Erratum to “Extensive chromosomal homologies and evidence of karyotypic stasis in Atlantic grunts of the genus Haemulon (Perciformes

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  Erratum to “Extensive chromosomal homologies and evidence of karyotypic stasis in Atlantic grunts of the genus Haemulon (Perciformes
  Extensive chromosomal homologies and evidence of karyotypic stasis in Atlanticgrunts of the genus  Haemulon  (Perciformes) Clóvis Coutinho Motta Neto a , Marcelo Belo Ciof  fi b , Luis Antônio Carlos Bertollo b , Wagner Franco Molina a, ⁎ a Departamento de Biologia Celular e Genética, Centro de Biociências, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil b Departamento de Genética e Evolução, Universidade Federal de São Carlos, São Carlos, SP, Brazil a b s t r a c ta r t i c l e i n f o  Article history: Received 7 January 2011Received in revised form 24 February 2011Accepted 28 February 2011 Keywords: Chromosomal evolutionKaryotypic conservatismMarine PerciformesMarker chromosomes Fish of the genus  Haemulon , known as grunts, are common in coral reefs, displaying a large trophic andeconomic importance. Recent evidence suggests the occurrence of sympatric speciation and hybridizationduring the differentiation process of some species. Chromosomal analyses based on conventional cytogeneticmarkers have revealed the presence of conserved karyotypes, with few identi fi able rearrangements in mostspecies. In this study, we compared the cytogenetic data of the species  Haemulon aurolineatum ,  Haemulon plumierii , and  Haemulon steindachneri , using different staining methods (C-, Ag-, and DAPI/CMA 3  banding),  insitu digestionbyrestrictionendonucleases(  Alu I, EcoR I, Pst  I,and Taq I)andchromosomalmappingofribosomalgenes (18S and 5S) by double-FISH. All species displayed the karyotype comprising 2n=48 acrocentricchromosomes and with accentuated chromosomal homologies, which suggest the maintenance of extensivesyntenic content evolutionarilymaintainedwithfewchanges. H. aurolineatum  and H. plumierii ,fromthecoastof Brazil, have subtle but identi fi able cytogenetic differences in relation to populations from other parts of theAtlantic, re fl ecting the disruptive role of the biogeographical barriers between these regions. Karyotypicconservatism in this genus shows a karyotypic stasis, probably related to intrinsic characteristics of thekaryotype itself and/or biological characteristics of this group of   fi sh.© 2011 Elsevier B.V. All rights reserved. 1. Introduction The  fi sh of the genus  Haemulon  (Haemulidae, Haemulinae)representagroupofPerciformesuniquetotheNewWorldthatconsistsof 15 species, seven of which ( Haemulon aurolineatum ,  Haemulonchrysagyreum ,  Haemulon melanurum ,  Haemulon parra ,  Haemulon plu-mierii , Haemulonsteindachneri ,and Haemulonsquamipinna )occuralongthe Brazilian coast (Floeter et al., 2003; Nelson, 2006). The Haemulidaespeciesusuallyformlargeshoalsthataredispersedamongrockydepthsand coral reefs, helping to maintain the latter in oligotrophic waters(Meyer et al., 1983), where they play a prominent trophic role aspredators and prey (Lindeman and Toxey, 2002; Ferreira et al., 2004). Due to their phylogenetic and biogeographic patterns,  Haemulon  hasbeenpresentedasamodelgroupforstudiesonevolutionandspeciationin the marine environment (Rocha et al., 2008). During the genus's evolutionary history, its species have undergone important changes ineating habits (Rocha et al., 2008), with possible repercussions on their spatial distribution, ecological adaptations, and genetic patterns.Haemulidae species, especially thoseof the genus  Haemulon , showa remarkable conservatism in their karyotypic macrostructure (Ronand Nirchio, 2005; Accioly and Molina, 2007). However, theavailability of chromosome markers that enable the detection of more subtle chromosomal homologies and changes are essential to amore critical analysis of the process of karyotypic evolution occurringin this group. This study compares the cytogenetic patterns of threespecies of Western Atlantic grunts,  H. aurolineatum ,  H. plumierii , and H. steindachneri , through C-banding, Ag – NOR, digestion with restric-tion enzymes, staining with AT and GC-speci fi c  fl uorochromes, andmapping of ribosomal sequences by double-FISH with 5S and 18SrDNA probes. The results allowed for the discussion of the possiblecausesofthesigni fi cantevolutionarystasisobservedforthespeciesof this genus. 2. Material and methods  2.1. Specimen collection and chromosome preparation Specimens of   H. aurolineatum  (n=17, 6 females, 9 males, and 2immature specimens),  H. plumierii  (n=19, 10 females, 7 males, and 2immaturespecimens),and H.steindachneri (n=18,8males,6females,and4immaturespecimens)were collectedonthe coastof thestateof RioGrandedoNorte(5°13 ′ 1.73 ″ S35°9 ′ 57.85 ″ W)ontheNortheasterncoast of Brazil.  Journal of Experimental Marine Biology and Ecology 401 (2011) 75 – 79 ⁎  Corresponding author at: Departamento de Biologia Celular e Genética, Centro deBiociências, Universidade Federal do Rio Grande do Norte, Campus Universitário, s/n,Lagoa Nova, CEP 59078-970, Natal, RN, Brazil. Tel.: +55 84 321119209; fax: +55 8432153346. E-mail address: (W.F. Molina).0022-0981/$  –  see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.jembe.2011.02.044 Contents lists available at ScienceDirect  Journal of Experimental Marine Biology and Ecology  journal homepage:  After the capture the specimens were subjected to mitoticstimulation  in vivo  with compound antigens (Molina, 2001; Molinaetal.,2010)for24 – 48 h.Afterthistimetheanimalswereanesthetizedwithcloveoil(Eugenol)andsacri fi cedforremovaloffragmentsoftheanterior kidney and sexedby macroscopicexaminationof the gonads.Mitotic chromosomes were obtained using the method of   in vitro interruption of the cell cycle (Gold et al., 1990).  2.2. Chromosome banding  The Ag – NOR and C-banding techniques followed the methodssuggested by Howell and Black (1980) and Sumner (1972), respec-tively. Double staining with CMA 3 /DAPI  fl uorochromes was used toshow GC-rich regions (Barros e Silva and Guerra, 2009). Brie fl y, slidesaged for three days were stained with CMA 3  (0.1 mg/ml) for 60 minand re-stained with DAPI (1  μ  g/ml) for 30 min. Then the slides weremounted in glycerol: McIlvaine buffer pH 7.0 (1:1) and analyzed withan epi fl uorescence microscope under the appropriate  fi lters.DNA restriction digests using  Alu I,  EcoR I,  Pst  I and  Taq I wereperformed using the speci fi c conditions established by previous teststo determine the optimal concentration, exposure time and incuba-tion temperature for each enzyme. After dilution to a total volume of 40  μ  l using the buffers indicated by the manufacturers, the endonu-cleases were deposited on freshly prepared slides, covered with acoverslip and incubated in a moist chamber for de fi ned periods atspeci fi c temperatures. The digestion patterns of the chromosomeswith  Alu I (AG/CT) were obtained with treatment using 0.3 units/ μ  l of the enzyme, at 37 °C for 3 h;  EcoR I (GAATTC), at 37 °C/10 h (0.5 U/ μ  l); Pst  I (C TGCA/G), at 37 °C/8 h (2 U/ μ  l); and  Taq I (TCGA), 65 °C for 8 h(0.5 U/ μ  l). After each treatment, the slides were washed in distilledwater, stained with 5% Giemsa diluted in phosphate buffer (pH 6.8)for 10 min and air dried.  2.3. Chromosome hybridization and karyotype analysis Fluorescent  in situ  hybridization (FISH) was performed on mitoticchromosome spreadsaccording toPinkelet al.(1986).The5Sand18S rDNA sequences were detected by double-FISH analysis. The tworibosomal sequences were isolated from the  Hoplias malabaricus (Teleostei,Characiformes)genome.The5SrDNArepeatcopyincluded120 base pairs (bp) of the 5S rRNA encoding gene and 200 bp of thenon-transcribed spacer (NTS) (Martins et al., 2006). The 18S rDNA probe corresponded to a 1400 bp-segment of the 18S rRNA gene,obtained via PCR from nuclear DNA (Ciof  fi  et al., 2009a). The 5S wereclonedintoplasmidvectorsandpropagatedinDH5 α E.coli competentcells (Invitrogen, San Diego, CA, USA). The 5S rDNA probe was labeledwith biotin-14-dATP by nick translation according to the manufac-turer'srecommendations (BioNick ™ Labeling System; Invitrogen, SanDiego, CA, USA). The 18S rDNA was labeled by nick translation withDIG-11-dUTP, according to the manufacturer's instructions (Roche,Mannheim, Germany).Approximately thirty metaphases were analyzed for each speci-men to con fi rm the modal diploid number.The best metaphaseswerephotographed using an Olympus BX50 epi fl uorescence photomicro-scope, equipped with an Olympus DP70 digital image capture. Themetaphase images were captured using CoolSNAP-Pro software(Media Cybernetic). The chromosomes were classi fi ed and sortedaccording to the nomenclature developed by Levan et al. (1964). 3. Results H. aurolineatum ,  H. plumierii , and  H. steindachneri  presentedmarkedsimilaritiesinkaryotypicstructure.Thesamekaryotypeformula,with 2n=48 acrocentric chromosomes (NF=48) with little sizedifferentiation between the chromosome pairs (where the largestchromosomes display approximately 3  μ  m and the smallest 1  μ  m), wasidenti fi ed in these species(Fig. 1a, c,and e). C-positive heterochromaticbands were observed in the centromeric/pericentromeric region of allchromosomes, as well as the presence of faint bands in the terminalregion of some pairs of the karyotype (Fig. 1b, d, and f).Simple Ag – NOR sites were observed in the interstitial position,coinciding with a secondary constriction present in pair no. 24 of allthespecies(Fig.1).Thesesiteswerecon fi rmedlaterbyequilocalsignswith 18S rDNA probes (Fig. 2b, d, and f). Coincidentally, thischromosome pair was the only one to exhibit a GC-rich DNA regionin the karyotype, coinciding with the NOR site and the C-positiveheterochromatic band (Fig. 2a, c, and e). Despite the great symmetryof chromosomes between species, the 5S rDNA sequences weretentatively mapped in the terminal position in pair no. 5 in the threespecies. In addition to this site, in  H. plumierii  the presence of an extracentromeric site was observed in pair no. 10 (Fig. 2b, d, and f).Mapping of the 18S and 5S rDNA sequences by double-FISH showedanabsenceofsyntenybetweentheseribosomalsubunitsinallspeciesanalyzed.Treatmentswith EcoR I, Pst  I, Taq I,and  Alu Ienzymesdemonstratedapattern largely similar among species, precluding their use for aprecise breakdown of the chromosomes between them (Fig. 3a – k).The  Eco RI enzyme revealed a small enzymatic action in theheterochromatic segments of the chromosomes of the three speciesanalyzed (Fig. 3a, e, and i). The enzymatic digestions indicated ingeneral that the heterochromatic blocks have a large number of cleavagesitesforthe Taq Ienzyme(Fig.3c,g,andk).The  Alu l(notusedin  H. steindachneri ) and  Pst  I endonucleases showeda diffuse digestionpattern, with an absence of cleavage sites on some telomeric regions(Fig. 3b, d, f, h, and j, respectively). Fig. 1.  Karyotype of the species  H. aurolineatum ,  H. plumierii , and  H. steindachneri stainedwithGiemsa(a,c,ande)andafterC-banding, indicatingthegeneral occurrenceof heterochromatic blocks in the centromeric region of the chromosomes and in theinterstitial region of some pairs of the karyotype (b, d, and f). Highlighted are theorganizing regions of the nucleolus, located on the 24th pair of the karyotype of all thespecies (box). Bar=5  μ  m.76  C.C.M. Neto et al. / Journal of Experimental Marine Biology and Ecology 401 (2011) 75 – 79  4. Discussion A marked chromosomal conservatism, both numerical andstructural, was seen among the  Haemulon  species analyzed, evenwith the use of a diverse set of cytogenetic techniques. Chromosomalconservatism is not exclusive to the genus  Haemulon , but a conditionpresent in several families of Perciformes (Galetti et al., 2000) thatshow remarkable evidence of evolutionary stasis with respect tokaryotype diversi fi cation (Molina, 2006).However, some characteristics that typically show higher rates of evolutionary change, such as ribosomal genes and heterochromaticregions, showed some secondary chromosomal differences through-out the evolutionary history of the species of this genus.Ag – NOR sites have been characterized as effective cytotaxonomicmarkers among some species of   fi sh. However, in  Haemulidae , thesesiteshaveahighlevelofconservatismthatgivesthemalimitedroleintaxonomic or phylogenetic inferences among closely related species.This fact had already been shown in the genus  Anisotremus  (Acciolyand Molina, 2007), where three Atlantic species,  Anisotremusvirginicus ,  Anisotremus moricandi , and  Anisotremus surinamensis ,share equilocal Ag – NOR regions in the interstitial position on thelong arm of chromosome pair no. 18. Likewise, apparently home-ologous ribosomal cistrons, located exclusively in pair no. 24, wereshown by using silver nitrate and by mapping with 18S rDNA probesin the three species being analyzed. This exclusive location was alsoobserved in two other species of the genus  Haemulon  ( Haemulon  fl avolineatum  and  Haemulon bonariense ) (Ron and Nirchio, 2005;Nirchio et al., 2007).Likewise,the5S rDNAsites, locatedon thelong armof pairno. 5of the three species analyzed here, appear to represent a symplesio-morphic condition consistently maintained in the genus  Haemulon . Inaddition to this location,  H. plumierii  shows an additional site on thecentromeric region of the 10th chromosome pair. Our data, combinedwith previous analyses in other  Haemulon  species (Nirchio et al.,2007), suggests that the occurrence of two chromosome pairscarrying 5S rDNA sequences could represent a plesiomorphiccondition for this genus.The lack of chromosomal markers in the genus  Haemulon  makesdif  fi cultacomparativepopulationanalysisofthenumberandlocationof the 18S and 5S ribosomal genes. However, the availablechromosome data support a homeologous pattern regarding thefrequency and placement of the 18S rDNA sites, demonstrating theevolutionary stabilityof the chromosomallocationof thesesequencesandhencetheirlimitationasamarkerinintra-andinterspeci fi clevelsin the genus. On the other hand, the distribution pattern of the 5S Fig. 2.  Karyotypes obtained by double staining with CMA 3  and DAPI  fl uorochromes for H. aurolineatum  (a),  H. plumierii  (c) and  H. steindachneri  (e), followed by double-FISHfor the same species in the same order (b, d, and f). The 18S rDNA sites (red) are clearlyevidencedinanon-syntenicposition tothe5SrDNAsites(green).The5Ssequencesarepresent in the 5th pair of all the species (b, d, and f) and in an additional site in the 10thpair exclusively in  H. plumierii  (d). The pairs carrying ribosomal sequences arehighlighted (boxes). Bar=5  μ  m. Fig.3. Treatmentof mitoticchromosomes with Eco RI, Pst  I,  Taq I,and  Alu Irestriction enzymesin H. aurolineatum ( Ha )(a – d), H. plumierii ( Hp )(e – h),and H. steindachneri  ( Hs )(i – k).In(l) C-banding in  H. steindachneri , showing the digestion pattern between species. Bar=5  μ  m.77 C.C.M. Neto et al. / Journal of Experimental Marine Biology and Ecology 401 (2011) 75 – 79  rDNA sequences may present sharply contrasting differences. This isthe case with the pattern observed in the  H. aurolineatum  populationof the coast of Northeastern Brazil and that presented by anotherpopulation of this same species from the Caribbean region, whichexhibits multiple sites in the karyotype (Nirchio et al., 2007). Similar differencesarealsofoundamong H.plumierii populationsofthesetworegions of the Western Atlantic. In this case, although in bothpopulations the 5S rDNA sites are numerous and located onchromosomes of similar size, their location is not equilocal, as theyare shown in the terminal position in the Brazilian population and inthe interstitial position in the Venezuelan population. Thus, contraryto the 18S rDNA sites, the 5S rDNA sites seem to be effectivepopulation markers for the Haemulidae species, where the  H.aurolineatum  and  H. plumierii  specimens from Brazil and theCaribbean display an evolution divergent from the chromosomallocation these sequences. These data suggest the occurrence of population structuring, considering also that these populations areseparated by a wide oceanic range, including the biogeographicalbarrier formed by the Amazon River delta.Evidence of marked genetic differentiations between geographi-cally distinct specimens of the same nominal  Haemulon  species hasbeen previously identi fi ed. In fact, phylogenetic analyses based onmitochondrial and nuclear DNA sequences presented genetic diver-gences suf  fi cient to suggest the division of   H. steindachneri , from thePaci fi c and the Atlantic, separated by the Isthmus of Panama into twodistinct species (Rocha et al., 2008). In addition, the Atlanticpopulations of Venezuela and Brazil also presented signi fi cant geneticdifferences, pointing to cryptic speciation and allowing us to treatthem as different evolutionary units.Contrary to what occurs in other groups of   fi sh, in which theprocess of evolution of repetitive sequences reveals profoundevolutionary features (Galetti et al., 1991; Mantovani et al., 2000), and where distribution and composition patterns in heterochromaticregionsmaybespecies-speci fi ccharacteristics,theseregionswerenotvery dynamic among  Haemulon  species. In fact, the heterochromaticregions displayed a relatively homogeneous distribution among thespecimens analyzed, revealing equally conspicuous blocks of similarsize on the centromeric regions and, in some pairs, on the telomericportions of the long arms.Compositionally, only the equilocal heterochromatic sites in thenucleolar organizing regions showed signi fi cant GC content in thekaryotype. Principal rDNA genes, interspersed with GC-rich hetero-chromatic sequences, are common among the Haemulidae analyzed,and widely distributed in various families of   fi sh (Martínez et al.,1996). On the other hand, the 5S rDNA sites, despite presentingthemselves as heterochromatic, displayed a particular compositionthat was not reactive to the AT and GC-speci fi c  fl uorochromesemployed, reinforcing an independent evolution of the principalribosomal sites.The banding pattern produced by digestion of the chromosomeswith restriction enzymes have led to the identi fi cation of differentialcleavage sites in the karyotype of several  fi sh species (Lloyd andThorgaard, 1988; Carvalho and Dias, 2005). However, in the three species of Haemulidae analyzed, chromosomal treatment with  Alu I, Pst  I,  Taq I, and  EcoR I enzymes demonstrated similar responses amongthem and to C-banding in some treatments, but with a lower level of intensity in general. The common response of the heterochromaticregions of each species to the enzymes employed reinforces therelative homogeneity of the repetitive portions of DNA in thesespecies. However, analysis of the composition of repetitive DNAsequences is needed to con fi rm the degree of homogeneity hetero-chromatin in these species.The chromosomal pro fi les in large part similar among the speciesanalyzed support the karyotypic orthoselection occurrence proposedfor some families of   fi sh (Molina, 2006). This process as srcinallyproposedwouldlead to fi x particularchromosomerearrangements inphylogeneticallyclosespeciesoverothers(Sumner,1972).Karyotypiccharacteristics particular to a group, such as paralogous regions of repetitive DNA that appear to exist in  Haemulon , could allow for theestablishmentofcertaintypesofchromosomalrearrangementsinthisgroup, which may lead to the formation of identical karyotypesamong related species of this genus, as well as among otherPerciformes. On the other hand, conserved karyotypes within agiven taxonomic group could also be derived from rapid and recentspeciation events (Sola et al., 1981), a situation that cannot bedismissed with respect to the genus  Haemulon , where electrophoreticstudies in hemoglobin and plasmatic proteins and analyses of DNAsequences indicate a monophyletic srcin with recent diversi fi cation(Cequea and Pérez, 1971; Rocha et al., 2008). Interpopulational karyotype divergences are determined mainlyunder conditions of allopatry. Identi fi cation in  H. aurolineatum  andpossiblyin H.plumierii ofvariantcytotypeswithrespecttothephysicalmapping of the 5S rDNA loci among populations of different areas of the Atlantic, Caribbean, and coast of Brazil, suggest this. Restrictedgene fl owamongtheselocationsispossiblyduetothein fl uenceofthedelta of the Amazon and Orinoco Rivers, which compose thegeographic barrier of the Amazon, classi fi ed as intermittent or semi-porous (Rocha, 2003) associated with ocean currents, as well as thegeographicaldistancebetweenthem.Thesebarriers,eitheraloneorincombination, have been identi fi ed as responsible for the profounddifferences in DNA sequences observed between the Brazilian coastandCaribbeanpopulationsofthespinylobster Panulirus argus (Sarveret al., 1998), the red lip blenny  Ophioblennius atlanticus  (Muss et al.,2001), and the ocean surgeon fi sh  Acanthurus bahianus  (Rocha et al.,2002), among others. Cytogenetic divergences suggest evidence of some level of, or at least ongoing, cryptic speciation among popula-tions of   H. aurolineatum  and  H. plumierii , suggesting us to treat thesespecimens as different evolutionary units. The absence of karyotypicdata for  H. steindachneri  from the Caribbean, identi fi ed as geneticallydistinct from the Brazilian population (Rocha et al., 2008), precludes,for now, the detection of possible divergent cytogenetic patternsbetween these populations.The effect of allopatry on karyotypic patterns in Haemulidae wasmuch smaller than what commonly occurs in freshwater speciessubjected to intense population fragmentations, such as in Erythrini-dae, where markedly distinct karyomorphs occur throughout itsgeographical distribution (Bertollo et al., 2000; Ciof  fi  et al., 2009b).Among the marine species that do not have adult migrants, such asHaemulidae, the potential source of dispersal and maintenance of theintra-speci fi c gene  fl ow stems in part from the extent of their pelagiclarval period in synergistic or antagonistic action with environmentalfactors (Molina, 2006). There are indications that this biologicalcharacteristic in marine reef   fi sh will occur in this environment as afunction inversely proportional to chromosomal diversi fi cation(Molina and Galetti, 2004; Sena and Molina, 2007). This cytogenetic characterization of   H. aurolineatum ,  H. plumierii ,and H. steindachneri ,usingawiderrangeof analysis methods,allowedfor access to the occurrence of a certain degree of populational orinterspeci fi c karyotypic diversi fi cation. However, a clear pattern thatis phylogenetically conserved and characteristic of the genus ismaintained, indicated by macrostructural characteristics of thekaryotype, by the relative heterochromatin homogeneity, and, inpart,bythe positionand frequency of the ribosomalsites. Themarkedkaryotypic conservatism present in these species is particularlyinteresting in view of the conspicuous ecomorphological patternsthat these species display. There is a clear asynchrony between theevolutionary differentiation levels of the chromosomes and bodymorphology in this and in some other groups of species (Molina et al.,2002). This condition reinforces the idea that diversi fi cation of thisgroup is linked, in some cases, to sympatric speciation events (Rochaetal.,2008),wheretheconspicuousmorphologicaladaptationswouldbe associated with rapid ecological changes for exploration of new 78  C.C.M. Neto et al. / Journal of Experimental Marine Biology and Ecology 401 (2011) 75 – 79  niches and habitats with few or no chromosomal divergences. Thishigh degree of cytogenetic similarity could provide a scenarioappropriate for the occurrence of hybridization events for the breachof pre-zygotic and post-zygotic reproductive barriers, which areapparently surmountable.  Acknowledgments The authors thank the Federal University of Rio Grande do Norte,CNPq (Process No. 556793/2009-9), IBAMA (Process No. 19135/1) forthe conditions for implementing this study, and José Garcia fortaxonomic identi fi cation of the specimens.  [RH]References Accioly, I.V., Molina, W.F., 2007. Contribuição à citogenética dos gêneros  Pomadasys  e  Anisostremus  (Haemulidae, Perciformes). Publica 3, 36 – 44.Barros e Silva, E., Guerra, M., 2009. The meaning of DAPI bands observed afterC-banding and FISH procedures. Biotech. Histochem. 4, 1 – 11.Bertollo, L.A.C., Born, G.G., Dergam, J.A., Fenocchio, A.S., Moreira-Filho, O., 2000. 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