research article sequential steps of chromosomal

Research ArticleSequential Steps of Chromosomal Differentiation inAtlantic Surgeonfishes Evolutionary Inferences

Paulo Roberto Antunes de Mello Affonso1 Maria Aparecida Fernandes2

Josivanda Santos Almeida1 and Wagner Franco Molina2

1 Departamento de Ciencias Biologicas Universidade Estadual do Sudoeste da Bahia 45206-150 Jequie BA Brazil2 Departamento de Biologica Celular e Genetica Universidade Federal do Rio Grande do Norte 59078-970 Natal RN Brazil

Correspondence should be addressed to Paulo Roberto Antunes de Mello Affonso paulomelloaffonsoyahoocombr

Received 4 May 2014 Accepted 25 July 2014 Published 12 August 2014

Academic Editor Joao P Barreiros

Copyright copy 2014 Paulo Roberto Antunes de Mello Affonso et al This is an open access article distributed under the CreativeCommons Attribution License which permits unrestricted use distribution and reproduction in any medium provided theoriginal work is properly cited

Surgeonfishes are a species-rich group and a major biomass on coral reefs Three species are commonly found throughout SouthAtlantic Acanthurus bahianus A chirurgus and A coeruleus In this paper we present the first cytogenetic data of these speciesrevealing a sequential chromosomal diversification A coeruleus was characterized by a relatively conserved karyotype evolved bypericentric inversions of some pairs (2119899 = 48 2sm + 4st + 42a) In contrast the karyotypes of A bahianus (2119899 = 36) and Achirurgus (2119899 = 34) were highly differentiated by the presence of six large metacentric pairs in A bahianus (12m + 2sm + 4st +18a) and A chirurgus (12m + 2sm + 4st +1 6a) probably derived by chromosomal fusions that corroborate their closer relationshipA discernible in tandem fusion represents an autapomorphic character to A chirurgus In spite of macrostructure variation singlenucleolar organizer regions (NORs) on short arms of a subtelocentric pair and similar distribution of C-bands were observed inthe three species Overlapping of chromosomal data with molecular phylogeny indicated pericentric inversions which took placenearly at 19Ma while centric fusions are as recent as 5Ma A physical mapping of coding and noncoding sequences in Acanthuruscould clarify the role of additional rearrangements during their chromosomal evolution

1 Introduction

Acanthuridae are a monophyletic fish family composed ofabout 80 species popularly known as surgeonfishes or tangs[1] This is an ancient group (nearly 54Ma) and most ofgenera (Acanthurus Naso Paracanthurus Zebrasoma andCtenochaetus) diverged between 17 and 21Ma in Early Mio-cene [2]

Apparently the Pacific Ocean is the center of origin ofsurgeonfishes retainingmost of Acanthuridae richness [3 4]Further colonization events resulted in distribution of thisfamily to virtually all tropical and subtropical seas of theworld but Mediterranean Sea [1 3] The genus Acanthurus isthe largest within the family but monophyly of the genus isstill controversial [2ndash5] This fish group is morphologicallyand ecologically diversified mainly in relation to foragingbehavior and dentition composing one of themost represen-tative herbivorous fish group on coral reefs [2 6]

A total of four species of Acanthuridae all belonging tothe genus Acanthurus are present in western Atlantic [7]Three species are common along the Brazilian coast (WesternSouth Atlantic) Acanthurus coeruleus (blue tang) A bahi-anus (barber surgeonfish) and A chirurgus (doctorfish) [78] Another Acanthurus species (A monroviae) was alsorecorded off southeastern coast of Brazil but it seems to bean occasional occurrence [9] Moreover A bahianus wasthought to range from USA to southern Brazil but mor-phological and genetic analyses have shown that populationsfrom Massachusetts to Caribbean actually refer to anotherspecies validated as A tractus [7]

In spite of the low diversity of Atlantic species when com-pared to Pacific and Indian oceans surgeonfishes are a dom-inant fish group forming large assemblages in several reefareas from South Atlantic [3] A coeruleus specimens areusually solitary due to their territoriality behavior while Abahianus and A chirurgus are commonly found in small to

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 825703 7 pageshttpdxdoiorg1011552014825703

2 The Scientific World Journal

Atlantic Ocean

Brazil

(a)

(b)

(c)

0

1

2

1500

(km)

N

EWS

Figure 1 Map of South America showing the collections sites of Acanthurus coeruleus (a) A bahianus (b) and A chirurgus (c) in the statesof Rio Grande do Norte (1) and Bahia (2) northeastern Brazil

large schools depending on the ontogenetic stage [10] Asmost acanthurids these species present relatively long pelagiclarval stages with a mean duration from 516 to 552 days [11]Usually wide-range reef fish species with long pelagic larvaldevelopment are characterized by a lack of genetic subdivi-sion among populations [12] and low rates of chromosomalvariation [13]

However reports about cytogenetic patterns of Acan-thuridae are still underrepresented (less than 5 of species)and restricted to Indo-Pacific species [14]The three analyzedspecies from Pacific Acanthurus triostegus Prionurus scal-prum [15] and Ctenochaetus striatus [16] all share a conser-vative Perciformes-like karyotype with 2119899 = 48a consideredbasal to this fish group [17]

In order to increase the karyotypic data of Acanthuridaeand to infer the chromosomal evolution of Atlantic speciescytogenetical analyses were carried out for three Acanthuri-dae species from Brazilian coast South Atlantic

2 Material and Methods

Nine individuals of Acanthurus coeruleus four individuals ofA bahianus and 17 individuals of A chirurgus were cytoge-netically studied Animals were collected using hand nets (60times 100 cm) by snorkeling at coastal reef areas from the states ofRio Grande do Norte (5∘461015840S 35∘121015840W) and Bahia (13∘001015840S38∘321015840W and 13∘521015840S 38∘561015840W) in northeastern Brazilianshore (Figure 1) Right after collection specimens were trans-ported in plastic bags with oxygen to the laboratories andplaced in 60 L tanks equipped with filtration and aerationsystems

Twenty-four hours prior to chromosomal preparationthe animals were inoculated via intramuscular with a solutionof antigen complexes (Munolan) for mitotic induction [18]After this period the specimens were anesthetized andeuthanized by immersion in in water at 0ndash4∘C up to completeinterruption of gill movements [19] To obtain mitotic chro-mosomes in vitro portions of anterior kidney were removedand transferred to RPMImedium (Cultilab) with about 50120583Lof 0025 colchicine followed by hypotonic treatment (KCL0075M) for 20 minutes at 37∘C and fixation in Carnoyrsquosfixative (methanol acetic acid 3 1) [20] Chromosomes werestained with 5 Giemsa in phosphate buffer (pH 68) forkaryotypic analyses Nucleolus organizer regions (NORs)were detected by silver nitrate staining (Ag-NORs) [21]whereas heterochromatic regions were evidenced by C-banding [22]

Metaphases were photographed using an Olympus BX51(Olympus Tokyo Japan) epifluorescence photomicroscopeequipped with digital capture system Chromosomes wereclassified as metacentric (m) submetacentric (sm) subtelo-centric (st) and acrocentric (a) based on arm ratio [23] Thepairswere arranged in decreasing order size according to eachmorphological category (m sm st and a) in karyotypes usingthe software Adobe Photoshop CS6 v 130

3 Results

The three Acanthurus species showed remarkable kary-otype diversification Acanthurus coeruleus presented 2119899 =48 composed of two submetacentric four subtelocentricand 42 acrocentric chromosomes (Figure 2(a)) The diploid

The Scientific World Journal 3

sm st

a

22 23

1 2 3 2

24

4 5 6 7 8 9

10 11 12 13 14 15

16 17 18 19 20 21

(a)

sm st

a

22 23

1 2 3

24

4 5 6 7 8 9

10 11 12 13 14 15

16 17 18 19 20 21

(b)

m

sm st

a

st

1 2 3 4 5 6

7 89 8

10 11 12 13 14 15

16 17 18

(c)

st

m

sm st

a

1 2 3 4 5 6

7 8 9

10 11 12 13 14 15

16 17 18

(d)

m

sm st

a

1 2 3 4 5 6

7 8 89

10 11 12 13 14 15

16 17

(e)

m

smst

a

1 2 3 4 5 6

7 8 9

10 11 12 13 14 15

16 17

(f)Figure 2 Karyotypes of Acanthurus coeruleus ((a) and (b)) with 2119899 = 48 A bahianus ((c) and (d)) with 2119899 = 36 and A chirurgus ((e) and(f)) with 2119899 = 34 after conventional Giemsa staining ((a) (c) and (e)) and C-banding ((b) (d) and (f)) The NOR-bearing chromosomesafter silver nitrate staining of each species are shown in boxes (pair 2 of A coeruleus and pair 8 of A bahianus and A chirurgus)

number ofA bahianus equals 2119899 = 36with a karyotype com-posed of 12 large metacentric two submetacentric four sub-telocentric and 18 acrocentric chromosomes (Figure 2(c))while A chirurgus was characterized by 12 large metacentrictwo submetacentric four subtelocentric and 16 acrocentricchromosomes (2119899 = 34) (Figure 2(e))

Small amounts of heterochromatin were detected mainlyat pericentromeric regions and interspersed with NORs instudied species (Figures 2(b) 2(d) and 2(f)) In A bahianus

terminal C-bands were also observed in some pairs(Figure 2(d))

Single nucleolar organizer regions (NORs) were locatedby silver nitrate staining on short arms of the largest sub-telocentric pair in the three Acanthuridae species (Figure 2inbox)

Based on chromosomal data idiogramswere generated tohighlight particular karyotype traits for each species and theinferred pathways of chromosomal differentiation based on


Basal karyotype

Steps of karyotype differentiation

A bahianus A chirurgus

In tandem fusion

Robertsonian translocations

Pericentric inversions

Ag-NORs

M

SM

M

(a)

(b)

(c) (d)

1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 10 11 12 13 14 15 16 17 18

19 20 21 22 23 2419 20 21 22 23 24

A coeruleus

2n = 48

2n = 36

2n = 34

1 2 3 4 5 6 7 8 9

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17

10 11 12 13 14 15 16 17

18

lowast

lowast

ST

SM ST

SM ST

Figure 3 Idiograms of chromosomes sets of Acanthurus coeruleus (a) A bahianus (b) and A chirurgus (c) showing the most conspicuousshared cytogenetic traits in boxes In (d) the basal karyotype of Perciformes (2119899 = 48a) and a phylogenetic hypothesis based on sequentialchromosomal rearrangements inferred in the three Acanthurus species

a phylogenetic hypothesis to Atlantic Acanthurus (Figures3(a)ndash3(d))

4 Discussion

It is assumed that the presence of 48 acrocentric chromo-somes represents a plesiomorphic feature within Perciformes[17 24]This condition is particularly frequent amongmarinefish and could be related to dispersal abilities (high gene flow)between populations thereby preventing the fixation of newchromosomal rearrangements and karyotypic divergence[13] In fact the low genetic structure in reef fish species has

been correlated to the production of planktonic eggs andorlarvae that can be dispersed over large distances [12]

This trend (2119899 = 48a) seems to be valid for Acanthuridaespecies from Indo-Pacific Ocean of different genera such asAcanthurus Ctenochaetus and Prionurus [14] Howeverinconsistent relationship between pelagic larval duration(PLD) and genetic connectivity or chromosomal patterns hasbeen reported in some marine species [11 25] In these casesecological and biogeographic aspects of each speciesmight bemore relevant to explain the genetic variation than PLD itselfas observed in the present study

As expected for widely distributed species with long PLDA coeruleus presented typical Perciformes-like features that


is a diploid number of 48 singleNORs and a large number ofacrocentric chromosomes (Figure 2(a))Thekaryotype of thisspecies (2sm + 4st + 42a) demonstrates the occurrence ofpericentric inversions in three chromosome pairs (1st 2ndand 3rd pairs) a common rearrangement in Perciformes thataccounts for most of karyotype diversification in marine fish[24] A similar set of three chromosomal pairs in bothmorphology and size is also observed in A bahianus and Achirurgus represented by a submetacentric pair and two sub-telocentric pairs including the NOR-bearing pair Because ofthe high resemblance of such pairs in the three Acanthurusspecies they are supposed to share a common origin beforethe differentiation of each lineage thereby indicating a sym-plesiomorphic trait Estimates of divergence time betweenthe subclade that comprises A coeruleus and that clusters Achirurgus and A tractus [2] a sister-species of A bahianussuggest that these putative homeologous pairs (sm and st) hadarisen at nearly 19Ma

On the other hand A bahianus and A chirurgus pre-sented an evolutionary chromosomal pattern rarely found intypical marine fishes The drastic reduction in diploid num-ber from 48 chromosomes to 2119899 = 36 and 2119899 = 34 respec-tively along the presence of large metacentric pairs is evi-dence of sequential Robertsonian rearrangements or centricfusions (Figures 2(c) and 2(e)) Indeed the uniqueness ofRobertsonian translocations in karyotypes ofA bahianus andA chirurgus (Figure 3) and similar size of metacentric pairsreinforces these rearrangements are a recently shared traitbetween both species An additional fusion representing anautapomorphic condition is presented in A chirurgus kary-otype since this species has an exclusive large sm pair (7th)and lacks the smallest acrocentric pair observed in the othertwo species (Figure 2(e))

The chromosomal speciation observed in Acanthurusspecies of South Atlantic is likely to reflect historical eventsExtensive analysis of biogeography and evolution of reef fishfrom Atlantic indicated that changes in ocean dynamics overthe past 10 Ma have determined the differential richness andendemism levels of fish genera and families of reef fish [26]As discussed by Galetti et al [24] the rate of chromosomalevolution in reef fish from Atlantic Ocean also seems to bestrongly related to habitat isolation of coastal areas duringglaciation periods followed by further sea level uprising

Unfortunately no reports about time of divergencebetween A bahianus and A chirurgus are available thus hin-dering theminimum time span afterRobertsonian rearrange-ments that gave rise to the large metacentric chromosomesherein referred as a single trait However estimates inferredfor A chirurgus and A tractus [2] being the latter a siblingspecies of A bahianus [7] point out that these rearrange-ments took place by at least 5Ma Even though the timeestimates for the occurrence of chromosomal fusions in bothAcanthurus species might require some bias correction theyare intermediary to periods of major biogeographic isolationevents in Atlantic Ocean such as Amazon outflow (sim10Ma)and uplifting of Panama isthmus (sim3Ma) [27] Nonethelessthe influence of these biogeographic barriers in the putativefixation of chromosomal rearrangements remains unclearand further cytogenetic studies in other species particularlyA tractus are highly encouraged

Different from pericentric inversions centric fusions orRobertsonian rearrangements are usually reported in non-Perciformes marine fish such as flatfish (Pleuronectiformes)[28] toadfish (Batrachoidiformes) [29] and some mullets(Mugiliformes) [30] Centric fusions are particularly com-mon in Batoidea (stingrays guitarfish and skates) the mostderived superorder of elasmobranchs [31] Conversely theserearrangements have been scarcely identified in Perciformesat a polymorphic stage in Pomacentridae [32] Gobiidae [3334] Lutjanidae [35] Apogonidae [36 37] andUranoscopidae[38] or else restricted to a particular taxon like Sparisoma(parrotfishes) [39] Therefore the karyotypes of A bahianusandA chirurgus can be regarded as highly derived in relationto basal karyotype suggested to Perciformes

While the macrostructure was variable the NOR-bearingchromosomes seem to be conserved in the three species(Figure 2 inbox) This pattern indicates that these chromo-somal regions as poor cytotaxonomicmarkers differing fromother Atlantic fishes in which the identification of ribosomalcistrons has proved to be efficient to distinguish apparentlyhomogeneous karyotypes as in Lutjanidae [40] Serranidae[41] and Gerreidae [42] or even population units [43 44]Similarly heterochromatin was virtually similar among Acoeruleus A bahianus and A chirurgus being mainly dis-persed over centromeres and NORs as commonly found inmost Perciformes [45] Therefore it is unclear if the deepdivergences in these fish karyotypes are followed by mic-rostructural changes Further analyses using other bandingmethodologies and mapping of sequences by fluorescence insitu hybridization (FISH) are required to evaluate the exten-sion of such apparent homogeneity of specific chromosomalregions in Acanthurus

The amount of chromosomal traits in the three Acanthu-rus species from Brazilian coast allows raising a phylogenetichypothesis to them Indeed the ordination and sharing ofthe traits show a closer phylogenetic relationship between Abahianus and A chirurgus than to A coeruleus This result iscorroborated by previous genetic analyses Indeed analysis ofCytB sequences showed a more basal condition between Acoeruleus in relation to the other two congeners [7]Recently a phylogenetic analysis of Acanthuridae based onsequence data of two mitochondrial and seven nuclear genes[2] corroborated the ancestral position of A coeruleus inrelation to A chirurgus and A tractus which replaces Abahianus in the Caribbean [7 46]

Thus for analyzed Atlantic species the chromosomaltraits show robust support to clarify the phylogenetic arrange-ment among them serving as useful markers to evolutionarystudies in Acanthuridae Moreover in agreement with phy-logeographic studies [11 26] the chromosomal differencesbetween Atlantic species of Acanthurus seem to be morerelated to ecology and evolutionary history than to dispersalpotential since the three species share a relatively long PLD

5 Conclusion

In conclusion the chromosomal analyses in Acanthurusallowed identifying sequential events related to speciation


process that differ frommost cytogenetical reports onmarinePerciformes where specific rearrangements are often unclearA step-by-step karyotype modification can be inferred fromthe most basal pattern involving few structural rearrange-ments (pericentric inversions inA coeruleus) to high derivedones originated by Robertsonian fusions in bothA bahianusandA chirurgus and additional in tandem fusion inA chirur-gus This scenario reveals a unique condition to tracing backthe order of chromosomal evolutionary changes in Atlanticsurgeonfish

Conflict of Interests

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

Acknowledgments

Theauthors thankCAPES CNPq (no 5567932009-9) INCTldquoCiencias do Marrdquo and FAPESB (no APP00642011) forthe financial support and ICMBioSISBIO (licenses 19135-1131360-1 and 27027-2) for the authorization in collectingspecimensThey are also grateful to Dr Jose Garcia Junior forthe taxonomic identification of specimens

References

[1] J S Nelson Fishes of the World John Wiley amp Sons New YorkNY USA 3rd edition 2006

[2] L Sorenson F Santini G Carnevale andM E Alfaro ldquoAmulti-locus timetree of surgeonfishes (Acanthuridae Percomorpha)with revised family taxonomyrdquo Molecular Phylogenetics andEvolution vol 68 no 1 pp 150ndash160 2013

[3] K L Tang P B Berendzen E OWiley J F Morrissey R Win-terbottom and G D Johnson ldquoThe phylogenetic relationshipsof the suborderAcanthuroidei (Teleostei Perciformes) based onmolecular and morphological evidencerdquoMolecular Phylogenet-ics and Evolution vol 11 no 3 pp 415ndash425 1999

[4] R C Guiasu and R Winterbottom ldquoOsteological evidence forthe phylogeny of recent genera of surgeonfishes (Percomorpha Acanthuridae)rdquo Copeia no 2 pp 300ndash312 1993

[5] KD Clements RDGray and JH Choat ldquoRapid evolutionarydivergences in reef fishes of the family Acanthuridae (Perci-formes Teleostei)rdquo Molecular Phylogenetics and Evolution vol26 no 2 pp 190ndash201 2003

[6] R B Francini-Filho C M Ferreira E O C Coni R L DeMoura and L Kaufman ldquoForaging activity of roving herbiv-orous reef fish (Acanthuridae and Scaridae) in eastern BrazilInfluence of resource availability and interference competitionrdquoJournal of the Marine Biological Association of the UnitedKingdom vol 90 no 3 pp 481ndash492 2010

[7] M A Bernal and L A Rocha ldquoAcanthurus tractus Poey 1860 avalid western atlantic species of surgeonfish (Teleostei Acan-thuridae) distinct from Acanthurus bahianus Castelnau 1855rdquoZootaxa vol 2905 pp 63ndash68 2011

[8] J L Figueiredo and N A MenezesManual de Peixes Marinhosdo Sudeste do Brasil VI Teleostei (5) Museu de ZoologiaUniversidade de Sao Paulo Sao Paulo Brazil 2000

[9] O J Luiz Jr S R Floeter J L Gasparini C E L Ferreira and PWirtz ldquoThe occurrence of Acanthurus monroviae (Perciformes

Acanthuridae) in the south-western Atlantic with commentson other eastern Atlantic reef fishes occurring in Brazilrdquo Journalof Fish Biology vol 65 no 4 pp 1173ndash1179 2004

[10] G L Lawson D L Kramer andW Hunte ldquoSize-related habitatuse and schooling behavior in two species of surgeonfish (Acan-thurus bahianus and A coeruleus) on a fringing reef in Barba-dos West Indiesrdquo Environmental Biology of Fishes vol 54 no 1pp 19ndash33 1999

[11] L A Rocha A L Bass D R Robertson and B W BowenldquoAdult habitat preferences larval dispersal and the compara-tive phylogeography of three Atlantic surgeonfishes (TeleosteiAcanthuridae)rdquo Molecular Ecology vol 11 no 2 pp 243ndash2522002

[12] J A Eble L A Rocha M T Craig and B W Bowen ldquoNot alllarvae stay close to home insights into marine population con-nectivity with a focus on the brown surgeonfish (Acanthurusnigrofuscus)rdquo Journal of Marine Biology vol 2011 Article ID518516 12 pages 2011

[13] W F Molina ldquoChromosomal changes and stasis in marine fishgroupsrdquo in Fish Cytogenetics E Pisano C Ozouf-Costaz FForesti and B G Kapoor Eds pp 69ndash110 Science PublishersEnfield NH USA 2007

[14] R Arai Fish Karyotypes A Check List Springer Tokyo Japan2011

[15] R Arai andM Inoue ldquoChromosomes of seven species of Poma-centridae and two species of Acanthuridae from Japanrdquo Bulletinof the National Science Museum A vol 2 pp 73ndash78 1976

[16] Y Ojima and K Yamamoto ldquoCellular DNA contents of fishesdetermined by flow cytometryrdquo Kromosomo vol 57 pp 1871ndash1888 1990

[17] M J I Brum and P M Galetti Jr ldquoTeleostei ground plankaryotyperdquo Journal of Comparative Biology vol 2 pp 91ndash1021997

[18] W F Molina D E Alves W C Araujo P A Martinez M FSilva and G W Costa ldquoPerformance of human immunostim-ulating agents in the improvement of fish cytogenetic prepara-tionsrdquo Genetics and Molecular Research vol 9 no 3 pp 1807ndash1814 2010

[19] J J Blessing J C Marshall and S R Balcombe ldquoHumane kill-ing of fishes for scientific research a comparison of twomethodsrdquo Journal of Fish Biology vol 76 no 10 pp 2571ndash25772010

[20] J R Gold C Lee N S Shipley and P K Powers ldquoImprovedmethods for working with fish chromosomes with a review ofmetaphase chromosome bandingrdquo Journal of Fish Biology vol37 no 4 pp 563ndash575 1990

[21] W M Howell and D A Black ldquoControlled silver-staining ofnucleolus organizer regions with a protective colloidal devel-oper a 1-step methodrdquo Experientia vol 36 no 8 pp 1014ndash10151980

[22] A T Sumner ldquoA simple technique for demonstrating cen-tromeric heterochromatinrdquo Experimental Cell Research vol 75no 1 pp 304ndash306 1972

[23] A Levan K Fredga and A A Sandberg ldquoNomenclature forcentromeric position on chromosomesrdquo Hereditas vol 52 pp201ndash220 1964

[24] P M Galetti Jr W F Molina P R A M Affonso and C TAguilar ldquoAssessing genetic diversity of Brazilian reef fishes bychromosomal andDNAmarkersrdquoGenetica vol 126 no 1-2 pp161ndash177 2006


[25] P R A De Mello Affonso and P M Galetti Jr ldquoChromosomaldiversification of reef fishes from genus Centropyge (Perci-formes Pomacanthidae)rdquo Genetica vol 123 no 3 pp 227ndash2332005

[26] S R Floeter L A Rocha D R Robertson et al ldquoAtlantic reeffish biogeography and evolutionrdquo Journal of Biogeography vol35 no 1 pp 22ndash47 2008

[27] L A Rocha ldquoPatterns of distribution and processes of specia-tion in Brazilian reef fishesrdquo Journal of Biogeography vol 30 no8 pp 1161ndash1171 2003

[28] J A Bitencourt I Sampaio R T C Ramos and P R A MAffonso ldquoChromosomal fusion in Brazilian populations of Tri-nectes inscriptus Gosse 1851 (Pleuronectiformes Achiridae)as revealed by internal telomere sequences (ITS)rdquo Journal ofExperimental Marine Biology and Ecology vol 452 pp 101ndash1042014

[29] G W W F Costa and W F Molina ldquoKaryoevolution of thetoadfish Thalassophryne nattereri (Batrachoidiformes Batra-choididae)rdquo Genetics and Molecular Research vol 8 no 3 pp1099ndash1106 2009

[30] MNirchio R CiprianoM Cestari andA Fenocchio ldquoCytoge-netical andmorphological features reveal significant differencesamong Venezuelan and Brazilian samples of Mugil curema(Teleostei Mugilidae)rdquo Neotropical Ichthyology vol 3 no 1 pp107ndash110 2005

[31] L Rocco I Liguori D Costagliola M A Morescalchi F Tintiand V Stingo ldquoMolecular and karyological aspects of Batoidea(Chondrichthyes Elasmobranchi) phylogenyrdquo Gene vol 389no 1 pp 80ndash86 2007

[32] W F Molina and P M Galetti Jr ldquoRobertsonian rearrange-ments in the reef fish Chromis (Perciformes Pomacentridae)involving chromosomes bearing 5s rRNA genesrdquo Genetics andMolecular Biology vol 25 no 4 pp 373ndash377 2002

[33] V Giles G Thode and M C Alvarez ldquoA new Robertsonianfusion in the multiple chromosome polymorphism of a Medi-terranean population of Gobius paganellus (Gobiidae Perci-formes)rdquo Heredity vol 55 pp 255ndash260 1985

[34] G Thode V Giles and M C Alvarez ldquoMultiple chromosomepolymorphism in Gobius paganellus (Teleostei Perciformes)rdquoHeredity vol 54 no 1 pp 3ndash7 1985

[35] MNirchio R Rondon COliveira et al ldquoCytogenetic studies inthree species of Lutjanus (Perciformes Lutjanidae Lutjaninae)from the Isla Margarita Venezuelardquo Neotropical Ichthyologyvol 6 no 1 pp 101ndash108 2008

[36] Y Ojima and K Kojima ldquoChromosomal polymorphisms inApogonidae fishesrdquo Proceedings of the Japan Academy B vol 61pp 79ndash82 1985

[37] K A Rivlin J W Rachlin and G Dale ldquoIntraspecific chromo-somal variation inApogon binotatus (Perciformes Apogonidae)from the Florida Keys and St Croixrdquo Annals of the New YorkAcademy of Sciences vol 494 pp 263ndash265 1987

[38] R Vitturi E CatalanoM R LoConte AMAlessi F P Amicoand D Colombera ldquoIntra-populational and intra-individualmosaicisms of Uranoscoper scaber L (Perciformes Uranoscop-idae)rdquo Heredity vol 67 pp 325ndash330 1991

[39] D C S Sena and W F Molina ldquoRobertsonian rearrangementsand pericentric inversions in Scaridae fish (Perciformes)rdquoGenetics andMolecular Research vol 6 no 3 pp 575ndash580 2007

[40] E C Rocha and W F Molina ldquoCytogenetic analysis in westernAtlantic snappers (Perciformes Lutjanidae)rdquoGenetics andMol-ecular Biology vol 31 no 2 pp 461ndash467 2008

[41] W F Molina F A Maia-Lima and P R A M Affonso ldquoDiver-gence between karyotypical pattern and speciation events inSerranidae fish (Perciformes)rdquo Caryologia vol 55 no 4 pp299ndash305 2002

[42] L L Calado L A C Bertollo G W W F D Costa and W FMolina ldquoCytogenetic studies of Atlantic mojarras (PerciformesGerreidae) chromosomal mapping of 5S and 18S ribosomalgenes using double FISHrdquo Aquaculture Research vol 44 no 5pp 829ndash835 2012

[43] D C S de Sena and W F Molina ldquoChromosomal rearrang-ements associated with pelagic larval duration in Labridae(Perciformes)rdquo Journal of Experimental Marine Biology andEcology vol 353 no 2 pp 203ndash210 2007

[44] I V Accioly L A C Bertollo G W W F Costa U P Jacobinaand W F Molina ldquoChromosomal population structuring incarangids (Perciformes) between the north-eastern and south-eastern coasts of Brazilrdquo African Journal of Marine Science vol34 no 3 pp 383ndash389 2012

[45] W F Molina G W W F Costa R X Soares et al ldquoExtensivechromosome conservatism in Atlantic butterflyfishes genusChaetodon Linnaeus 1758 implications for the high hybridiza-tion successrdquo Zoologischer Anzeiger vol 253 pp 137ndash142 2013

[46] J Castellanos-Gell A Robainas-Barcia D Casane P Chevalier-Monteagudo F Pina-Amargos and E Garcıa-Machado ldquoThesurgeonfish Acanthurus bahianus has crossed the Amazon-Orinoco outflow barrierrdquo Marine Biology vol 159 no 7 pp1561ndash1565 2012

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Atlantic Ocean

Brazil

(a)

(b)

(c)

0

1

2

1500

(km)

N

EWS

Figure 1 Map of South America showing the collections sites of Acanthurus coeruleus (a) A bahianus (b) and A chirurgus (c) in the statesof Rio Grande do Norte (1) and Bahia (2) northeastern Brazil

large schools depending on the ontogenetic stage [10] Asmost acanthurids these species present relatively long pelagiclarval stages with a mean duration from 516 to 552 days [11]Usually wide-range reef fish species with long pelagic larvaldevelopment are characterized by a lack of genetic subdivi-sion among populations [12] and low rates of chromosomalvariation [13]

However reports about cytogenetic patterns of Acan-thuridae are still underrepresented (less than 5 of species)and restricted to Indo-Pacific species [14]The three analyzedspecies from Pacific Acanthurus triostegus Prionurus scal-prum [15] and Ctenochaetus striatus [16] all share a conser-vative Perciformes-like karyotype with 2119899 = 48a consideredbasal to this fish group [17]

In order to increase the karyotypic data of Acanthuridaeand to infer the chromosomal evolution of Atlantic speciescytogenetical analyses were carried out for three Acanthuri-dae species from Brazilian coast South Atlantic

2 Material and Methods

Nine individuals of Acanthurus coeruleus four individuals ofA bahianus and 17 individuals of A chirurgus were cytoge-netically studied Animals were collected using hand nets (60times 100 cm) by snorkeling at coastal reef areas from the states ofRio Grande do Norte (5∘461015840S 35∘121015840W) and Bahia (13∘001015840S38∘321015840W and 13∘521015840S 38∘561015840W) in northeastern Brazilianshore (Figure 1) Right after collection specimens were trans-ported in plastic bags with oxygen to the laboratories andplaced in 60 L tanks equipped with filtration and aerationsystems

Twenty-four hours prior to chromosomal preparationthe animals were inoculated via intramuscular with a solutionof antigen complexes (Munolan) for mitotic induction [18]After this period the specimens were anesthetized andeuthanized by immersion in in water at 0ndash4∘C up to completeinterruption of gill movements [19] To obtain mitotic chro-mosomes in vitro portions of anterior kidney were removedand transferred to RPMImedium (Cultilab) with about 50120583Lof 0025 colchicine followed by hypotonic treatment (KCL0075M) for 20 minutes at 37∘C and fixation in Carnoyrsquosfixative (methanol acetic acid 3 1) [20] Chromosomes werestained with 5 Giemsa in phosphate buffer (pH 68) forkaryotypic analyses Nucleolus organizer regions (NORs)were detected by silver nitrate staining (Ag-NORs) [21]whereas heterochromatic regions were evidenced by C-banding [22]

Metaphases were photographed using an Olympus BX51(Olympus Tokyo Japan) epifluorescence photomicroscopeequipped with digital capture system Chromosomes wereclassified as metacentric (m) submetacentric (sm) subtelo-centric (st) and acrocentric (a) based on arm ratio [23] Thepairswere arranged in decreasing order size according to eachmorphological category (m sm st and a) in karyotypes usingthe software Adobe Photoshop CS6 v 130

3 Results

The three Acanthurus species showed remarkable kary-otype diversification Acanthurus coeruleus presented 2119899 =48 composed of two submetacentric four subtelocentricand 42 acrocentric chromosomes (Figure 2(a)) The diploid


sm st

a

22 23

1 2 3 2

24

4 5 6 7 8 9

10 11 12 13 14 15

16 17 18 19 20 21

(a)

sm st

a

22 23

1 2 3

24

4 5 6 7 8 9

10 11 12 13 14 15

16 17 18 19 20 21

(b)

m

sm st

a

st

1 2 3 4 5 6

7 89 8

10 11 12 13 14 15

16 17 18

(c)

st

m

sm st

a

1 2 3 4 5 6

7 8 9

10 11 12 13 14 15

16 17 18

(d)

m

sm st

a

1 2 3 4 5 6

7 8 89

10 11 12 13 14 15

16 17

(e)

m

smst

a

1 2 3 4 5 6

7 8 9

10 11 12 13 14 15

16 17








Basal karyotype



In tandem fusion



Ag-NORs

M

SM

M

(a)

(b)

(c) (d)

1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 10 11 12 13 14 15 16 17 18

19 20 21 22 23 2419 20 21 22 23 24

A coeruleus

2n = 48

2n = 36

2n = 34

1 2 3 4 5 6 7 8 9

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17

10 11 12 13 14 15 16 17

18

lowast

lowast

ST

SM ST

SM ST



4 Discussion














5 Conclusion






Acknowledgments


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


sm st

a

22 23

1 2 3 2

24

4 5 6 7 8 9

10 11 12 13 14 15

16 17 18 19 20 21

(a)

sm st

a

22 23

1 2 3

24

4 5 6 7 8 9

10 11 12 13 14 15

16 17 18 19 20 21

(b)

m

sm st

a

st

1 2 3 4 5 6

7 89 8

10 11 12 13 14 15

16 17 18

(c)

st

m

sm st

a

1 2 3 4 5 6

7 8 9

10 11 12 13 14 15

16 17 18

(d)

m

sm st

a

1 2 3 4 5 6

7 8 89

10 11 12 13 14 15

16 17

(e)

m

smst

a

1 2 3 4 5 6

7 8 9

10 11 12 13 14 15

16 17








Basal karyotype



In tandem fusion



Ag-NORs

M

SM

M

(a)

(b)

(c) (d)

1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 10 11 12 13 14 15 16 17 18

19 20 21 22 23 2419 20 21 22 23 24

A coeruleus

2n = 48

2n = 36

2n = 34

1 2 3 4 5 6 7 8 9

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17

10 11 12 13 14 15 16 17

18

lowast

lowast

ST

SM ST

SM ST



4 Discussion














5 Conclusion






Acknowledgments


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Basal karyotype



In tandem fusion



Ag-NORs

M

SM

M

(a)

(b)

(c) (d)

1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 10 11 12 13 14 15 16 17 18

19 20 21 22 23 2419 20 21 22 23 24

A coeruleus

2n = 48

2n = 36

2n = 34

1 2 3 4 5 6 7 8 9

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17

10 11 12 13 14 15 16 17

18

lowast

lowast

ST

SM ST

SM ST



4 Discussion














5 Conclusion






Acknowledgments


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










5 Conclusion






Acknowledgments


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Acknowledgments


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article sequential steps of chromosomal

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