Heredity 69 (1992 50—56 Received 10 August 1991 Genetical Society of Great Britain

B chromosomes in Astyanax scabripinnis (Pisces, )

LARA BELLINTANI SALVADOR & ORLANDO MOREIRA-FILHO Universidade Federal de São Car/os, Departmento de Genét/ca e Evo/ucao, Caixa Postal 676, 13560 São Car/os, SP, Bras/I

Akaryotypic analysis was carried out using conventional staining and C-banding in 32 specimens of Aslyanax scabripinnis (Pisces, Characidae) from Campos do Jordão (São Paulo State, Brazil). Twenty-eight individuals (87.5 per cent of the sample studied) showed 1—2 extra B-chromosomes, similar in size and shape (metacentrics) to pair 1 of the karyotype, with a high intra-individual constancy. Two types of B could be identified on the basis of their constitutive heterochromatin patterns. The hypothesis of the origin of the B-chromosome from non-disjunction of chromosome 1, followed by a heterochromatinization process, is postulated.

Keywords:Aslyanaxscabripinnis, B-chromosomes, C-banding, karyotype.

Introduction some level which permitted their distinction and were therefore proposed to be distinct . Thegrowing number of papers on cytogenetics This species complex is restricted to the headwaters published over the last 20 years have significantly con- of small tributaries. Thus, its diversity should be tributed to our knowledge of the karyotype structure of initially interpreted as a function of each microbasin in many fish species. Among the many discoveries particular and later analysed in a comparative manner reported are cases of accessory or B chromosomes. between larger draining systems (Moreira-Filho & Accessory chromosomes were probably mentioned by Bertollo, 1991). Taylor (1967) in a study of Eptatretus stoutii. Pauls & Bertollo (1983) reported the first detection of a B chromosome among Ostariophysi, in Prochilodus scrofa. Since this report, other cases have been Materialsand methods detected in 18 species belonging to seven different Thirty-twospecimens (22 females and 10 males) of As- families of bony fish (Table 1). Some of these cases can lyanax scabripinnis (Fig. 1) were collected from the be characterized as B chromosomes, whereas others Córrego das Pedras' stream, municipality of Campos are reports of more sporadic occurrences of extra do Jordão, State of São Paulo, Brazil. This region chromosomes. Karyotype studies conducted on presents a considerably irregular relief with altitudes Aslyanax scabripinnis have demonstrated chromosome ranging from 1800 to 730 m, the collection site being diversity in this species, with the detection of diploid located at 172Dm. numbers equal to 46, 48 and 50 in different popula- Mitotic cells were obtained by the teclmique of tions (Moreira-Filhb et al., 1978; Morelli et al., 1983; Egozcue (1971), adapted by Bertollo (1978), and C Martins et al., 1984; Moreira-Filho & Bertollo, 1986; banding was obtained by the method of Sumner Rocon-Stange & Passamani, 1988; Maistro et al., (1972). 1990; Salvador & Moreira-Filho, 1990). The chromosomes were measured with the aid of a Moreira-Filho & Bertollo (1991) conducted karyo- dry-tip compass and pachymeter. The length of their typic and morphological studies on seven populations short and long arms and the total length were obtained. of A. scabripinnis. Some populations were distinguish- The mean values were then calculated for each able by morphological traits, others by karyotypic chromosome pair. The relative length (per cent) of the traits, and still others by both types of characteristics. different pairs was calculated from these values in Six of the seven populations studied presented unique relation to the total length of the haploid lot. B chromo- of exclusive traits at the morphological and/or chromo- somes were included in the calculation.

50 B CHROMOSOMES IN A. SCABRIPINNIS 51

Table I Supernumerary chromosomes in Osteichthyes fish

Individuals Number of Individual with extra extra species analysis chromosomes 2n chromosomes Size Type Reference

Prochilodontidae Prochilodus scrofa 62 61 54 0—5 Micro — 1 P.scrofa 54 0—7 Small M 2 P. cearenis 7 5 54 0—2 Micro — 1 Curimatidae Cyphocharax modesta 10 1 3x =81 1 Small 3 cit. Curimata nwdesta Cyphocharax modesta 17 1 54 1 Micro 4 Parodontidae Apareiodonpiracicabae 20 1 54 0—1 Large M 5 Characidae Oligosarcus pintoi cit. 19 2 50 0—1 Large M 5 Paroligosarcus pintoi Moenkhausia intermedia 14 8 50 0—1 Small — 6 — M. sanctafilomenae 30 30 50 1—8 Micro 7 Aslyanax eigenmanniorum 3 1 50 0—1 Large M 8 — A. scabripinnis 29 3 50 0—2 Small 9 A. scabripinnis 15 1 50 0—1 Large M 10 A. scabripinnis 32 28 50 0—2 Large M 11 Characidium cfzebra 28 1 50 0—1 Small A 12 — Piabina argentea 12 1 52 0—1 Small 13 Pimelodidae — Pimelodella kronei 5 1 50 0—1 Micro 14 — Bergiaria westermani 7 7 56 0—5 Small 15 — Rhadia quelen 30 13 58 0—2 Small 16 — R. hilarii 51 50 58 0—5 Small 17 Callichthyidae 60 0—3 Small — 18 Cotydorasaeneus 33 9 Middle 19 Callichthys callichthys 27 18 58 0—16 M/SM Loricariidae Micro lepidogaster 20 M. leucofrenatus 34 9 54 0—2 Large M Cichlidae Micro — 21 Gymnogeophagusbalzanii 4 3 48 0-4

References: M = 1. Pauls & Bertollo (1990) 11. Present paper metacentric 2.Cavallaro & Bertollo (1990) 12. Miyazawa(1991) A acrocentric 3. Venere & Galetti Jr (1985) 13. Wasko,(1991) M/SM =metacentricor submetacentric 4. Venere(1991) 14. Almeida Toledo etal.(1985) 5. Falcão etal.(1984) 15.Dias(1987) 6.Portela etal. (1988) 16. Hochberg & Erdtmann(1988) 7. Foresti etal.(1989) 17. Fenocchio & Bertollo (1990) 8. Stripeck etal. (1985) 18. Oliveira etal.(1986) 9. Rocon-Stange & Passamani(1988) 19. Erdtmann etal.(1990) 10. Maistro etal. (1990) 20. Andreata (1991) 21. Feldberg & Bertollo (1984) with twoextra chromosomes(Fig.5). Both chromosometypeswere presentintheindividual specimens withasinglesupernumerary chromosome. other ofthesechromosome typeswasobservedinthe with distalheterochromatin blocks(Fig.5).Oneorthe euchromatiri segmentoneach sideofthecentromere, heterochromatic andthe other presentedamedian the heterochromatinpattern.Oneofthemwasfully two typesofsupernumerarychromosomesinterms chromosome wasdetectedinanyofthecellsexamined. individuals, twofemalesandmales,noextra obtained fromonefemale(Table3).Intheremaining were detectedin100percentofcompletemetaphases chromosomes ofsimilarmorphologyandsize(Fig.4) karyotype complement(Fig. and slightlysmallerthanthefirstpairinbasic chromosome isofthemetacentrictype,largeinsize in 100percentoftheirmetaphases(Table3).This males presentingonlyoneofthiskindchromosome specimens investigated,with19femalesandeight equal to88(Table2andFig.2). pairs ofacrocentrics,withafundamentalnumber(FN) submetacentrics, fivepairsofsubtelocentrics,andsix three pairsofmetacentricchromosomes,11 fication showedthatthebasickaryotypeconsistsof site is2n= number ofA.scabripinnisfromtheCamposdoJordão of thetotalnumberexamined.Thus,diploid have 50chromosomes,correspondingto73.1percent females wereanalysed,and367cellsfoundto A Results AR> 7.01). centrics (ST:AR=3.01—7.00),andacrocentrics(A: submetacentrics (SM:AR= and classifiedasmetacentrics(M:AR=1.00—1.70), ratio (AR)criteriaproposedbyLevarietat.(1964) 52 C-banding permittedthecharacterizationofatleast Extra chromosomesweredetectedin28ofthe32 total of502metaphasepreparationsfrommalesand The chromosomeswereidentifiedusingthearm t;I•% / L. BELLINTANISALVADOR&0.MOREJRA-FILHO 50. Chromosome measurementsandclassi- 1.71—3.00), 3). Twoaccessory subtelo- Table ChromosomemeasurementsandtypesinAslyanax two chromosomes typestakentogether represented 13 haploid lot,andthesecond, 6.43percent.Thus,the per centparticipationin therelativelengthof p = Pair sea bripinnisfromCamposdoJordão,SP,Brazil AR armratio. RL (%)= q = B2 B' 25 24 23 22 21 20 19 18 14 13 12 TL totallength. 17 16 15 11 10 9 8 7 6 5 4 3 2 1 The firstsupernumeracychromosome typehad6.67 long short p Pair means 11.67 10.45 arm. 9.65 0.50 0.80 0.75 2.37 2.10 2.57 2.57 3.00 2.69 2.70 3.40 3.30 3.20 3.25 3.70 3.65 6.02 5.15 5.92 1.20 1.10 1.25 1.60 1.97 arm. relative q 11.85 12.45 12.22 10.95 15.67 11.85 10.52 10.35 7.45 9.05 7.15 7.45 8.50 7.75 8.40 5.41 5.30 7.07 7.07 7.22 7.50 7.95 7.95 8.07 8.15 7.32 7.42 Standard length= Campos doJordão,SP,Brazil. from the'CdrregodasPedras'stream, Fig. 1Astyanaxscabripinnisspecimen length. 27.34 21.50 22.30 10.25 11.32 11.45 13.20 13.47 10.10 10.32 10.37 10.62 10.80 11.15 11.20 11.77 11.80 16.97 12.47 13.34 TL 7.95 9.52 9.55 7.98 8.30 9.76 9.77 B2 = B' = A= acrocentric M = ST = SM = B B metacentric Subtelocentric Submetacentric 8.17 6.43 6.67 2.38 3.06 3.38 3.42 3.94 4.02 2.85 2.85 3.02 3.08 3.10 2.39 2.48 2,92 2.92 3.33 3.35 3.52 3.53 5.07 3.73 3.99 RL (%) chromosome type2 3.17 3.23 chromosome type1 8.0 cm. AR 13.15 14.90 16.60 7.54 9.41 9.77 3.01 3.54 5.31 3.01 4.26 2.10 2.62 2.62 2.12 2.27 2.45 2.44 2.18 2.23 1.34 1.22 1.13 1.76 1.82 1.42 1.25 M M A A A A A A ST ST SM SM SM SM SM SM SM SM M M M Type ST ST ST SM SM SM B CHROMOSOMES IN A. SCABRIPINNIS 53

M IX$A SMkI $1 a k't' U A* aI sT ! * I! Fig. 2 Basic karyotype of an Aslyanax scabripinnis male from Campos do Jordão, SP, Brazil. AN 2!2324 25

Table 3 Frequency of B chromosome per cell in females and males of A. scabripinnis from Campos do Jordão, SP, Brazil

Number of B chromosomes/cell Number of cells Fish 0 1 2 Number of B analysed Cells with B (%) 6164923 — — 0 23 0 61669 — 31 — 1 31 100 61679 — 16 — 1 16 100 61689 17 — — 0 17 0 61769 — 02— 1 02 100 69119 — 08— 1 08 100 69129 — 05 — 1 05 100 69169 — 12 — 1 12 100 69179 — 05 — 1 05 100 69449 — 17 — 1 17 100 69569 — 14 — 1 14 100 69779 — 06— 1 06 100 69799 — 20— 1 20 100 69859 — 06— 1 06 100 69869 — 02— 1 02 100 69879 — 04— 1 04 100 69889 — 07 — 1 07 100 69899 — 09— 1 09 100 69909 — — 20 2 20 100 69919 — 24 — 1 24 100 75509 — 09— 1 09 100 75819 — 07— 1 07 100 6169d — 15 — 1 15 100 6175d — 15 — 1 15 100 6919d — 06— 1 06 100 6943c5 22 — — 0 22 0 6945d — 08 — 1 08 100 6972d — 06 — 1 06 100 6973d — 04— 1 04 100 6980d — 05 — 1 05 100 6981d — 02— 1 12 100 7107d 20 — — 0 20 0

Total flumber of cells analysed 367 chromosomes. characteristics permitus toconsiderthembeB chromosomes atthepopulation level,aswelltheir chromosome initskaryotype. Theoccurrenceofthese Campos doJordãoshows thepeculiarityofextra chromosome classes.However, A.scabripinnisfrom present anARclosetotheclassifyinglimitofthesetwo part tothefactthatseveralofthesechromosomes ST andAchromosomes.Thisdifferencemaybeduein State ofSãoPaulo,Brazil)butdiffersinthenumber tions fromdifferentsites(SalesópolisandSãoCarlos: Moreira-Filho andBertollo(1992)intwopopula- karyotype complementissimilartothosedetectedby a diploidnumberequalto50chromosomesandits A. Discussion (Table 2). per 54

scabripinnis fromCamposdoJordão(SP)presents CD U) cent ofthehaploidcomplementspecies

- — L. BELLINTANISALVADOR&0.MOREIRA-FILHO K) K) • 0 a as ______S K)— -eK)0 a —o1 . —c °'s ", —— K)S —S,S— -J S° a , - S K)S0 5 S MS S aSK)S tD, 5 as -.oac Ms lie S Os-a -.5 -s a -sS genetic inactivation. chromosome non-disjunction followedbyaprocessof chromosomes mayresult fromautosomeorsex tral karyotype.Theother proposesthatthese ments thatoccurredduring theevolutionofances- chromosomes mayberelicsofstructuralrearrange- the originofBchromosomes.Oneproposesthat analysed) butalsototheirintra-individualconstancy. in thepopulation(87.5percentofindividuals characteristics relatednotonlytotheirhighincidence chromosomes ofA.scabripinnispresentinteresting chromosomes duringmeiosis.Onthisbasis,the B possible toconductastudyonthebehaviourofthese karyotype complement,althoughithasnotyetbeen to insurethestabilityofthesechromosomesin (Table 3)indicatesthatsomemechanismmustexist cent ofthecellsfromindividualsbearingthem TM5 Volobujev (1981)raisedtwohypothesestoexplain

The constancyofthesechromosomesin100 - (a-s -a' as-p chromosome. Jordão, SP,Brazil,showingaB scabripinnis malefromCamposdo Fig. 3KaryotypeofanAstyanax chromosomes. Jordão, SP,Brazil,showingtwoB Fig. 4KaryotypeofanAstyanax scabripinnis femalefromCamposdo per B CHROMOSOMES IN A. SCABRIPINNIS 55 4 '1 I. ,, '. It b 4- a - 'SI' • 1 I- F 4 —-a' St.. 4 I 49 Si' *1. S. a I t 4' a S —3 .$ a 0 4 U

Fig. 5 Types of B chromosomes I I, detected by C-banding in Astyanax "4 scabripinnis from Campos do Jordão, #.4. a SP, Brazil. (a) Metaphase showing a . fully heterochromatic B chromosome - .._4 F-S w (arrow). (b) Metaphase with a partially

euchromatic B chromosome in the ,.a I

middle region (arrow). (c) Metaphase 'C p.

with two B types present (arrows). The 0 0 fl inset at lower right gives details of the ea two B types observed.

Scheel (1973) and later Morelli et al. (1983) and Falcão & Bertollo (1985) have emphasized as a widely Acknowledgements diffuse characteristic of Characidae the presence of a Theauthors are grateful to Dr Heraldo Britski for first metacentric chromosome pair which is higher than taxonomic identification. Funds to support this study the remaining chromosomes in the karyotype comple- were provided by FAPESP and CNPq. ment. This chromosome pair is also present in the basic karyotype of A. scabripinnis from Campos do Jordão. In turn, the B chromosomes detected in this References population are similar in both size and shape to the first pair in the complement. On this basis, it may be ALMEIDA-TOLEDO,L. F., TRAJANO, E., TOLEDO-FILHO, 5. A. AND F0RESTI, F. 1985. Análise citogenética comparativa entre suggested that they probably originated from non- peixe cego Pimelodella kronei, das grutas de Iporanga (SP) disjunction of one of these chromosomes followed by e seu possIvel ancestral. Pimelodella transitoria. Absir. heterochromatinization, in agreement with the second Cienc. Cult., 37, 777. hypothesis raised by Volobujev(1981). ANDREATA, A. A. 1991. Estudos Citogenéticos no Subfamilia According to Rejon et al. (1987), the heterochro- Hypopoptopomatinae (Pisces siluriformes, horicariidae) matinization of the B chromosome may be of a selec- Master's Thesis. Departamento de Biologia, Instituto de tive evolutionary nature because this chromosome may Biociências da Universidade de São Paulo, Brazil. have harmful effects on the organism which would be BERT0LLO, L. A. C. 1978. Estudos Citogenéticos no Gênero minimized by a process of heterochromatinization. Hoplias Gill, 1903 (Pisces, Ergthrinidae) Ph. D. Thesis. The heterochromatin patterns presented by the B Faculdade de Medicina de Ribeirão Preto, Universidade chromosomes of A. scabripinnis from Campos do de São Paulo, Brazil. CAVALLARO, Z. I. AND BERTOLLO, L. A. C. 1990. Estudo compara- Jordão (Fig. 5) may represent distinct stages of hetero- tivo dos cromossomos B de Prochilodus scrofa (Teleostei, chromatinization and consequently of genetic inactiva- , Prochilodontidade) do rio Mogi-Guacu e tion of these chromosomes. The high incidence of B in de amostras cultivadas. Abstr. III Simp. Citogen. Evolut. the population analysed may be justified by the specific Aplic. Peixes Neotrop., 22. characteristics of the scabripinnis group, which forms DIAS, A. L. 1987. Análises Citogeneticas de Peixes da Familia small isolated populations and reproduces at different Pimelodidae (Pisces-Siluriformes) Master's Thesis. times of the year, a fact that would favour an easier Departamento de Ciências Biológicas. Universidade fixation of these chromosomes in the population. Federal de São Carlos, Brazil. 56 L. BELLINTANI SALVADOR & 0. MOREIRA-FILHO

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