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Zoologischer Anzeiger 254 (2015) 62–66

Contents lists available at ScienceDirect

Zoologischer Anzeiger

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tlantic surgeonfishes bear only minor microstructural changesn highly derived karyotypes

aria Aparecida Fernandes a, Paulo Roberto Antunes de Mello Affonso b,arcelo de Bello Cioffi c, Luiz Antonio Carlos Bertollo c,d,ideão Wagner Werneck Félix Costa a, Wagner Franco Molina a,∗

Department of Cellular Biology and Genetics, Biosciences Center, Federal University of Rio Grande do Norte, 59078-970 Natal, RN, BrazilDepartment of Biology Science, State University of Southwest of Bahia, Jequié, Bahia, BrazilDepartment of Genetics and Evolution, Federal University of São Carlos, 13565-905 São Carlos, SP, BrazilProfessor Senior at Universidade Federal de São Carlos, Brazil

r t i c l e i n f o

rticle history:eceived 3 September 2014eceived in revised form 7 November 2014ccepted 17 November 2014vailable online 20 November 2014

eywords:arine fishes

equential rearrangementsobertsonian rearrangementscanthuridaeelomeres

a b s t r a c t

Acanthurus is a representative and widespread genus of marine fish that plays a key role in ecologicaldynamics of coral reefs. Three species are common along coastal reefs of Western Atlantic: A. coeruleus,A. bahianus, and A. chirurgus. The cytogenetic patterns of these species indicated sequential steps ofchromosomal rearrangements dating back 19–5 millions of years ago (M.a.) that accounted for their inter-specific differences. A. coeruleus (2n = 48; 2sm + 4st + 42a), A. bahianus (2n = 36; 12m + 2sm + 4st + 18a), andA. chirurgus (2n = 34; 12m + 2sm + 4st + 16a) share an older set of three chromosomal pairs that were orig-inated through pericentric inversions. A set of six large metacentric pairs formed by Robertsonian (Rb)translocations found in A. bahianus and A. chirurgus and a putative in tandem fusion found in A. chirurgusare more recent events. In the present study, new cytogenetic data are being reported for these threeAcanthurus species based on mapping of repetitive sequences such as ribosomal 18S and 5S rDNA andtelomeric repeats to improve their karyotype evolutionary analyses. The lack of interstitial telomericsequences (ITS) in spite of several centric fusions in A. bahianus and A. chirurgus might be related to the

long period of time after their occurrence (estimated in 5 M.a.). Furthermore, the homeologies amongthe chromosome pairs bearing ribosomal genes, in addition to other structural features, highlight largeconserved chromosomal regions in the three species. Our findings indicate that macrostructural changesoccurred during the cladogenesis of these species were not followed by conspicuous microstructuralrearrangements in the karyotypes.

© 2014 Elsevier GmbH. All rights reserved.

. Introduction

The Acanthuridae family (Perciformes) is a monophyletic andelatively ancient group of marine fishes (nearly 54 millions ofears ago) with fast evolutionary divergence (Clements et al., 2003;orenson et al., 2013). Nowadays, this family is divided into sixenera and comprises about 80 species, popularly known as sur-eonfishes (Nelson, 2006).

The genus Acanthurus Forsskål (1775) is one of the mostmblematic groups of surgeonfishes closely associated to coastaleefs (Bellwood and Wainwright, 2002; Nelson, 2006). This genus is

∗ Corresponding author. Tel.: +55 84 32119209; fax: +55 84 32153346.E-mail addresses: molinawf@pq.cnpq.br, molinawf@yahoo.com.br

W.F. Molina).

ttp://dx.doi.org/10.1016/j.jcz.2014.11.003044-5231/© 2014 Elsevier GmbH. All rights reserved.

widespread over tropical and subtropical seas, playing a major rolein the ecology and evolution of coral reefs due to their herbivoryhabit (Valentine and Heck, 1999). The large assemblages of Acan-thuridae and other grazers (e.g., parrotfishes and damselfishes)influence the coral reef structure by promoting bioerosion and con-trolling the spread of macroalgae cover (Horn, 1989; Paddack et al.,2006).

Four Acanthurus species are recorded in Western Atlantic, beingthree of them commonly found throughout Brazilian coast; A.coeruleus Bloch and Schneider (1801), A. bahianus Castelnau (1855),and A. chirurgus (Bloch, 1787) (Bernal and Rocha, 2011; Menezesand Figueiredo, 1985). These three species present highly differ-

entiated karyotypes (2n = 48, 36, and 34, respectively) in whichRobertsonian rearrangements reduced the diploid values from2n = 48 to 2n = 36 in A. bahianus and 2n = 34 in A. chirurgus. Infact, six large metacentric pairs, which are absent in A. coeruleus,

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ere derived from centric fusions and are apparently homeolo-ous between A. bahianus and A. chirurgus, corroborating their closehylogenetic relationship. Such remarkable differences in the kary-type macrostructure are rare in marine fishes. Comparing theseesults to molecular data available for Acanthuridae, it was possi-le to infer that centric fusions have taken place about 5 M.a. whilehe pericentric inversions shared by the three species were morencient events (nearly 19 M.a.) (Affonso et al., 2014).

In view of the unusual cytogenetic variation in Atlantic Acanthu-us, chromosomal banding and cytogenetic mapping of ribosomalenes and telomeric sequences were performed in A. coeruleus, A.ahianus, and A. chirurgus. Our purpose was to infer if microstruc-ural chromosome differentiation also followed the diversificationn the karyotype macrostructure.

. Material and methods

Cytogenetic analyses were carried out in specimens of A.oeruleus (N = 23), A. bahianus (N = 12), and A. chirurgus (N = 17)ollected along the shoreline of Natal, Rio Grande do Norte state5◦46′S, 35◦12′W) and Salvador, Bahia state (13◦00′S, 38◦32′W),ortheastern Brazil. Mitotic stimulation was performed by the

noculation of antigen complexes in the specimens (Molina, 2001;olina et al., 2010). After 24 h, the animals were anesthetized with

rave oil (1 mL/15 L of salt water) and euthanized for removal ofnterior kidney. The experiments and euthanasia of specimensere authorized by the committee of animal ethics (CEUA/UESB)

rom Universidade Estadual do Sudoeste da Bahia (#32/2013).Chromosomal spreads were obtained by interrupting the cell

ycle in vitro (Gold et al., 1990). The active nucleolar organizeregions (NORs) were detected by silver nitrate staining (Howell andlack, 1980). As heterochromatin patterns have been previouslyeported in the analyzed species (Affonso et al., 2014), chromo-omes were stained with base-specific fluorochromes to locateC- and AT-rich sites by mithramycin (MM) and 4′-6-diamino-2-

enilindole (DAPI), respectively (Schweizer, 1980).FISH was performed according to Pinkel et al. (1986) using 18S,

S, and telomeric probes. The probes of 5S rDNA (nearly 200 bp)nd 18S rDNA (nearly 1400 bp) were obtained from DNA samplesf A. coeruleus via PCR using the primers A 5′-TAC GCC CGA TCTGT CCG ATC-3′/B 5′-CAG GCT GGT ATG GCC GTA AGC-3′ (Pendást al., 1994) and NS1 5′-GTA GTC ATA TGC TTG TCT C-3′/NS8 5′-CC GCA GGT TCA CCT ACG GA-3′ (White et al., 1990), respectively.oth probes were labeled by nick translation (Roche, Mannheim,ermany). The 5S rDNA probes were labeled with biotin-14-dATP

invitrogen) and detected with avidin-FITC (Sigma); while the 18SDNA probes were labeled with digoxigenin-11-dUTP (Roche) andetected with anti-digoxigenin-rhodamin (Roche). The (TTAGGG)nequences were mapped by FISH using Telomere PNA FISH Kit/FITCccording to manufacturer’s instructions (DakoCitomation).

The micrographs were obtained using an epifluorescence pho-omicroscope OlympusTM BX51 (Olympus, Tokyo, Japan) withppropriate filters equipped with digital capture system OlympusP73 using the software CellSens (Olympus). The karyotypes wererranged as previously defined by Affonso et al. (2014) with iden-ification of metacentric (m), submetacentric (sm), subtelocentricst), and acrocentric (a) chromosomes based on arm ratio. An idio-ram was built based on the structural features of chromosomalairs of each species combining the results of mapped sequences.

. Results

The karyotypic patterns agree with the previous results reportedor the three Acanthurus species (Affonso et al., 2014). There-ore, A. coeruleus presented 2n = 48 (2sm + 4st + 42a; NF = 54), A.

Anzeiger 254 (2015) 62–66 63

bahianus 2n = 36 (12m + 2sm + 4st + 18a; NF = 54) and A. chirurgus2n = 34 (12m + 2sm + 4st + 16a; NF = 52) (Fig. 1a–c). Active NORswere located at terminal position on short arms of the largest st pairof each species, corresponding to pairs 2, 8, and 8 of A. coeruleus, A.bahianus, and A. chirurgus, respectively, (Fig. 1, inbox). The GC-richsites (MM+/DAPI−) were identified only at active NORs while AT-rich heterochromatin segments were absent in the three species.

The 18S rDNA sites were coincident with Ag-NORs in all species,except for A. coeruleus that presented an additional and probablyinactive NOR site (identified by the lack of silver nitrate staining) onpair 13. On the other hand, the 5S rDNA sites were invariably locatedin the largest acrocentric pair of the three acanthurids, correspond-ing to pair 4 in A. coeruleus, and pair 10 in both A. bahianus and A.chirurgus. The (TTAGGG)n sequences were located exclusively atterminal chromosomal regions (Fig. 1). The two-color FISH withribosomal probes showed lack of synteny between the 18S and 5SrDNA sites.

4. Discussion

Previous cytogenetic reports allowed tracing back the occur-rence of chromosomal rearrangements in the Atlantic surgeonfishA. coeruleus, A. bahianus, and A. chirurgus (Affonso et al., 2014). Theseauthors verified that a set of sm–st chromosomes (pairs 1–3 in A.coeruleus and 7–9 in A. bahianus and A. chirurgus) shared by thesespecies derived from relatively ancient pericentric inversions whencompared to the basal karyotype of Perciformes (2n = 48a). On theother hand, a set of large metacentric pairs (1–6), associated withthe drastic reduction in 2n values of A. bahianus and A. chirurgus,indicated a more recent event of Rb translocations that corrobo-rates the closer phylogenetic relationship between both species inrelation to A. coeruleus.

This approach was favored by the rare possibility of datingchromosomal rearrangements in this group. The divergence periodestimated for the three Acanthurus species (considering A. tractusPoey (1860), as sister-group of A. bahianus) by molecular analy-ses (Sorenson et al., 2013) provides a reliable indication that theset of six metacentric chromosomes related to Rb rearrangementsand shared by A. bahianus and A. chirurgus has arisen at least 5 M.a.(Affonso et al., 2014). Given the karyotype macrostructure varia-tion in Acanthuridae from Brazilian coast, the mapping of repetitivesequences is particularly useful to the identification of possibleshared chromosomal similarities as well as refining the under-standing about the interspecific differentiation after the origin ofchromosomal rearrangements.

In the three Acanthurus species, the heterochromatin is mainlydistributed over pericentromeric regions and interspersed to NORs.Following this trend, the qualitative analysis of heterochromaticsegments presented in this study, also revealed similar base com-position with homogenous amounts of GC and AT content afterfluorochrome staining. The only exceptions were the GC-rich sites(MM+/DAPI− signals) at NORs, as usually detected in teleosteans.

This reduced and quite homogenous heterochromatic patternof Acanthuridae (Affonso et al., 2014) has been considered as anobstacle to evolutionary changes in karyotypes of marine Perci-formes (Molina, 2007). Indeed, the mapping of repetitive sequencesin several families of this order, characterized by stable kary-otypes, revealed a reduced phylogenetic variation (Motta-Netoet al., 2011a, b; Calado et al., 2013). On the other hand, heterochro-matin heterogeneity has been reported in some fish groups withremarkable karyotypic changes such as Gobiidae (Caputo et al.,

1997; Lima-Filho et al., 2014; Molina et al., 2014), Characidae(Mantovani et al., 2000), and Erythrinidae (Cioffi et al., 2009; Blancoet al., 2011). For instance, some species of marine Perciformeslike Centropyge aurantonotus Burgess (1974) (Pomacanthidae),

64 M.A. Fernandes et al. / Zoologischer Anzeiger 254 (2015) 62–66

Fig. 1. Karyotypes and metaphases of A. coeruleus (A), A. bahianus (B), and A. chirurgus (C) after Giemsa staining (left column), two-color FISH (middle column) with 18S rDNA( n sequn m. (Fw

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magenta signals), and 5S rDNA (green signals) probes and distribution of (TTAGGG)itrate staining (left column) and fluorochrome staining (middle column). Bar = 5 �eb version of this article.)

resented unusual karyotype for the family and a unique richnessf C-bands and GC-rich sites (Affonso and Galetti, 2005). There-ore, the absence of heterochromatin variation in the differentiatedaryotypes of Acanthurus species in the present work representsn unusual condition. However, it should be pointed out that het-rochromatin might be more complex than previously thoughtfter analyses of other repetitive sequences (histones, transposons,etrotransposons, and microsatellites), as revealed in some Perci-ormes species (Costa et al., 2013, 2014).

The location of 18S and 5S rRNA genes has been proved to befficient cytotaxonomic markers in distinct marine fish groups likeerreidae (Calado et al., 2013, 2014), Lutjanidae (Rocha and Molina,008), and Nototheniidae (Pisano and Ghigliotti, 2009). Usually,oth families of these ribosomal genes are not syntenic (Gornung,013), as observed in Acanthurus (Fig. 1). Nonetheless, the distri-ution of both 18S and 5S rDNA sites were quite similar among thehree species, being apparently located in homeologous pairs.

The NOR-bearing chromosomes identified by silver nitrate anduorochrome staining as well as by FISH with 18S rDNA probesere included in “sm–st chromosomes set” possibly formed by

ericentric inversions that took place before the split of the threecanthurus species around 19 M.a. (Sorenson et al., 2013). In spitef the conservatism of NORs in these acanthurids, an additional 18S

ences (right column). Inbox, the NOR-bearing chromosomes are shown after silveror interpretation of the reference to color in the text, the reader is referred to the

rDNA site was located on pair 13 in the karyotype of A. coeruleus,regarded as the most basal taxon among the analyzed species(Bernal and Rocha, 2011; Castellanos-Gell et al., 2012; Sorensonet al., 2013). The ribosomal site in pair 13 lacks argyrophylic natureindicating an inactive copy of ribosomal gene (Santoro, 2005) orpseudogenes (Affonso et al., 2005; Lima-Filho et al., 2014).

The lack of conspicuous microstructural changes in karyotypesof Acanthurus contrasts with the remarkable variation in chromoso-mal number and morphology found in A. bahianus and A. chirurgus,particularly caused by Robertsonian (Rb) translocations or centricfusions that formed the “m-chromosomes set” pairs in both species.

Previous reports in fishes suggested that Rb rearrangementsare favored by the presence of terminal 18S or 5S rDNA sitesinterspersed with heterochromatin in formerly acrocentric chro-mosomes (Fujiwara et al., 1998; Molina and Galetti, 2002).This seems to be the case of Selene setapinnis (Mitchill, 1815)(Carangidae) in which a Rb translocation was inferred to explaininternalized 18S rDNA regions previously located at terminalregions (Jacobina et al., 2013). Similarly, biarmed chromosomesderived from centric fusions in the pomacentrids Chromis flavi-

cauda (Günther, 1880) and C. jubauna Moura (1995), shared 5SrDNA sites (Molina and Galetti, 2002). The mapping of 18S rDNAin A. bahianus and A. chirurgus did not show these sequences in the

M.A. Fernandes et al. / Zoologischer Anzeiger 254 (2015) 62–66 65

F ), andp some( indic

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ig. 2. Specimens of A. coeruleus (A), A. bahianus (B), and A. chirurgus (C) (bar = 2 cmhysical mapping of ribosomal and telomeric sequences. The homeologous chromom-chromosomes set) are highlighted in boxes. The asterisk in pair 7 of A. chirurgus

-chromosomes set. However, the presence of an extra rDNA site inhe terminal position of an acrocentric pair of A. coeruleus, a speciesarrying the basal karyotype 2n = 48, does not excludes the role ofhis DNA family in the Rb processes occurred in the Acanthurusenus.

The mapping of telomeric sequences by FISH is highly indicatedo species or populations with reduced chromosomal numbers byentric fusions (Reed and Phillips, 1995). This approach is able toeveal internal telomere sequences (ITS) that serve as evidence ofb rearrangements (Bitencourt et al., 2014; Meyne et al., 1990).owever, several cases of centric fusions are not followed by theresence of these ectopic sites. In vertebrates, this lack of corre-

ation between Rb rearrangements and ITS has been related toosses during centric fusions or modification of telomeric sequencesfter long periods of internalization (Nanda et al., 1995; Ocalewicz,013). Likewise, ITS were absent in the fused chromosomes of A.ahianus and A. chirurgus. Taking into account that Rb rearrange-ents in both species have probably taken place at nearly 5 M.a., it

eems plausible to infer that the telomeric-like sequences internal-zed by fusions accumulated enough mutations or were lost duringhis long period of time.

A more recent chromosomal rearrangement was inferred in A.hirurgus and representing an autapomorphic feature. Putatively,he reduction of the chromosomal number (2n = 34) in this speciesas derived from a tandem fusion of a small acrocentric pair

present in A. bahianus) on the short arms of the chromosomal pair in A. chirurgus (Fig. 2). Usually, ectopic telomeric sequences couldlso be preserved after tandem fusions (Cioffi et al., 2011; Hartmannnd Scherthan, 2004). However, internal (TTAGGG)n sequencesere not identified in this case and could have been probably lost

uring the chromosomal rearrangements.In conclusion, the refined analysis of karyotypic features in the

hree species of Acanthurus showed little microstructural changesespite of their long-term evolutionary history of chromosomalearrangements. The coincident distribution of ribosomal genes, asell as the presence of homeologous and phylogenetically related

respective idiogram combining the results from NOR/GC-rich region location ands derived from pericentric inversions (sm–st chromosomes set) and centric fusionsates a putative autapomorphy (in tandem fusion) to this species.

sets of sm–st chromosomes (3 pairs) and m chromosomes (6 pairs)reveal the conservatism of large syntenic chromosomal regions inthese species. This condition has been reported in other marinePerciformes (Molina, 2007; Molina et al., 2013; Motta-Neto et al.,2011a), revealing that chromosomal rearrangements have proba-bly taken place during cladogenesis of these groups and remainedfixed ever since.

Acknowledgments

The authors thank CNPq (#556793/2009-9; 301409/2009-9),and INCT AmbTrop - Brazilian National Institute of Sciencesand Technology for Tropical Marine Enviroments, and FAPESB(#565054/2010-4; 8936/2011) for the financial support, CAPES forthe MSc scholarship granted to MAF, and ICMBio/SISBIO (licenses19135-1, 131360-1, and 27027-2) for the authorization in collect-ing specimens. We are also grateful to Dr. José Garcia Júnior for thetaxonomic identification of specimens.

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