research article karyotype patterns of hypsolebias antenori...

Research ArticleKaryotype Patterns of Hypsolebias antenori(Cyprinodontiformes Rivulidae) An Endangered Killifish ofthe Semiarid Region of Brazil

Wallace Silva do Nascimento1 Juliana Galvatildeo Bezerra2 Paulo Augusto Lima-Filho3

Maria Emiacutelia Yamamoto1 Sathyabama Chellappa1 and Wagner Franco Molina2

1 Programa de Pos-Graduacao em Psicobiologia Centro de Biociencias Universidade Federal do Rio Grande do NorteAvenida Salgado Filho 3000 Lagoa Nova 59072-970 Natal RN Brazil

2 Departamento de Biologia Celular e Genetica Centro de Biociencias Universidade Federal do Rio Grande do NorteAvenida Salgado Filho 3000 Lagoa Nova 59072-970 Natal RN Brazil

3 Instituto Federal de Ciencia e Tecnologia do Rio Grande do Norte 59500-000 Macau RN Brazil

Correspondence should be addressed to Sathyabama Chellappa chellappasathyabama63gmailcom

Received 23 November 2013 Accepted 31 December 2013 Published 11 February 2014

Academic Editors W Oetting and I V Sharakhov

Copyright copy 2014 Wallace Silva do Nascimento 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

Annual fish which belong to the order Cyprinodontiformes constitute an excellent model for evolutionary studies their short lifecycle distribution in ecologically dynamic environments and low agility make them favorable for genetic analyses The speciesHypsolebias antenori (Rivulidae) encountered in seasonal pools located in the semiarid region of Northeastern Brazil has beenthe object of surveys with a view to study its ecological and behavioral aspects This study reports on the karyotype patterns ofthis species which represents the first contribution to the cytogenetics of this genus The karyotype of this species is composedof 2n = 48 chromosomes (6m + 4sm + 36st NF = 96) the heterochromatic regions are located in centromeric or pericentromericposition and aremore pronounced in the nucleolar organizer regions Two sites Ag-NORsCMA+DAPIwere identified in the shortarms of pairs 2 (metacentric) and 21 (subtelocentric) Unlike the other species of this family which show an evolution modulatedby events of centric fusions H antenori shows the maintenance of a basal diploid number and the large number of bibrachialelements indicates karyotypic diversification derived by pericentric inversions Cytogenetic analyzes in this species will providenew taxonomic markers capable of being utilized in conservation issues and systematics

1 Introduction

The family Rivulidae pertaining to the order Cyprinodon-tiformes is one of the largest families of freshwater fish ofthe Neotropical region It is a diverse group of annual fishmost popularly known as ldquokillifishrdquo which occur in seasonalfreshwater pools of tropical and subtropical areas of SouthAmericaThis groupof fish exhibit short life cycles thus limit-ing the process of sexualmaturation and completion of repro-ductive cycle between specific seasons of a year To surviveunder these extreme conditions the fish eggs are depositedin the sediment of the pools which go through diapausestages during which the embryonic development becomes

temporarily arrestedWith the onset of the next rainy seasonthese eggs hatch out and a new generation is formed [12] The suborder Aplocheiloidei is represented by manyspecies whose distribution extends over vast geographic areascovering the southeastern central and northeastern SouthAmerica [3] It has been suggested that from an evolutionarypoint of view that annualism in the suborder Aplocheiloideioriginated and was later lost however it was subsequentlyregained [4] Thus it demonstrates to be more plastic than itwas thought previously [5]

The family Rivulidae is taxonomically composed of 30genera and 350 valid species withmanymore to be identifiedand described [3 6 7] In the semiarid region of Brazil they

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 862434 6 pageshttpdxdoiorg1011552014862434

2 The Scientific World Journal

Cear a

South America

Brazil

Russas

Atlantic Ocean

Rio Grandedo Norte

4∘30

998400

4∘45

998400

5∘00

998400

5∘15

998400

5∘30

998400

4∘30

998400

4∘45

998400

5∘00

998400

5∘15

998400

5∘30

998400

38∘00

99840037

∘45

99840037

∘30

99840037

∘15

99840037

∘00

99840036

∘45

998400

38∘00

99840037

∘45

99840037

∘30

99840037

∘15

99840037

∘00

99840036

∘45

998400

0 20 40 60 80

(km)

E

S

Mossoro

N

O

Figure 1 Location map showing the study area Russas in Ceara Brazil

are found in the seasonal freshwater pools [2 3] Hypsolebiasantenori [8] was first collected in 1945 from Ceara statein the Northeastern Brazil and was described in the genusSimpsonichthys [9]

A recent ecological study has included H antenori inthe group of endangered organisms This species is currentlyunder severe anthropogenic pressure due to environmentaldegradation urbanization agricultural and ceramic produc-tion activities and absence of conservation measures [2]Thoughmany species of the family Rivulidae in South Amer-ica are not necessarily real evolutionary units they demon-strate a clear pattern of morphometric and meristic differen-tiation [10]

Small populations are particularly subjected to constantanthropogenic changes of the environment [11]Their biolog-ical characteristics such as small populations lentic habitslow vagility and hence reduced gene flow render them asexcellent models for chromosome studies among fish

Cytogenetic analyses in Neotropical freshwater fisheshave been increasingly employed in the elucidation of tax-onomic questions phylogenetic identification of crypticspecies and delineation of populations [12 13] Howeverchromosome analysis in theNeotropical fish species of Rivul-idae is scarce [14] and largely limited to species with morenortherly distribution including Colombia Venezuela andFrench Guyana [15ndash18] or to south of the continent coveringareas of southern Brazil and Uruguay [19 20] Besides thedeficiency of sampled species analyzed much of the chro-mosome information available is mainly derived from clas-sical cytogenetic techniques which limit the understanding

of structural aspects of the chromosomes of the speciesCytogenetic data for H antenori of the semiarid regions ofNortheastern Brazil are unknown

In order to contribute to a better understanding ofchromosomal evolution of Neotropical Rivulidae this workpresents cytogenetic data for the first time for a representativeof the genus Hypsolebias The fish species H antenori wasanalyzed byGiemsa staining C-banding silver impregnationtechnique of argentophilic ribosomal sites (Ag-NORs) andAT and GC base-specific fluorochromes The results helpto establish cytotaxonomic markers which are useful assubsidies for the recognition of several evolutionary units andthe biological conservation of species

2 Material and Methods

Cytogenetic analyses were performed on 16 specimens ofmales and females of H antenori captured from temporarypools located in the municipality of Russas Ceara(04∘57101584039810158401015840 S and 37∘54101584026210158401015840W) Northeastern Brazil(Figure 1) Fish were captured utilizing small hand trawl nets(50 times 150 cm) and sieves (60 times 60 cm) of 2mm mesh sizeThe fish captured were transported to the laboratory underintense aeration The sex was identified by macroscopic andmicroscopic examination of the fresh gonads The medialportion of the gonad was placed with two drops of distilledwater on a slide covered with cover slip and lightly pressedand immediately analyzed by an optical microscope at 200xmagnification The taxonomical identification of the fishspecies was verified and confirmed [8]

The Scientific World Journal 3

m1 2 3 4 5

6 7 8 9 10 11 12 13

14 15 16 17 18 19 20 21

22 23 24

st

sm

(a)

m1 2 3 4 5

6 7 8 9 10 11 12 13

14 15 16 17 18 19 20 21

22 23 24

st

sm

(b)

m1 2 3 4 5

6 7 8 9 10 11 12 13

14 15 16 17 18 19 20 21

22 23 24

st

sm

(c)

Figure 2 Karyotype and a male of Hypsolebias antenori (Bar = 1cm) (a) stained with Giemsa (b) C-banding and (c) sequential stainingwith fluorochromes CMA

3

and DAPI Ag-NORs sites are shown highlighted in box (bar = 5120583m)

Adult specimens were subjected to mitotic stimulationovernight by intraperitoneal and intramuscular injection ofantigens complex according to the methodology proposedby Molina [21] and Molina et al [22] After this periodthe specimens were anesthetized with clove oil (1mL15 Lwater) and sacrificed for removal of the anterior kidneyChromosome preparations were obtained by cell cycle arrestin vitro according to Gold et al [23] The heterochromatinand nucleolar organizer regions (NORs) were identifiedrespectively by using the techniques of Sumner [24] andHowell and Black [25]

In addition to the conventional staining the chromo-some preparations were also stained with chromomycin(CMA

3) and 410158406-diamidino-2-phenylindole (DAPI) fluo-

rochromes [26] in order to identify regions rich in GC-or AT- respectively Briefly slides were aged for three daysand stained with CMA

3(01mgmL) for 1 h and restained

with DAPI (1 120583gmL) for 30min The slides were thenmounted in glycerol McIlvaine buffer pH 70 (1 1) and exam-ined under epifluorescence light microscope (Olympus TMBX50) together with appropriate filters coupled to an imagecapture digital system (Olympus DP73) Images of the samemetaphase sequentially stained with fluorochromes CMA

3

and DAPI were superimposed using Adobe Photoshop CS5The diploid number was established by analysis of thirty

metaphases for each individual The best metaphases werephotographed and used in the construction of the karyotypeThe chromosomemorphologywas determined in accordancewith chromosome arms ratio [27]

3 Results

Males and females of H antenori presented 2n = 48 chro-mosomes with a karyotype composed of 6 metacentrics 4submetacentrics and 36 subtelocentrics (NF = 96 number ofchromosome arms) The chromosomes show little differencein size between the larger and smaller pairs and there is noevidence of structural or numerical differentiation indicativeof the existence of sex chromosomes for this species

The distribution of heterochromatin in the chromosomesshowed asymmetry Thus while few chromosome pairs (12 4 20 21) showed conspicuous heterochromatic blocks inmost of them heterochromatin is reduced and is distributedpreferentially in centromeric portions of chromosomes Inthe pairs 2 20 and 21 the short arms are entirely heterochro-matic (Figure 2)

Analysis of double staining with CMA3and DAPI flu-

orochromes revealed GC-rich regions coincident with Ag-NORs sites located on the short arms of pairs 2 metacentricand 21 subtelocentric (Figure 2 boxes)

4 Discussion

Morphological features based on analyses of osteologicalcharacters scales color patterns behavior ecology and thesurface characteristics of the chorion have been used ascharacters useful in the diagnosis of clades in Rivulidae[28ndash34] Phylogenetic analyses of South American Rivulidaespecies based onmolecular data have produced inconsistent


hypotheses inmany cases with those based onmorphologicalaspects [9] Sources of additional characters may provideimportant insights to elucidate evolutionary aspects andkinship relations in this family

Cytogenetic data has been widely used as a tool forunderstanding relationships of differentiation in groups ofNeotropical fishes [12 13 35 36] In this aspect the annualfish H antenori presents a karyotype with 2n conserved inrelation to considered basal karyotype for Cyprinodontif-ormes (2n = 48 NF = 48) [37] However the high numberof chromosome arms (NF = 96) involving all chromosomesof the karyotype suggests the occurrence of evolutionarygenomic reorganization for this species

In some genera of South American CyprinodontiformeslikeAustrolebias the speciesmay present from lowkaryotypicdiversification as suggested by the occurrence of karyotypeswith 2n = 48 and large numbers of acrocentric elements uptomore diversified with low chromosome numbers over thatconsidered as basic to the order or exhibiting large structuralchanges evidenced by a large variation in the number ofchromosome arms between species [19 38] Unlike otherCyprinodontiformesH antenori showed no evidence of AT-rich regions in the karyotype as hypothesized due to intensestructural rearrangements occurring in chromosomes ofsome species [39] This condition associated with the occur-rence of twoGC-rich regions coincident with nucleolar orga-nizer regions suggests lower participation of Robertsoniantranslocation events frequent in several species [15 19 38ndash40] The chromosomal variability in annual fish correspondsto the most extreme conditions observed among the variousgroups of fish The killifishes of the genus Aphyosemionappear to be one of the most remarkable examples known ofvariability and inter-and intraspecific chromosome diversifi-cation In this genus reduction in the chromosome numberis not entirely restricted phylogenetically [41] This conditionsupports the idea suggested for other groups of fish [42] theoccurrence of orthoselection karyotype process [43] wherethe propensity to karyotypes with Robertsonian translo-cations has favored its appearance multiple times throughindependent events On the other hand superposition ofthe cytogenetic data with molecular phylogeny available forsome species of Aphyosemion demonstrated support to thehypothesis of Scheel [41] In fact as pointed out by thehypothesis basal karyotypes in general have a greater num-ber of chromosomes and high number of acrocentric chro-mosomes whereas more derived species show a reductionin the number of chromosomes resulting from Robertsoniantranslocation events [5 44]

More recently extreme evolutionary dynamics involv-ing chromosomal rearrangements has also been identi-fied in annual African fish species of the genus Chroma-phyosemion where substantial cytogenetic changes relatedto chromosome morphology besides banding patterns andor diploid number between analyzed populations wereobserved [39] The karyotypic diversification among popula-tions and species of this genus is strongly modeled bymecha-nisms of Robertsonian translocation as well as other comple-mentary processes including heterochromatinization [40]Volker et al [39] suggest that fixation of high cytogenetics

variability in species of Chromaphyosemion could be due tohigh rate of chromosomal mutations as well as ecologicalcharacteristics and the reproductive system of this groupIn H antenori pericentric inversions modulate primarily thekaryotypic diversification In this family the pericentricinversions alongwith Robertsonian translocations representthe main mechanisms that have been operating in thekaryotype evolution [15 19]

Despite the evolutionary dynamics that exist among thekillifishes H antenori appears to have a karyotype with pre-dominantly basal traits highlighted by the diploid numberand frequency of major ribosomal genes Yet for all the kary-otype composed entirely of bibrachial chromosomes consti-tutes a derived condition apparently originated by pericen-tric inversions and heterochromatinization processes in somepairsThis chromosomemorphology considered structurallydivergent from the basal pattern in which acrocentric chro-mosomes prevail appear to have contributed towards thereduction of the karyotypic diversification frequently foundin other species of Rivulidae

Phylogenetic relationships derived from molecular datafor Rivulidae suggest greater proximity of some species ofthe genusHypsolebias (previously Simpsonichthys) with Aus-trolebias [45] From the phylogenetic perspective amore pro-nounced degree of karyotypic similarity would be expectedbetween H antenori and Austrolebias species In fact thecytogenetic data of the species as basal diploid valuewith 48 chromosomes heterochromatin distribution largelyrestricted to centromeric regions and the presence of a pairof chromosomes bearing ribosomal sites shared with severalspecies of Austrolebias seem to support a close phylogeneticrelationship with the genus [19 38]

Despite the predictable shortcomings of cytogeneticinformation for some genera of the New World the kary-otypic survey for the species pertaining to 12 genera ofSouth America reveals some common chromosomal patternsamong them [14] Thus diploid values with 2n = 48 arepresent in most species of Austrolebias and KriptolebiasModal diploid numbers with gt48 chromosomes appear to bea rare condition and are only found in one species of the genusAphyolebias (2n = 54) Other genera reveal few species with2n = 48 and most with 2n lt 48 or just 2n lt 48 fixed withintheir species

The exclusive presence of bibrachial chromosomes inH antenori should have possibly contributed to preventingnumerical karyotypic changes and a low detectable changeinvolving Ag-NORs sites In fact numeric polymorphismsor position of the inter or intraindividual Ag-NOR sites wasnot identified in this populationThis contrasts with the highlevel of polymorphism of these chromosomal regions foundin some species of Aplocheiloidei [39]

Cytogenetic analysis on eight putative species of Aus-trolebias identified a range between 3 and 6 active Ag-NORssites The heterochromatic regions in these species have beenidentified in telomeric centromeric and interstitial positions[19] The absence of extensive translocation mechanismsas in some species of Austrolebias can contribute to thelast position being absent in the chromosomes ofH antenoriand also interstitial NORs The combination of cytogenetic


characteristics of H antenori and species of Austrolebiassuggest about ancestral states of some characters in chro-mosomes of the speciesfamily The extensive occurrence ofmultiple NORs present in two or three chromosome pairsseems to indicate that this is a symplesiomorphic conditionfor the family

The data presented for H antenori represent the firstknown information for this species in South AmericaAlthough the ribosomal sites are not polymorphic in Hantenori the data available for several species of Rivulidae[14 19 38] indicate polymorphic condition and diversifiedsites which are potentially useful as cytotaxonomic markersof populations

Evolutionarily karyotypic variations can represent effi-cient postzygotic barriers in killifish Experiments of crossbreeding between populations and species showed partial orcomplete reproductive isolation between cytogenetically dif-ferent populations and species [41] Chromosomal rearrange-ments such as pericentric inversions can cause cladogenesisevents in populations of highly polymorphic killifish Patternrecognition of cytogenetically divergent South Americankillifishes has been used to propose models of conservationin threatened species Austrolebias [38] Analyses in newpopulations of H antenori as well as in other species of thegenus could establish the level of karyotypic divergence aswell as contributing information for biological conservationof this peculiar group of fish

5 Conclusion

Killifishes are considered as intriguing biological models dueto their adaptive and ontogenetic peculiarities Several groupswithin the suborder Aplocheiloidei have shown surprisingkaryotype diversification due to allopatric factors and pos-sibly intrinsic characteristics of the karyotype Hypsolebiasantenori an endemic fish from the semiarid region of North-eastern Brazil presents a mixture of karyotypic patternsconsidered basal and derivative Considering the extensivekaryotype diversity in the family Rivulidae the peculiarkaryotypic data identified for the species which constitutethe first cytogenetic records for the genus may provide auseful tool in the interpopulational analysis of variation andkaryotype evolution of this group

Conflict of Interests

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

Acknowledgments

This study was supported by the National Council for Sci-entific and Technological Development of the Ministry ofScience and Technology of Brazil (CNPqMCT) in the formof research grants and by the Postgraduate Federal Agencyof the Ministry of Education and Culture of Brazil (CAPESMEC)

References

[1] J PWourms ldquoDevelopmental biology of annual fishes I Stagesin the normal development of Austrofundulus myersi DahlrdquoThe Journal of Experimental Zoology vol 182 no 2 pp 143ndash1671972

[2] W S Nascimento M E Yamamoto and S Chellappa ldquoPro-porcao sexual e Relacao peso-comprimento do peixe anualHypsolebias antenori (Cyprinodontiformes Rivulidae) de pocastemporarias do Semiarido brasileirordquo Biota Amazonia vol 2pp 37ndash44 2012

[3] W J E M Costa ldquoFamily Rivulidae (South American annualfishes)rdquo in Check List of the Freshwater Fishes of South and Cen-tral America R E Reis S O Kullander and C J Ferraris JrEds pp 526ndash548 Edipucrs Porto Alegre Brazil 2003

[4] W J Murphy and G E Collier ldquoPhylogenetic relationshipswithin the aplocheiloid fish genus Rivulus (Cyprinodontif-ormes Rivulidae) implications for Caribbean and CentralAmerican biogeographyrdquoMolecular Biology and Evolution vol13 no 5 pp 642ndash649 1996

[5] W J Murphy and G E Collier ldquoPhylogenetic relationships ofAfrican killifishes in the generaAphyosemion and Fundulopan-chax inferred from mitochondrial DNA sequencesrdquo MolecularPhylogenetics and Evolution vol 11 no 3 pp 351ndash360 1999

[6] W J EM Costa ldquoComparativemorphology phylogenetic rela-tionships and historical biogeography of plesiolebiasine sea-sonal killifishes (Teleostei Cyprinodontiformes Rivulidae)rdquoZoological Journal of the Linnean Society vol 162 no 1 pp 131ndash148 2011

[7] R Fricke and W N Eschmeyer A Guide to Fish Collections inthe Catalog of Fishes Database 2012

[8] W J E M Costa ldquoTaxonomic revision of the seasonal SouthAmerican killifish genus Simpsonichthys (Teleostei Cyprin-odontiformes Aplocheiloidei Rivulidae)rdquo Zootaxa no 1669pp 1ndash134 2007

[9] W J E M Costa Peixes anuais brasileiros diversidade econservacao UFPR Curitiba Brazil 2002

[10] T Hrbek D C Taphorn and J EThomerson ldquoMolecular phy-logeny of Austrofundulus Myers (Cyprinodontiformes Rivuli-dae) with revision of the genus and the description of four newspeciesrdquo Zootaxa no 825 pp 1ndash39 2005

[11] A A Agostinho SMThomaz and L C Gomes ldquoConservacaoda biodiversidade em aguas continentais do BrasilrdquoMegadiver-sidade vol 1 no 1 pp 70ndash78 2005

[12] L A C Bertollo C Oliveira W F Molina et al ldquoChromosomeevolution in the erythrinid fish Erythrinus erythrinus (Tele-ostei Characiformes)rdquo Heredity vol 93 no 2 pp 228ndash2332004

[13] M B Cioffi W F Molina R F Artoni and L A C BertolloldquoChromosomes as tools for discovering biodiversitymdashthe caseof Erythrinidae fish familyrdquo in Recent Trends in CytogeneticStudiesmdashMethodologies and Applications P Tirunilai Ed pp125ndash146 InTech Rijeka 2012

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

[15] J J Scheel ldquoRivuline karyotypes and their evolution (RivulinaeCyprinodontidae Pisces)rdquoZeitschrift fur Zoologische Systematikund Evolutionsforschung vol 10 pp 180ndash209 1972

[16] P K Zoch B GHanks and J R Gold ldquoThe standard andNOR-stained karyotype ofRivulus agilae (Rivulidae Teleostei)rdquoTexasJournal of Science vol 41 pp 104ndash106 1989


[17] J F Elder Jr B J Turner J E Thomerson and D C TaphornldquoChromosomal divergence and heterogameity in two annualkillifishes of the genus Pterolebiasrdquo Genome vol 34 no 4 pp674ndash676 1991

[18] J F Elder Jr B J Turner J E Thomerson and D C TaphornldquoKaryotypes of nine Venezuelan annual killifishes (Cyprin-odontidae) with comments on karyotype differentiation inannual killifishesrdquo Ichthyological Exploration of Freshwaters vol4 no 3 pp 261ndash268 1993

[19] G Garcia E Scvortzoff M C Maspoli and R Vaz-FerreiraldquoAnalysis of karyotypic evolution in natural populationsof Cynolebias (Pisces Cyprinodontiformes Rivulidae) usingbanding techniquesrdquo Cytologia vol 58 pp 85ndash94 1993

[20] G Garcia E Scvortzoff and A Hernandez ldquoKaryotypic het-erogeneity in South American annual killifishes of the genusCynolebias (Pisces Cyprinodontiformes Rivulidae)rdquoCytologiavol 60 pp 103ndash110 1995

[21] W F Molina ldquoAn alternative method of mitotic stimulation infish cytogeneticsrdquo Chromosome Science vol 5 no 3 pp 149ndash152 2001

[22] 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

[23] 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

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

[25] 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

[26] D Schweizer ldquoSimultaneous fluorescent staining of R bands andspecific heterochromatic regions (DA-DAPI bands) in humanchromosomesrdquo Cytogenetics and Cell Genetics vol 27 no 2-3pp 190ndash193 1980

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

[28] W J E M Costa ldquoAnalise filogenetica da famılia Rivulidae(Cyprinodontiformes Aplocheiloidei)rdquo Revista Brasileira deBiologia vol 50 pp 65ndash82 1990

[29] W J E M Costa ldquoPhylogeny and classification of Rivulidaerevisited origin and evolution of annualism and miniaturiza-tion in rivulid fishes (Cyprinodontiformes Aplocheiloidei)rdquoJournal of Comparative Biology vol 3 no 1 pp 33ndash92 1998

[30] W J E M Costa ldquoRelationships and redescription of Fundu-lus brasiliensis (Cyprinodontiformes Rivulidae) with descrip-tion of a new genus and notes on the classification of theAplocheiloideirdquo Ichthyological Exploration of Freshwaters vol15 pp 105ndash120 2004

[31] W J E M Costa ldquoTaxonomy and phylogenetic relationshipsamong species of the seasonal internally inseminating SouthAmerican killifish genus Campellolebias (Teleostei Cyprino-dontiformes Rivulidae) with the description of a new speciesrdquoZootaxa no 1227 pp 31ndash55 2006

[32] W J E M Costa ldquoDescriptive morphology and phylogeneticrelationships among species of the Neotropical annual killifish

generaNematolebias and Simpsonichthys (CyprinodontiformesAplocheiloidei Rivulidae)rdquo Neotropical Ichthyology vol 4 no1 pp 1ndash26 2006

[33] D F Belote and W J E M Costa ldquoReproductive behaviorpatterns in the Neotropical annual fish genus SimpsonichthysCarvalho 1959 (Cyprinodontiformes Rivulidae) descriptionand phylogenetic implicationsrdquoBoletim doMuseuNacional vol489 pp 1ndash10 2002

[34] D Fava and M Toledo-Piza ldquoEgg surface structure in theannual fishes Simpsonichthys (subgenera Ophthalmolebias andXenurolebias) and Nematolebias (Teleostei Cyprinodontif-ormes Rivulidae) Variability and phylogenetic significancerdquoJournal of Fish Biology vol 71 no 3 pp 889ndash907 2007

[35] L A C Bertollo G G Born J A Dergam A S Fenocchio andO Moreira-Filho ldquoA biodiversity approach in the neotropicalErythrinidae fishHoplias malabaricus Karyotypic survey geo-graphic distribution of cytotypes and cytotaxonomic consider-ationsrdquo Chromosome Research vol 8 no 7 pp 603ndash613 2000

[36] L A C Bertollo ldquoChromosome evolution in the NeotropicalErythrinidae fish family an overviewrdquo in Fish Cytogenetics EPisano C Ozouf-Costaz F Foresti and B G Kapoor Eds pp195ndash211 Enfield Conn USA 2007

[37] A W Ebeling and T R Chen ldquoHeterogamety in teleosteanfishesrdquo Transactions of the American Fisheries Society vol 99pp 131ndash136 1970

[38] G Garcıa ldquoMultiple simultaneous speciation in killifishes of theCynolebias adloffi species complex (Cyprinodontiformes Rivul-idae) from phylogeography and chromosome datardquo Journal ofZoological Systematics and Evolutionary Research vol 44 no 1pp 75ndash87 2006

[39] M Volker R Sonnenberg P Rab and H Kullmann ldquoKary-otype differentiation in Chromaphyosemion killifishes (Cyprin-odontiformes Nothobranchiidae) II Cytogenetic and mito-chondrial DNA analyses demonstrate karyotype differentiationand its evolutionary direction in C riggenbachirdquo Cytogeneticand Genome Research vol 115 no 1 pp 70ndash83 2006

[40] M Volker R Sonnenberg P Rab and H Kullmann ldquoKary-otype differentiation in Chromaphyosemion killifishes (Cyprin-odontiformes Nothobranchiidae) III Extensive karyotypicvariability associated with low mitochondrial haplotype differ-entiation in C bivittatumrdquo Cytogenetic and Genome Researchvol 116 no 1-2 pp 116ndash126 2007

[41] J J Scheel Atlas of Killifishes of the Old World TFH Publica-tions New Jersey 1990

[42] 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 2007

[43] M J D White Animal Cytology and Evolution CambridgeUniversity Press Cambridge UK 3rd edition 1973

[44] W J Murphy J E Thomerson and G E Collier ldquoPhylogenyof the Neotropical Killifish Family Rivulidae (Cyprinodon-tiformes Aplocheiloidei) Inferred from Mitochondrial DNASequencesrdquo Molecular Phylogenetics and Evolution vol 13 no2 pp 289ndash301 1999

[45] T Hrbek and A Larson ldquoThe evolution of diapause in the killi-fish family Rivulidae (Atherinomorpha Cyprinodontiformes)A molecular phylogenetic and biogeographic perspectiverdquo Evo-lution vol 53 no 4 pp 1200ndash1216 1999

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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BioinformaticsAdvances in

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Stem CellsInternational



Enzyme Research



Microbiology


Cear a

South America

Brazil

Russas

Atlantic Ocean

Rio Grandedo Norte

4∘30

998400

4∘45

998400

5∘00

998400

5∘15

998400

5∘30

998400

4∘30

998400

4∘45

998400

5∘00

998400

5∘15

998400

5∘30

998400

38∘00

99840037

∘45

99840037

∘30

99840037

∘15

99840037

∘00

99840036

∘45

998400

38∘00

99840037

∘45

99840037

∘30

99840037

∘15

99840037

∘00

99840036

∘45

998400

0 20 40 60 80

(km)

E

S

Mossoro

N

O

Figure 1 Location map showing the study area Russas in Ceara Brazil

are found in the seasonal freshwater pools [2 3] Hypsolebiasantenori [8] was first collected in 1945 from Ceara statein the Northeastern Brazil and was described in the genusSimpsonichthys [9]

A recent ecological study has included H antenori inthe group of endangered organisms This species is currentlyunder severe anthropogenic pressure due to environmentaldegradation urbanization agricultural and ceramic produc-tion activities and absence of conservation measures [2]Thoughmany species of the family Rivulidae in South Amer-ica are not necessarily real evolutionary units they demon-strate a clear pattern of morphometric and meristic differen-tiation [10]

Small populations are particularly subjected to constantanthropogenic changes of the environment [11]Their biolog-ical characteristics such as small populations lentic habitslow vagility and hence reduced gene flow render them asexcellent models for chromosome studies among fish

Cytogenetic analyses in Neotropical freshwater fisheshave been increasingly employed in the elucidation of tax-onomic questions phylogenetic identification of crypticspecies and delineation of populations [12 13] Howeverchromosome analysis in theNeotropical fish species of Rivul-idae is scarce [14] and largely limited to species with morenortherly distribution including Colombia Venezuela andFrench Guyana [15ndash18] or to south of the continent coveringareas of southern Brazil and Uruguay [19 20] Besides thedeficiency of sampled species analyzed much of the chro-mosome information available is mainly derived from clas-sical cytogenetic techniques which limit the understanding

of structural aspects of the chromosomes of the speciesCytogenetic data for H antenori of the semiarid regions ofNortheastern Brazil are unknown

In order to contribute to a better understanding ofchromosomal evolution of Neotropical Rivulidae this workpresents cytogenetic data for the first time for a representativeof the genus Hypsolebias The fish species H antenori wasanalyzed byGiemsa staining C-banding silver impregnationtechnique of argentophilic ribosomal sites (Ag-NORs) andAT and GC base-specific fluorochromes The results helpto establish cytotaxonomic markers which are useful assubsidies for the recognition of several evolutionary units andthe biological conservation of species

2 Material and Methods

Cytogenetic analyses were performed on 16 specimens ofmales and females of H antenori captured from temporarypools located in the municipality of Russas Ceara(04∘57101584039810158401015840 S and 37∘54101584026210158401015840W) Northeastern Brazil(Figure 1) Fish were captured utilizing small hand trawl nets(50 times 150 cm) and sieves (60 times 60 cm) of 2mm mesh sizeThe fish captured were transported to the laboratory underintense aeration The sex was identified by macroscopic andmicroscopic examination of the fresh gonads The medialportion of the gonad was placed with two drops of distilledwater on a slide covered with cover slip and lightly pressedand immediately analyzed by an optical microscope at 200xmagnification The taxonomical identification of the fishspecies was verified and confirmed [8]


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3 Results





4 Discussion

















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Acknowledgments


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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







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article karyotype patterns of hypsolebias antenori...

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