6])Dosinia exoleta (Dosiniinae clade A4 in [6]) and Venusverrucosa (Venerinae clade A2 in [6]) The anchoring of thechromosomal mapping data obtained together with previ-ously published data for Polititapes aureus and P rhomboides[18] (Tapetinae clade A1 in [6]) and Mercenaria mercenaria[16] (Chioninae clade A3 in [6]) to the phylogenetic treeproposed by Chen et al [6] allows us to shed some light onthe chromosomal changes that occurred during the evolutionof this bivalve family

2 Materials and Methods

21 Biological Material Specimens of the pullet carpetshell Venerupis corrugata (pullastra) (Gmelin 1791) thegrooved carpet shell Ruditapes decussatus (Linnaeus 1758)the Japanese carpet shell Ruditapes philippinarum (Adamsand Reeve 1850) the rayed artemisDosinia exoleta (Linnaeus1758) and the warty venus Venus verrucosa (Linnaeus 1758)were collected from field and cultured populations in Rıa dePontevedra and Rıa de Vigo (NWSpain) Identification of thespecies level was performed on the basis of themorphology oftheir shells and the degree of separation of their siphons Thenomenclature used for these taxa followed theWorld RegisterofMarine Species database (httpwwwmarinespeciesorg)

Male and female juvenile individuals were maintained inthe laboratory in 10 L tanks of aerated filtered sea water at18 plusmn 1∘C The animals were fed on a suspension of algal cells(Isochrysis galbana) for at least 10 days in order to promoteboth somatic growth and gonadic maturation Mature speci-mens were also collected during the reproductive season

22 Probe Preparation Total genomic DNA was extractedfrom ethanol-preserved adductor muscles [19] Approxi-mately 3mg of tissue was homogenized in hexadecyltrimeth-ylammoniumbromide (CTAB) buffer and digested overnightwith pronase (15mgmL) at 60∘C DNA was extracted withchloroformisoamyl alcohol (241) and stored at 4∘C

FISH probes were obtained by polymerase chain reaction(PCR) amplifications performed in 20 120583L of a solutioncontaining 50 ng DNA 1xPCR buffer 50120583M each dNTP25mM MgCl

2 1 120583M each primer and 1U BIOTAQ DNA

polymerase (Bioline) Universal primers were used to amplifythe whole internal transcribed spacer (ITS) region of themajor rDNA cluster (ITS4 51015840-TCCTCCGCTTATTGATAT-GC-31015840 ITS5 51015840-GGAAGTAAAAGTCGTAACAAGG-31015840[20]) For the amplification of the whole repeat unit of the 5SrDNA we designed primers (5SD 51015840-CAACGTGATATG-GTCGTAGAC-31015840 5SR 51015840-AACACCGGTTCTCGTCCG-ATC-31015840) from the sequence of the 5S rRNA ofMytilus edulis[21]

After 5min of denaturation at 95∘C 30 cycles of ampli-fication were performed (95∘C 30 s 48∘C 30 s 72∘C 30 sfor the ITS 50 s for the 5S rDNA) A final extension step of7min at 72∘C was applied All reactions were performed in aGeneAmp PCR system 9700 (Applied Biosystems) and PCRproducts were examined by electrophoresis on 2 agarosegels

The amplified ITSs obtained from the five species and the5S rDNA from Venerupis corrugata were labeled with biotin-16-dUTP (Roche Applied Science) andor digoxigenin-11-dUTP (10xDIG Labeling Mix Roche Applied Science) usinga nick translation kit (RocheApplied Science)The remaining5S rDNAs were directly labeled by PCR with either biotin-16-dUTP (20120583M) or digoxigenin-11-dUTP (5 120583M)

23 Mitotic Chromosome Synaptonemal Complex andRelease Chromatin Fiber Preparation Mitotic chromosomepreparations were obtained according to Pasantes et al[22] Juvenile specimens were housed in 05 L beakers andexposed to colchicine (0005) for 12 h Gills were excisedand immersed in 50 and 25 sea water for 1 h and fixedwith ethanolacetic acid for 1 h Small pieces of tissue weredissociated in 60 acetic acid and the cell suspension wasdropped onto slides heated to 50∘C

Surface spreading of synaptonemal complexes (SCs) wasperformed as indicated by Hurtado and Pasantes [13] Freshmale gonadic tissue was minced in 04M NaCl and cen-trifuged several times to remove the sperm One drop of thecellular suspension was spread for 1ndash5min on clean slidescovered by a spreading medium (01M sucrose 05 TritonX-100) The slides were then flooded with paraformaldehyde(4) and the liquid mixture allowed settling overnight Afterdraining briefly the slides were rinsed in distilled water andair-dried

Chromatin fibers were released as described by Fidlerovaet al [23] In brief the cellular suspension obtained follow-ing the protocol for mitotic chromosome preparation wascentrifuged for 10min at 1200 rpm and the supernatant wasdiscarded The pellet was resuspended in fresh fixative anddropped on clean slides After leaving to evaporate brieflyslideswere immersed in 1x PBS for 1min and chromatin fiberswere released with 005M NaOH in 30 ethanol

24 Fluorescent In Situ Hybridization (FISH) FISH SC-FISH and Fiber-FISH experiments using biotin and digox-igenin labeled species-specific ITS and 5S rDNA probeswere performed following the methods published elsewhere[13 18] Slides were pretreated with RNase and pepsinbefore denaturation Preparations were denatured for 2minat 70∘C (mitotic chromosomes and released chromatin fibers)or 80∘C (meiotic chromosomes) Hybridization was per-formed overnight at 37∘C Signal detection was carried outwith fluorescein avidin and biotinylated anti-avidin for thebiotinylated probes and with mouse anti-digoxigenin andanti-mouse TRITC for the probes labeled with digoxigeninSlides were counterstained for 8min with 41015840-6-diamidino-2-phenylindole (DAPI 014 120583gmLminus1 in 2x SSC) and mounted

BioMed Research International 3

in antifade (Vectashield Vector) In addition we also per-formed FISH using a vertebrate telomeric (C

3TA2)3peptide

nucleic acid (PNA) probe (Applied Biosystems) following theprotocol indicated by the supplier

Slide visualization and photography were performedusing a Nikon Eclipse-800 microscope equipped with an epi-fluorescence system Aminimum of 5 individuals per speciesand 20 metaphases per individual were recorded for eachprobe on mitotic chromosomes For SC-FISH a minimumof 5 individuals per species and 30 SC spreads per individualwere recorded for each probe Separated images for eachfluorochrome were obtained with a DS-Qi1Mc CCD camera(Nikon) controlled by the NIS-Elements software (Nikon)The merging of the images was performed with AdobePhotoshop

3 Results

All specimens of the five clam species presented mitoticmetaphase plates showing 38 chromosomes and meioticprophase I plates with 19 bivalents (Figures 1 and 2)

Single- and double-color FISH experiments showed thatmajor rDNAsmapped to a single locus in all five clam species(Figures 1 and 2) Intercalary signals appeared in mitoticmetaphase chromosomes and meiotic prophase I bivalentsin both Venerupis corrugata (Figures 1(a) and 1(b)) andRuditapes decussatus (Figures 2(a) and 2(b))The signals werelocated on the long arms of metacentric and submetacentricchromosome pairs respectively On the other hand signalswere subterminal to short arms in R philippinarum (Figures1(c) and 1(d)) Venus verrucosa (Figures 1(e) and 1(f)) andDosinia exoleta (Figures 1(g) and 1(h))

Minor rDNA signals mapped to a single locus inVenerupis corrugata and Venus verrucosa and to two locion different chromosomes in Ruditapes philippinarum Rdecussatus and Dosinia exoleta The single 5S rDNA clus-ter was subterminal to the long arms of a subtelocentricchromosome in V corrugata (Figures 1(a) and 1(b)) butalmost centromeric on the short arms of a metacentricchromosome in V verrucosa (Figures 1(e) and 1(f)) The two5S rDNA clusters present in the remaining three specieswere subterminal and intercalary to the long arms of twosubtelocentric chromosomes in R philippinarum (Figures1(c) and 1(d)) subterminal to the short arms and almostcentromeric to the long arms of two submetacentric chromo-somes in D exoleta (Figures 1(g) and 1(h)) and intercalaryand subterminal to the long arms of submetacentric andsubtelocentric chromosomes in R decussatus (Figures 2(a)and 2(b))

Double-color FISH experiments showed that major andminor rDNAs mapped to different chromosome pairs inVenerupis corrugata (Figures 1(a) and 1(b)) Ruditapes philip-pinarum (Figures 1(c) and 1(d)) Venus verrucosa (Figures1(e) and 1(f)) and Dosinia exoleta (Figures 1(g) and 1(h))However in R decussatus one of the minor rDNA geneclusters and the major rDNA cluster were located in thesame positions on the same chromosome pair resulting in

overlapping signals (Figures 2(a) and 2(b)) In order to deter-mine whether these signals were really overlapping or closetogether but distinct double-color FISH experiments wereperformed on extended chromatin fibers of R decussatusAs clearly shown in Figures 2(c) and 2(d) all fibers showingmajor rDNA signals also presented signals corresponding to5S rDNA When the fibers lack enough extension these redand green signals overlap but when stretched the signalswere clearly interspersed On the same slides other fibersonly showed 5S rDNA signals

Sequential FISH experiments using a vertebrate telomeric(C3TA2)3PNA probe followed by a second FISH using

major rDNA or 5S rDNA probes showed terminal sig-nals at the ends of the sister chromatids of every mitoticchromosome and of the SCs in every prophase I biva-lent (Figure 3) No intercalary signals were detected Aspreviously reported in Dosinia exoleta [13] telomeric sig-nals appeared to be quite condensed and tightly associatedat the ends of the SCs in pachytene chromosomes Onthe other hand rDNA signal dots on SC spreads werealways associated with chromatin that loops away from theSC

Figure 4 shows a summary of the rDNA mapping resultscurrently available for the family Veneridae The specieswere ordered according to the phylogenetic tree proposed byChen et al [6] and assigned to both their clade groups (A1A2 A3 and A4) and the traditional subfamily classification(Tapetinae Chioninae Venerinae and Dosiniinae)

4 Discussion

To date karyological data are available for only a few speciesof the family Veneridae [10 11 24] Both the chromosomenumbers and the karyotypes determined in this work forRuditapes philippinarum R decussatus Venerupis corrugataVenus verrucosa and Dosinia exoleta are in agreement withprevious results [12 13 25 26] and further confirm thatunlike other families within the order Veneroida in whichinterspecific differences in chromosome numbers have beendetected all Veneridae species have the same chromosomenumber 2n = 38 Nevertheless the high variation in kary-otype composition detected in these species indicates thatspeciation in Veneridae was accompanied by some chromo-some rearrangements

Ribosomal markers have been successfully applied inspecies identification and consequently have provided valu-able information regarding chromosome and genome evo-lution [3 4] However very little is known about thesesequences in the species belonging to the family Veneridaein which major rDNA sequences have only been mapped tochromosomes of six species [13 16ndash18] and 5S rDNA in fourspecies [17 18]

The presence of major rDNA signals on just a sin-gle chromosome pair in Venerupis corrugata Ruditapesdecussatus Ruditapes philippinarum Venus verrucosa andDosinia exoleta is coincidental with previous results [13 17]


VCO

(a)

5SITS

(b)

RPH

(c) (d)

VVE

(e) (f)

DEX

(g) (h)

Figure 1 Chromosomal location of 5S rDNA and major rDNA to mitotic chromosomes ((a) (c) (e) and (g)) and surface spreadsynaptonemal complexes ((b) (d) (f) and (h)) ofVenerupis corrugata (VCO (a) and (b)) Ruditapes philippinarum (RPH (c) and (d))Venusverrucosa (VVE (e) and (f)) and Dosinia exoleta (DEX (g) and (h)) counterstained with DAPI Major rDNA signals (ITS digoxigeninRhodamine red) are intercalary to the long arms of a pair of metacentric chromosomes (bivalents) in Venerupis corrugata ((a) and (b)) butterminal to the short arms of a metacentric chromosome pair (bivalent) in Ruditapes philippinarum ((c) and (d)) and Dosinia exoleta ((g)and (h)) and a submetacentric chromosome pair (bivalent) in Venus verrucosa ((e) and (f)) 5S rDNA signals (5S biotin fluorescein green)are subterminally located on the short arms of a subtelocentric chromosome pair (bivalent) in Venerupis corrugata ((a) and (b)) close to thecentromere on the short arms of a metacentric chromosome pair (bivalent) in Venus verrucosa ((e) and (f)) intercalary to the long arms ofsubmetacentric and subtelocentric chromosome pairs (bivalents) in Ruditapes philippinarum ((c) and (d)) terminal to the short arms andcentromeric on the long arms of two submetacentric chromosome pairs (bivalents) in Dosinia exoleta ((g) and (h)) Note that all signals areon different chromosome pairs (bivalents) Scale bars 5 120583m

Within the family Veneridae a single major rDNA locushas been also detected in Polititapes (Venerupis) aureus andP (Tapes) rhomboides [18] whereas Mercenaria mercenariapresents two major rDNA loci [16] Therefore our results for

V corrugata and V verrucosa increase the number of Veneri-dae species showing a single major rDNA cluster to seven outof a total of eight studied These sequences were subterminalin P rhomboides R philippinarum V verrucosa and D


5SITS

RDE

(a) (b)

(c)

(d)

Figure 2 Two-color FISH of major rDNA (ITS digoxigenin Rhodamine red) and 5S rDNA (5S biotin fluorescein green) probes to mitoticchromosomes (a) surface spread synaptonemal complexes (b) and released chromatin fibers ((c) and (d)) of Ruditapes decussatus (RDE)Major rDNA red signals are intercalary on the long arms of a submetacentric chromosome pair (bivalent) overlapping 5S rDNA green signalstherefore giving yellow signalsThe remaining 5S rDNA signals are terminal to the long arms of a subtelocentric chromosome pair (bivalent)The green and red signals that overlap in mitotic and meiotic chromosomes are clearly distinct on the released chromatin fibers and show aninterspersion pattern ((c) and (d)) Scale bars 5 120583m

exoleta but intercalary in P aureusV corrugata andR decus-satus Furthermore the two major rDNA clusters presentin M mercenaria also showed subterminal and intercalarylocations [16]

The occurrence of single 5S rDNA clusters in Venerupiscorrugata and Venus verrucosa matches that of Polititapesaureus and P rhomboides [18] On the other hand ourstudy also revealed the existence of two 5S rDNA clusters inRuditapes decussatus R philippinarum and Dosinia exoletawhich contrasts with previous findings [17 18] The chromo-somal location of 5S rDNA clusters also showed interspecificdifferences Subterminal clusters appeared in P aureus Vcorrugata R decussatus P rhomboides R philippinarumand D exoleta intercalary clusters in R decussatus and Rphilippinarum and pericentromeric ones in V verrucosa andD exoleta

Chromosomal mapping of both 5S and major rDNAsequences is known in a total number of 21 species of bivalves[17 18 24 27ndash31] Seventeen of these species show 5S andmajor rDNA clusters located on different chromosome pairsand three of them also show nonlinked clusters but locatedon the same chromosome pair finally Ruditapes decussatusis the only one in which overlapping signals for both kindsof rDNA sequences were detected [17] The data presentedhere also showed the presence of separated 5S and majorrDNA signals in Venerupis corrugata Dosinia exoleta andVenus verrucosa confirmed the absence of linkage betweenthese sequences in R philippinarum [17] and corroboratedthe presence of overlapping signals in R decussatus [17]Additionally the occurrence of alternate 5S and major

rDNA signals on extended chromatin fibers of R decussatussuggests that these sequences are intercalated Our resultsshowing the existence of linkage betweenmajor and 5S rDNAsequences in R decussatus but not in its congeneric speciesR philippinarum or in any of the other species within thesubfamily Tapetinae Polititapes aureus Venerupis corrugataand P rhomboides support the suggestion that 5S and majorrRNA gene linkages have been repeatedly established and lostduring the evolution of eukaryotic genomes [1 2] These dataare also in agreement with previous results reported in a wideanalysis ofmetazoan genomes [32] inwhich though 5S rDNAwas found to be linked to various noncoding RNA genes inseveral clades no evidence of evolutionary-conserved linkageamong them was detected

Detection of the vertebrate (T2AG3)n repeat at chromo-

some ends in Venerupis corrugata Ruditapes decussatus Rphilippinarum andVenus verrucosa coincides with the resultsobtained inMercenaria mercenaria [15] Dosinia exoleta [13]Polititapes aureus and P rhomboides [18] Sequential FISHexperiments using telomeric and rDNA probes on the sameSC spreads also showed that in these species telomericsequences are separated from rDNA sequences not onlywhenthe rDNA signals are intercalary but even in those specieswith subterminal rDNA (major or 5S) clusters These resultsdiffer from those reported for other species and contradictthe idea of Vitturi et al [33] that in invertebrate speciesmajor rDNA clusters are closely associated with telomericsequences

The molecular phylogenies available for the species ofVeneridae [5 6 8 9] show that Polititapes aureus Venerupis


VCO 5STEL

(a)

RDE 5STEL

(b)

RPH ITSTEL

(c)

ITSTEL

VVE

(d)

Figure 3 Two-color FISH of telomeric (TEL fluorescein green) and 5S or major rDNA (5S or ITS digoxigenin Rhodamine red) probes tosurface spread synaptonemal complexes of Venerupis corrugata (VCO (a)) Ruditapes decussatus (RDE (b)) Ruditapes philippinarum (RPH(c)) andVenus verrucosa (VVE (d)) counterstained with DAPI Note that all telomeric signals appear as tightly packed single or double greendots attached to the SCs at both ends of each bivalent in the four clam species On the other hand the subterminal red signals correspondingto 5S (VCO (a) RDE (b)) and ITS (RPH (c) VVE (d)) rDNA probes surround the telomeric signals radiating away from the SCs in all clamsScale bars 5120583m

corrugata Ruditapes decussatus P rhomboides and R philip-pinarum cluster together asmembers of the subfamily Tapeti-nae whereasMercenaria mercenaria (Chioninae) Venus ver-rucosa(Venerinae) and Dosinia exoleta (Dosiniinae) formseparated clades However previous studies [6 8] have alsoshown results that disagree with the classical arrangementsof many currently accepted taxa [34ndash37] In this regard Rdecussatus is closer to the species coming from the samegeographic area (P aureus V corrugata and P rhomboides)than to its congeneric R philippinarum while P aureus ismore closely related to V corrugata and R decussatus thanto P rhomboides

The rDNA chromosomal mapping results schematizedin Figure 4 together with a simplified representation of thephylogenetic tree proposed by Chen et al [6] indicatedthat the existence of single subterminal major and minorrDNA clusters on different chromosome pairs as in Politi-tapes rhomboides is most likely the ancestral condition Thephylogenetic relationships among the rest of the species ofthe subfamily Tapetinae (cladeA4) could be explained on theone hand as a consequence of the translocation of the singlemajor rDNA locus to an intercalary location in a common

ancestor of P aureus V corrugata and Ruditapes decussatusfollowed by an invasion of the major rDNA by some 5SrDNA sequences in R decussatus On the other hand anindependent translocation of some copies of the 5S rDNAto an intercalary location could explain the mapping datain R philippinarum In an independent event an ancestorof Venus verrucosa suffered the translocation of the whole5S rDNA cluster to an intercalary position The presence ofan additional intercalary major rDNA cluster in Mercenariamercenaria and an additional centromeric 5S rDNA cluster inDosinia exoleta could also be the result of this kind of eventbut in this case only affecting some of the clustered copies ofthese genes

In summary this study provides further insights into thechromosomal distribution of ribosomal RNA gene clustersin species of the family Veneridae Although the chromoso-mal mapping of major and minor rDNAs in these speciesdid not clarify the phylogenetic relationship among venusclams the striking similarity and clear differences among thechromosomal distribution of these rDNA clustersmake thempromising markers for the further study of chromosomeevolution within Veneridae


VenerinaeChioninaeTapetinae

Ruditapes philippinarum

Ruditapes decussatus

Polititapes aureus(Tapes aurea)

Venerupis corrugata

Venerupis galactites

Polititapes rhomboides (Tapes rhomboides)

A

(V pullastra)

(R decussata)

Venus verrucosa

2n Major rDNA 5S rDNAChioninae

Tapetinae

Venerinae

ChioninaeVenerinaeChioninae

Venerinae

Chioninaesm m

1t 1pc

sm sm1ic 1t

m1t

sm sm1t 1pc

mst1t 1t

st1ic 1t

m

m st38 1ic 1t

sm38 1ic 1t 1ic

st sm

st st1t 1ic

m1t38

Chioninae

VenerinaeTapetinae

38

38

38

38

38

A1

Mercenaria mercenaria

Dosinia exoleta (Pectunculus exoletus)

A4

A3

A2

Dosiniinae

Figure 4 Schematic representation of the distribution of major (red) and 5S (green) rDNA clusters on chromosomes of Veneridae Yellowmarks indicate linkage of major and minor rDNAsThe species are organized according to the phylogenetic tree (clade A) proposed by Chenet al [6] (left) and also assigned to subfamilies according to the accepted traditional classification (right)

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Concepcion Perez-Garcıa and Ninoska S Hurtado con-tributed equally to this work

Acknowledgments

The authors wish to thank P Alvarino and N Santamarıafor their technical assistance M Lastra A Simon and SPereira for kindly providing the clams and R Fallon andMJI Briones for correcting the paper This work was partlyfunded by Grants fromXunta de Galicia and Fondos FEDERldquoUnha maneira de facer Europardquo (PGIDIT03PXIC30102PN08MMA023310PR Grupos de Referencia Competitiva201080 Grupos con Potencial Crecimiento GPC2013-011)

and Universidade de Vigo (64102C124) Ninoska S Hurtadowas partly funded by aMutis fellowship from AECI (Spain)

References

[1] G Drouin and M M de Sa ldquoThe concerted evolution of 5Sribosomal genes linked to the repeat units of other multigenefamiliesrdquoMolecular Biology and Evolution vol 12 no 3 pp 481ndash493 1995

[2] S Wicke A Costa J Munoz and D Quandt ldquoRestless 5S there-arrangement(s) and evolution of the nuclear ribosomal DNAin land plantsrdquo Molecular Phylogenetics and Evolution vol 61no 2 pp 321ndash332 2011

[3] V Roy L Monti-Dedieu N Chaminade et al ldquoEvolution of thechromosomal location of rDNAgenes in twoDrosophila speciessubgroups ananassae and melanogasterrdquo Heredity vol 94 no4 pp 388ndash395 2005

[4] P M Datson and B G Murray ldquoRibosomal DNA locusevolution in Nemesia transposition rather than structuralrearrangement as the key mechanismrdquo Chromosome Researchvol 14 no 8 pp 845ndash857 2006


[5] P M Mikkelsen R Bieler I Kappner and T A RawlingsldquoPhylogeny of Veneroidea (Mollusca Bivalvia) based on mor-phology and moleculesrdquo Zoological Journal of the LinneanSociety vol 148 no 3 pp 439ndash521 2006

[6] J Chen Q Li L Kong and X Zheng ldquoMolecular phylogenyof venus clams (Mollusca Bivalvia Veneridae) with emphasison the systematic position of taxa along the coast of mainlandChinardquo Zoologica Scripta vol 40 no 3 pp 260ndash271 2011

[7] A Canapa I Marota F Rollo and E Olmo ldquoPhylogeneticanalysis of Veneridae (Bivalvia) comparison of molecular andpaleontological datardquo Journal ofMolecular Evolution vol 43 no5 pp 517ndash522 1996

[8] A Canapa S Schiaparelli I Marota andM Barucca ldquoMolecu-lar data from the 16S rRNA gene for the phylogeny of Veneridae(Mollusca Bivalvia)rdquo Marine Biology vol 142 no 6 pp 1125ndash1130 2003

[9] I Kappner and R Bieler ldquoPhylogeny of venus clams (BivalviaVenerinae) as inferred from nuclear and mitochondrial genesequencesrdquo Molecular Phylogenetics and Evolution vol 40 no2 pp 317ndash331 2006

[10] C Thiriot-Quievreux ldquoAdvances in cytogenetics of aquaticorganismsrdquo in Genetics and Evolution of Aquatic Organisms AR Beaumont Ed pp 369ndash388 Chapman and Hall LondonUK 1994

[11] C Thiriot-Quievreux ldquoReview of the literature on bivalvecytogenetics in the last ten yearsrdquo Cahiers de Biologie Marinevol 43 no 1 pp 17ndash26 2002

[12] A-B M Ebied and F M Aly ldquoCytogenetic studies onmetaphase chromosomes of six bivalve species of familiesmytilidae and veneridae (Nucinelloidea Mollusca)rdquo Cytologiavol 69 no 3 pp 261ndash273 2004

[13] N S Hurtado and J J Pasantes ldquoSurface spreading of synap-tonemal complexes in the clam Dosinia exoleta (MolluscaBivalvia)rdquo Chromosome Research vol 13 no 6 pp 575ndash5802005

[14] A Leitao R Chaves D Matias S Joaquim F Ruano andH Guedes-Pinto ldquoRestriction enzyme digestion chromosomebanding on two commercially important venerid bivalvespecies Ruditapes decussatus and Cerastoderma edulerdquo Journalof Shellfish Research vol 25 no 3 pp 857ndash863 2006

[15] Y Wang and X Guo ldquoChromosomal mapping of the vertebratetelomeric sequence (TTAGGG)

119899

in four bivalve molluscs byfluorescence in situ hybridizationrdquo Journal of Shellfish Researchvol 20 no 3 pp 1187ndash1190 2001

[16] Y Wang and X Guo ldquoChromosomal mapping of major ribo-somal rRNA genes in the hard clam (Mercenaria mercenaria)using fluorescence in situ hybridizationrdquo Marine Biology vol150 no 6 pp 1183ndash1189 2007

[17] N S Hurtado C Perez-Garcıa P Moran and J J PasantesldquoGenetic and cytological evidence of hybridization betweennative Ruditapes decussatus and introduced Ruditapes philip-pinarum (Mollusca Bivalvia Veneridae) in NW Spainrdquo Aqua-culture vol 311 no 1ndash4 pp 123ndash128 2011

[18] J Carrilho C Perez-Garcıa A Leitao I Malheiro and J J Pas-antes ldquoCytogenetic characterization and mapping of rDNAscore histone genes and telomeric sequences in Venerupis aureaand Tapes rhomboides (Bivalvia Veneridae)rdquo Genetica vol 139no 6 pp 823ndash831 2011

[19] B Winnepenninckx T Backeljau and R de Wachter ldquoExtrac-tion of high molecular weight DNA from molluscsrdquo Trends inGenetics vol 9 no 12 article 407 1993

[20] T J White T Burms S Lee and J W Taylor ldquoAmplificationand direct sequences of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide to Methods andApplications M A Inmus D H Guelfand J J Sminsky andT J White Eds pp 315ndash322 Academic Press New York NYUSA 1990

[21] B-L Fang R de Baere A Vandenberghe and R de WachterldquoSequences of three molluscan 5 S ribosomal RNAs confirm thevalidity of a dynamic secondary structure modelrdquoNucleic AcidsResearch vol 10 no 15 pp 4679ndash4685 1982

[22] J J Pasantes M J Martınez-Exposito A Martınez-Lage andJ Mendez ldquoChromosomes of Galician musselsrdquo Journal ofMolluscan Studies vol 56 no 1 pp 123ndash126 1990

[23] H Fidlerova G SengerM Kost P Sanseau andD Sheer ldquoTwosimple procedures for releasing chromatin from routinely fixedcells for fluorescence in situ hybridizationrdquo Cytogenetics andCell Genetics vol 65 no 3 pp 203ndash205 1993

[24] A Arias-Perez A Insua R Freire J Mendez and J Fernandez-Tajes ldquoGenetic studies in commercially important species ofVeneridaerdquo in Clam Fisheries and Aquaculture F da CostaGonzalez Ed pp 73ndash105 Nova Science Publishers Haup-pauge NY USA 2013

[25] P Borsa and CThiriot-Quievreux ldquoKaryological and allozymiccharacterization of Ruditapes philippinarum R aureus and Rdecussatus (Bivalvia Veneridae)rdquo Aquaculture vol 90 no 3-4pp 209ndash227 1990

[26] A Insua and C Thiriot-Quievreux ldquoKaryotypes of Cerasto-derma edule Venerupis pullastra and Venerupis rhomboıdes(Bivalvia Veneroidea)rdquo Aquatic Living Resources vol 5 no 1pp 1ndash8 1992

[27] A Leitao and R Chaves ldquoBanding for chromosomal identi-fication in bivalves a 20-year historyrdquo Dynamic BiochemistryProcess Biotechnology and Molecular Biology vol 2 no 1 pp44ndash49 2008

[28] C Perez-Garcıa J M Cambeiro P Moran and J J PasantesldquoChromosomal mapping of rDNAs core histone genes andtelomeric sequences in Perumytilus purpuratus (BivalviaMytil-idae)rdquo Journal of Experimental Marine Biology and Ecology vol395 no 1-2 pp 199ndash205 2010

[29] C Perez-Garcıa J Guerra-Varela P Moran and J J PasantesldquoChromosomalmapping of rRNAgenes core histone genes andtelomeric sequences in Brachidontes puniceus and Brachidontesrodriguezi (Bivalvia Mytilidae)rdquo BMC Genetics vol 11 article109 2010

[30] C Perez-Garcıa P Moran and J J Pasantes ldquoCytogeneticcharacterization of the invasive mussel species Xenostrobussecuris Lmk (Bivalvia Mytilidae)rdquo Genome vol 54 no 9 pp771ndash778 2011

[31] A Gonzalez-Tizon V Rojo J Vierna K T Jensen E Egea andA Martınez-Lage ldquoCytogenetic characterisation of the razorshells Ensis directus (Conrad 1843) and E minor (Chenu 1843)(Mollusca Bivalvia)rdquo Helgoland Marine Research vol 67 no 1pp 73ndash82 2013

[32] J Vierna S Wehner C Honer zu Siederdissen A Martınez-Lage and M Marz ldquoSystematic analysis and evolution of 5Sribosomal DNA inmetazoansrdquoHeredity vol 111 no 5 pp 410ndash421 2013

[33] R Vitturi M S Colomba A M Pirrone and M MandriolildquorDNA (18S-28S and 5S) colocalization and linkage betweenribosomal genes and (TTAGGG)n telomeric sequence in the


earthworm Octodrilus complanatus (Annelida OligochaetaLumbricidae) revealed by single- and double-color FISHrdquoJournal of Heredity vol 93 no 4 pp 279ndash282 2002

[34] M Huber Compendium of Bivalves A Full-Color Guide to 3300of the Worldrsquos Marine Bivalves A Status on Bivalvia after 250years of research ConchBooks Hackenheim Germany 2010

[35] S Gofas ldquoRuditapes Chiamenti 1900rdquo World Register ofMarine Species httpwwwmarinespeciesorgaphiaphpp=taxdetailsampid=231748

[36] P Bouchet and S Gofas ldquoPolititapes Chiamenti 1900rdquo WorldRegister of Marine Species httpwwwmarinespe-ciesorgaphiaphpp=taxdetailsampid=246149

[37] S Gofas ldquoVenerupis Lamarck 1818rdquo World Register ofMarine Species httpwwwmarinespeciesorgaphiaphpp=taxdetailsampid=138647

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


In order to advance the chromosomes characterizationof bivalve species and gain some insights on their evolutionwe hybridized major rDNA 5S rDNA and telomeric probesto mitotic chromosomes and surface spread synaptonemalcomplexes of five species of clams of different subfamiliesof the family Veneridae Venerupis corrugata Ruditapesphilippinarum and R decussatus (Tapetinae clade A1 in [6])Dosinia exoleta (Dosiniinae clade A4 in [6]) and Venusverrucosa (Venerinae clade A2 in [6]) The anchoring of thechromosomal mapping data obtained together with previ-ously published data for Polititapes aureus and P rhomboides[18] (Tapetinae clade A1 in [6]) and Mercenaria mercenaria[16] (Chioninae clade A3 in [6]) to the phylogenetic treeproposed by Chen et al [6] allows us to shed some light onthe chromosomal changes that occurred during the evolutionof this bivalve family

2 Materials and Methods

21 Biological Material Specimens of the pullet carpetshell Venerupis corrugata (pullastra) (Gmelin 1791) thegrooved carpet shell Ruditapes decussatus (Linnaeus 1758)the Japanese carpet shell Ruditapes philippinarum (Adamsand Reeve 1850) the rayed artemisDosinia exoleta (Linnaeus1758) and the warty venus Venus verrucosa (Linnaeus 1758)were collected from field and cultured populations in Rıa dePontevedra and Rıa de Vigo (NWSpain) Identification of thespecies level was performed on the basis of themorphology oftheir shells and the degree of separation of their siphons Thenomenclature used for these taxa followed theWorld RegisterofMarine Species database (httpwwwmarinespeciesorg)

Male and female juvenile individuals were maintained inthe laboratory in 10 L tanks of aerated filtered sea water at18 plusmn 1∘C The animals were fed on a suspension of algal cells(Isochrysis galbana) for at least 10 days in order to promoteboth somatic growth and gonadic maturation Mature speci-mens were also collected during the reproductive season

22 Probe Preparation Total genomic DNA was extractedfrom ethanol-preserved adductor muscles [19] Approxi-mately 3mg of tissue was homogenized in hexadecyltrimeth-ylammoniumbromide (CTAB) buffer and digested overnightwith pronase (15mgmL) at 60∘C DNA was extracted withchloroformisoamyl alcohol (241) and stored at 4∘C

FISH probes were obtained by polymerase chain reaction(PCR) amplifications performed in 20 120583L of a solutioncontaining 50 ng DNA 1xPCR buffer 50120583M each dNTP25mM MgCl

2 1 120583M each primer and 1U BIOTAQ DNA

polymerase (Bioline) Universal primers were used to amplifythe whole internal transcribed spacer (ITS) region of themajor rDNA cluster (ITS4 51015840-TCCTCCGCTTATTGATAT-GC-31015840 ITS5 51015840-GGAAGTAAAAGTCGTAACAAGG-31015840[20]) For the amplification of the whole repeat unit of the 5SrDNA we designed primers (5SD 51015840-CAACGTGATATG-GTCGTAGAC-31015840 5SR 51015840-AACACCGGTTCTCGTCCG-ATC-31015840) from the sequence of the 5S rRNA ofMytilus edulis[21]

After 5min of denaturation at 95∘C 30 cycles of ampli-fication were performed (95∘C 30 s 48∘C 30 s 72∘C 30 sfor the ITS 50 s for the 5S rDNA) A final extension step of7min at 72∘C was applied All reactions were performed in aGeneAmp PCR system 9700 (Applied Biosystems) and PCRproducts were examined by electrophoresis on 2 agarosegels

The amplified ITSs obtained from the five species and the5S rDNA from Venerupis corrugata were labeled with biotin-16-dUTP (Roche Applied Science) andor digoxigenin-11-dUTP (10xDIG Labeling Mix Roche Applied Science) usinga nick translation kit (RocheApplied Science)The remaining5S rDNAs were directly labeled by PCR with either biotin-16-dUTP (20120583M) or digoxigenin-11-dUTP (5 120583M)

23 Mitotic Chromosome Synaptonemal Complex andRelease Chromatin Fiber Preparation Mitotic chromosomepreparations were obtained according to Pasantes et al[22] Juvenile specimens were housed in 05 L beakers andexposed to colchicine (0005) for 12 h Gills were excisedand immersed in 50 and 25 sea water for 1 h and fixedwith ethanolacetic acid for 1 h Small pieces of tissue weredissociated in 60 acetic acid and the cell suspension wasdropped onto slides heated to 50∘C

Surface spreading of synaptonemal complexes (SCs) wasperformed as indicated by Hurtado and Pasantes [13] Freshmale gonadic tissue was minced in 04M NaCl and cen-trifuged several times to remove the sperm One drop of thecellular suspension was spread for 1ndash5min on clean slidescovered by a spreading medium (01M sucrose 05 TritonX-100) The slides were then flooded with paraformaldehyde(4) and the liquid mixture allowed settling overnight Afterdraining briefly the slides were rinsed in distilled water andair-dried

Chromatin fibers were released as described by Fidlerovaet al [23] In brief the cellular suspension obtained follow-ing the protocol for mitotic chromosome preparation wascentrifuged for 10min at 1200 rpm and the supernatant wasdiscarded The pellet was resuspended in fresh fixative anddropped on clean slides After leaving to evaporate brieflyslideswere immersed in 1x PBS for 1min and chromatin fiberswere released with 005M NaOH in 30 ethanol

24 Fluorescent In Situ Hybridization (FISH) FISH SC-FISH and Fiber-FISH experiments using biotin and digox-igenin labeled species-specific ITS and 5S rDNA probeswere performed following the methods published elsewhere[13 18] Slides were pretreated with RNase and pepsinbefore denaturation Preparations were denatured for 2minat 70∘C (mitotic chromosomes and released chromatin fibers)or 80∘C (meiotic chromosomes) Hybridization was per-formed overnight at 37∘C Signal detection was carried outwith fluorescein avidin and biotinylated anti-avidin for thebiotinylated probes and with mouse anti-digoxigenin andanti-mouse TRITC for the probes labeled with digoxigeninSlides were counterstained for 8min with 41015840-6-diamidino-2-phenylindole (DAPI 014 120583gmLminus1 in 2x SSC) and mounted



3TA2)3peptide



3 Results









4 Discussion





VCO

(a)

5SITS

(b)

RPH

(c) (d)

VVE

(e) (f)

DEX

(g) (h)





5SITS

RDE

(a) (b)

(c)

(d)










VCO 5STEL

(a)

RDE 5STEL

(b)

RPH ITSTEL

(c)

ITSTEL

VVE

(d)











Venerupis corrugata



A

(V pullastra)

(R decussata)

Venus verrucosa


Tapetinae

Venerinae


Venerinae

Chioninaesm m

1t 1pc

sm sm1ic 1t

m1t

sm sm1t 1pc

mst1t 1t

st1ic 1t

m

m st38 1ic 1t

sm38 1ic 1t 1ic

st sm

st st1t 1ic

m1t38

Chioninae

VenerinaeTapetinae

38

38

38

38

38

A1



A4

A3

A2

Dosiniinae






Acknowledgments



References

















119899

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



3TA2)3peptide



3 Results









4 Discussion





VCO

(a)

5SITS

(b)

RPH

(c) (d)

VVE

(e) (f)

DEX

(g) (h)





5SITS

RDE

(a) (b)

(c)

(d)










VCO 5STEL

(a)

RDE 5STEL

(b)

RPH ITSTEL

(c)

ITSTEL

VVE

(d)











Venerupis corrugata



A

(V pullastra)

(R decussata)

Venus verrucosa


Tapetinae

Venerinae


Venerinae

Chioninaesm m

1t 1pc

sm sm1ic 1t

m1t

sm sm1t 1pc

mst1t 1t

st1ic 1t

m

m st38 1ic 1t

sm38 1ic 1t 1ic

st sm

st st1t 1ic

m1t38

Chioninae

VenerinaeTapetinae

38

38

38

38

38

A1



A4

A3

A2

Dosiniinae






Acknowledgments



References

















119899

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


VCO

(a)

5SITS

(b)

RPH

(c) (d)

VVE

(e) (f)

DEX

(g) (h)





5SITS

RDE

(a) (b)

(c)

(d)










VCO 5STEL

(a)

RDE 5STEL

(b)

RPH ITSTEL

(c)

ITSTEL

VVE

(d)











Venerupis corrugata



A

(V pullastra)

(R decussata)

Venus verrucosa


Tapetinae

Venerinae


Venerinae

Chioninaesm m

1t 1pc

sm sm1ic 1t

m1t

sm sm1t 1pc

mst1t 1t

st1ic 1t

m

m st38 1ic 1t

sm38 1ic 1t 1ic

st sm

st st1t 1ic

m1t38

Chioninae

VenerinaeTapetinae

38

38

38

38

38

A1



A4

A3

A2

Dosiniinae






Acknowledgments



References

















119899

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


5SITS

RDE

(a) (b)

(c)

(d)










VCO 5STEL

(a)

RDE 5STEL

(b)

RPH ITSTEL

(c)

ITSTEL

VVE

(d)











Venerupis corrugata



A

(V pullastra)

(R decussata)

Venus verrucosa


Tapetinae

Venerinae


Venerinae

Chioninaesm m

1t 1pc

sm sm1ic 1t

m1t

sm sm1t 1pc

mst1t 1t

st1ic 1t

m

m st38 1ic 1t

sm38 1ic 1t 1ic

st sm

st st1t 1ic

m1t38

Chioninae

VenerinaeTapetinae

38

38

38

38

38

A1



A4

A3

A2

Dosiniinae






Acknowledgments



References

















119899

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


VCO 5STEL

(a)

RDE 5STEL

(b)

RPH ITSTEL

(c)

ITSTEL

VVE

(d)











Venerupis corrugata



A

(V pullastra)

(R decussata)

Venus verrucosa


Tapetinae

Venerinae


Venerinae

Chioninaesm m

1t 1pc

sm sm1ic 1t

m1t

sm sm1t 1pc

mst1t 1t

st1ic 1t

m

m st38 1ic 1t

sm38 1ic 1t 1ic

st sm

st st1t 1ic

m1t38

Chioninae

VenerinaeTapetinae

38

38

38

38

38

A1



A4

A3

A2

Dosiniinae






Acknowledgments



References

















119899

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






Venerupis corrugata



A

(V pullastra)

(R decussata)

Venus verrucosa


Tapetinae

Venerinae


Venerinae

Chioninaesm m

1t 1pc

sm sm1ic 1t

m1t

sm sm1t 1pc

mst1t 1t

st1ic 1t

m

m st38 1ic 1t

sm38 1ic 1t 1ic

st sm

st st1t 1ic

m1t38

Chioninae

VenerinaeTapetinae

38

38

38

38

38

A1



A4

A3

A2

Dosiniinae






Acknowledgments



References

















119899

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













119899

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Evolutionary Dynamics of rDNA Clusters in …downloads.hindawi.com/journals/bmri/2014/754012.pdf · 2019-07-31 · Research Article Evolutionary Dynamics of rDNA