-- --I.--.._____ CARYOLOGIA 48, n. 299.307, 1995 ---I_-.- __ Vol. 3.4:

Karyotypes of three <(small>>Barbus species () from Republic of Guinea (Western Africa) with a review on karyology of African small -{ - PETR RAB, ANNIE MACHORDOM *, ANABEL PERDICES and JEAN-FRANCOIS GUEGAN ** ..---. Institute of Physiology and Genetics, Department of Genetics, Laboratory of Fish Genetics, Acndemy of Sciences of Czech Republic, 277 21 Libechod Czech Republic; Consejo Superior de Investigaciones Cientificns, Museo Naciond de Ciencias N$rurales, Laborarory Biodiversitas, 28006 Madrid, Spain; ** Antenne ORSTOM. Laboratoire d’Ichtfiyologie Génirale ct Appliquée, Muséum National d’Histoire Naturelle, 75231 Paris Cedex 05, Fri&.

SUMMARY - Karyotyp up of asmalln African from the Republic of me (Zn)and chromo- 96, for 8 ablober 2~ I respectivelv The first ly larger, and it can be rype characteristics of the diploid group of s Burbur sensu stric[o

INTRODUCTION

Numerous African species assigned t us Barbus involve in fact 230 two distinct groups: ctsmalL (about s dargen (about 70 species) -. et L‘. L‘. t barbels (SKELTON al. 1991). These tw r especially in adult size 1: (i.e. about 150 mm SL and 700-900 m

(e.g. cal VERVOORT1980; RAB 1981; V COLLARES-PEREIRAand MADEIRA 1990, et al. LUBTSOV and KRYSANOV1993; RAB AN et aì 1995) investiga-

.. 300 . KAU, MAClIOKDOM, PERDICES 2nd CUECAN KARI'L tions demonstrated that only chose species wich evolutionary tetraploid (2s = 100) and hexaploid (2n = 148-1501 Ievels,,are assignable to the Barbus sensu stricto (and/or to che broader cntegbry of barbin lineage), while chose (2~ possessing a diploid chromosome numbqr = f 50) belong CO distinct lineages of cyprinine cyprinids (sensu HOW,ES1991) such as Puntitrs and related genera (MAGTOONand ARAI 1989, 1990; ARAIand MAGTOON1991; Yu et al. 1987, 1989). For African Barbus specie's, che existence of a polyphyletic assemblage was stressed and discussed by RAB(1981) and put in the limelight by GOLUBTSOV and KRYSANOV(1993) again. From a karyological view point, this African group of ccsmalla barbels, similarly as nearly all other African ichthyofauna, it remains practically unknown. Tab. 1 shows that there are 4 reports on the chromosome numbers and/or karfgtypes €or 10 African usmalln Barbus species only. This present report deals wikh the description of karyotypes of three species of wnalh barbels, namely Barbus bigomei (Lévèque, Teugels and Thys van den Audenaerde, 1988), B. ablaber (Blekeer, 1863) and E. macrops BOU- lenger, 1911, all of them caughc in Republic of Guinea (Western Africa). The karyology of this poorly studied African cyprinid group is finally reviewed and discussed.

MATERIALS AND METHODS

The specimens used in this srudy represenr a parr of large sample during a joint French-Spaniard expedition in Republic of Guinea in 1993. All specimens karyotyped i were preserved and are deposited as vouchers in che Museo Nacional de Ciencias Naturales (MNCN) ar Madrid (Spain). The analyzed mncerial consisted of 3 specimens (1 male, 2 females) of Barbus bigonzei (No. 83838-40 MNCN) from the Mongo river (Upper Little Scarcies basin) ar Marela, 1 specimen (female1 of Barbus abbbes (No. 83857) from the Kaba River (Upper Little Scarcies basin) at Kouloundala, and 1 specimen (sex unknown) of Barbus macrops (No. 83960) from the Samou river (Kon- kouré basin) at Débélé. Chromosome preparations were made directly in field conditions according to the method described in DOUSSALIDE'BAZIGNAN and OZOUF-COSTAZ(1985). Fixed cell suspensions were kept in deep freezer unril rheir analysis in the laboratory. Because cell suspensions were fixed with ethanol (instead of methanol) acetic acid fixative and such suspensions did not provide suitable metaphase plates, the prococol was modified as follows. The suspensions were refixed in cold, freshly made mechanol-acetic acid fixarive ar least five times. The chromosome preparations were made by dropping of cell suspension either onto dry slides or, if unsuccessful, onto slides wetted with chloroform. After drying, che slides were srained with 5% of Giemsa stain and, if necessary and to get a better contrast, they were slightly counter-stained with 50% of silver nitrate. Selected and photographed metaphases were destained and nucleolar

.- - TABLE 1 - Comparison of current and previously published data on the chromosome number (2n). haploid karyotype characteristics and chromosome arm number (N F 1of species of mnalln African Barbus and related genera < 2n Haploid No. and sex .e-i Species karyotype N.F. of material Locality Reference CI examined O - _.._ - Barbus uiuiparur 24' not reported - .-I not Natal, aquarium stock POST 1965 Weber. 1897 m5 Barbus bariIiordes 48 16m+8sm 96 ¡uv. Angola, aquarium stock RAß 1981 Boulenger, 1914 ** 3 : Barbus hoIotaenia 50 12m+l3sm I&29 Zaire, aquarium RAB 1981 Boulenger, 1904 100 stock Fr o Barbus anema 50 2lm,sm+4a 92 3 ? Alvero R., Ethiopia KRYSANOV& GOLUIITSOV 1993 5 Boulenger. 1903 B 84 R Barbus kenlenit 50 17m, sn+8a 17 ? L. Abaya, Harr R., KRYSANOV& Co~uursov1993 Peter, 1868 Silc R.. Ethiopia i ßurbur paludinorus 50 23m. sm + 2a ? Bulbula R , Ethiopia KRYSANOV& GoLuursov 1993 hers, 1852 96 IS

KRYSANOV & GOt.UWlSt)V 1903 KRYSANOV& Go1 UIIrwv 19') 3 Hare R , Kulfo R , KKYSANOV& GOI 1111 I WV 1993 ? Mongo R I Guinea this report

Barbus bigomei 48 Levequi, Teugels 9m+15sm 98 I.!, 2cf Kaba K.. Guinea this report . and Thys vin den Audenarde. 1988 Barbus macrops 50 7m, t 14sm t 4st-a 92 I ? ßoulenger, I9 I I Samour R., Guinea this report

Caecobarbrir gecrtrii 50 6m. + 14sm + 551 100 2x 39 cavc near Thysville. Zaire VERVOOKT1980 ßoulenger, I92 I . . ~ . Explanations. III . nlciaccntric. sm . submetacentric. il . acroceniric cliromosumcs; * only haploid chromosome nunlber rc1,orLcd; *' thc il:rinc is il I of syiiuiiymc 8arhnr /urdolt~trrsGunthcr, 1868. i \r o i i I_

I ..

..,. , . .I

302 RAB, MACHOHDOM, PERDlCES an¿ CUECAN .. '. organizer regions (NORs) were analyzed by *e colloidal silver nitrate method of HOWELLan¿ wes classified according to LEVANet al. BLACK (1980). Chromosomes -v (1964). .8- 2'

. ...' ir, RESULTS , :.t I *! .".:i i Barbus Gigomgi, - Diploid ,chromosome number 2n = 48. The karyotype consists of 9 pairs of metacentric and 15 pairs of submetacentric chromosomes, NF = 96 (Fig. IA). NORs are located telomerically in one middle-sized

submetacentric pair (Figs. 2A, B). ,, Barbus ablabes. --Diploid chromosome number 2n = 50. The karyotype consists of 9 pairs of metacenrric/l5'pairs of submetacentric and 1 pair of subtelocentric to acrocentric cliiomosomes, NF = 98 (Fig. 1B). NORs are located telomerically in one middle- sized submetacentric pair (Figs. 2C, D). Barbus macrops. - Diploid chromosome number 27.3 = 50. The karyotype consists of 7 pairs of metacentric, 14 pairs of submetacentric and 4 pairs of subtelocentric to acrocentric chromosomes, NF = 92 (Fig. IC). The location of NORs could not be precisely located but interphase nuclei displayed 2 positive signals (Fig. 2E). Resulrs for all three species are summarized in Tab. 1.

DISCUSSION

Karyotypes of cyprinids, both evolutionary diploid and polyploid, are generally characterized by the presence of small elements with their centromere position placed gradually from a median to a nearly terminal position. This previous morphological characteristic plus the effect of chromosame arm con- rraction during mitosis due TO temporal and dose colchicine treatment make difficult precise assignment of ai number of chromosome pairs [o particular categories (RAB and ROTH 1989). Moreover and in spite of difficulties in ,. preparing chromosome suspensions in the field leading to their relative poor .. I quality, the karyotype features of these three barbels may however permit to discuss about the composition of their chromosomal sets. This is more especial- ly the case of E. macrops for whïch we had chance to analyze a limited number of metaphases (Fig. IC). Interestingly, the first pair of metacentric chromo- somes in the karyorypes of all three species was distinctly larger, and it can be

(A), (C); Fig. 1. - Karyotypes of Barbus bigomei B. ablaber (BI and B. macrops karyogram of B. mocropr is a camera lucida interpretation of,meraphase plate displayed in the inset. m . metacentric, sm . submctaccnrric, st . sub- telocentric and a. acrocentric chromosomes. Scale bars equal 5 pm. RAB, MACHORDOM, PERDICES and GUECAN -+ ' &e colloidal silver nitrate method of KAHYOTYI'ES OF TtIItEE ccSMhL1.n HAKIIU?: classified according to LEVAN al. Les..)+ ef A.

2.

.? s... .I .i: ne number 2n = 48. The karyotype )airs of submetacentric chromosomes, d telomerically in one middle-sized ne number 2n = 50. The karyotype airs of submetacentric and 1 pair of les, NF = 98 (Fig. 1B). NORs are submetacentric pair (Figs. 2C, D). me number 2n = 50. The karyotype irs of submetacentric and 4 pairs of es, NF = 92 (Fig. 1C). The location d but interphase nuclei displayed 2 marked in Tab. 1.

lutionary diploid and polyploid, are ' small elements with their centromere i to a nearly terminal position. This is the effect of chromosame arm con- and dose colchicine treatment make :r of chromosome pairs to particular cover and in spite of difficulties in le field leading to their relative poor three barbels may however permit to 1- romosomal sets. This is more especial- id chance to analyze a limited number the first pair of metacentric chromo- ies was distinctly larger, and it can be

(CI: blnbcs (B)and B. macropr karyogram of E. e plate displayed in the inset. m. metacentric, sm itric chromosomes. Scale bars equal 5 gm.

Y" e.

3- ..." , ~ .^ .. ,'...... t .:...... :::, . ''( : ! . .~., , ,. . .:.. , . .,..

-_.-..-_ ..

504 and RAD, MAC!{OKDOh!, I'EKDICES GUECAN

- E)

.-

I'., Fig. 2 - The metaphase plares (A . G)&d inrerphase nuclci (E)C)of Burbur bigomei (A, B), B. ablobes D!snd E. macrop (E)stained sequcntiallv wich Giemsa and silver (B, or nonsequenIia]ly .C.-:h 1.4, [A, D) (c, ilver iE1. The NOR bearing chromosome pairs in ßarbus bigomei B) and B. &,ber D)are iramed and also enlarged in the insers. &&phase nuclei of B. mucrops display IWO positivc signals. ..i ,. and KAU, MACI{OKDOfvl, I'EKDICES CUECAN KAKYOTYPES OF THKEE cISMhLLn UAXMUS 305 considered as a ctmarkeru element for [hese species. Anyway, the deeper interspecific comparison of karyotypes either between African ((small), Barbus and Asian Punfius species, or within c,Sma\ln African barbels, is pracrically impossible because of the absence of chromosome banding data. The actual

s on the number and location of NORs is, therefore, the their karyotypes more precisely. We paired NORs. This could be the case barbels and such an information can

hexaploid African Barbus (work in determination of ploidy level by means of checking the number ng very simple silver staining pensive and can be performed .. diploid status of three new '. II)) African barbels usually classified into this ((catch-all) phylogenetically to the true polyploid genus Barbus sensu Asian genus Punlius (and/or r to chis African group of up. GoLußsro\j and

KRYSANOV(1993) clearly stated: (( idea to relate small African of Barbus with this group is not new ( in LÉVÊQUE and DACET1984). karyological data seem to be mos ence supporting chis hypothe-

92 except for B. kerslenii where NF er remarkable contrast concerns the distribution of the two d ome numbers 2n = 48 (A. nrerphasc nuclci (E) of Barbus bigom?i BI, 11. ablaber = with Gicmsa and silver nonsequ and 50. The formula 2n 48 was foun f Puntius which is about L48C) (A. (B,D) or e pairs in Rubus bigomei BI and B. abhbes (C, -, ".. 9% of the total number of karyotyped )hasr nuclci of ß. macrops display two positive sifinals. ((Barbusa representing almost 24% of ka cates that the 2n = 48 formula might be

. . - -. .. . ._...,.

..

itnll, MACIIORDOM, IJPIWICES wid CUECAN KARYOTYPES OF THREE (>BARBUS 307 eculations about relationships between Asian Punkius from a cytocaxonomical LÉVÊQUEC., PhUCY D.and TEIJGELSC. G.G., 1990. - Fairve des poissons d'eaux douces cl saumdtrer de dative and, undoubtedly, we do need 1'Alrique de 1'0uesf. Ed. by Lévtque, D. Paugy, and G.G.Tcugels, vol. 1, Paris, ORSTOM and Tervuren, MRAC. :y prinids . MAGTOONW. and ARAIR., 1989. - Karyotypes 01 /ive puntjrrs species an¿ one Cyclocheilichfysspecies IPisces, Cyprinidne) /rom Thailand. Bull. Natnl. Sci. MUS., 15: 167.175. and Guinea) and CNSHB (Guinea) for support in -, 1990. - Karyotypes of three cyprinid fishes, Osteochilus hasselti, O. hasselti, and Labcobarbus lineatus, /rom Thailand. Jpn. J. Ichthyol.. 36: iks are due to R. Bigorne and B. Hugueny for theirC. 483-487. . Diop for his scientific assistance on the field, OELLERMANL.K. and SKELTONP.H., J. 1990. - Hexaploidy in ye/locufirh species (Barbus, Pisces. lg field protocol of chromosome preparations, and Cyprinidae)from southem Africa. Fish Biol., 37: 105.1 15. ome suspensions. This study was supported by an POSTA., 1965. - Vergleichende Untersuchungen der Ch~mosomenzahlbei 5usswasser.Teleosleen. Z. .cch Republic No 645107 (P.R.). and a PICASSO 2001. Syst. Evol. . Forsch., 3: 47-93. ireign Of[iCKS (A.M., A.P., ]..F.C.). RAB P., 1981. - Karyotypes O/ two African barbels Barbus barilojdes and Barbus boiotaenia. Folia Zool.. 30: 181-190. RAB P. and ROTH P., 1989. - Chromosome studies in.European leuciscine fishes (Pisces, Cyprinidne). Karyotypes of Rutilus pigus virgo an¿ R. rutilus. Folia Zwl., 38: 239-245. RABP., OZOUF.COSTAZC. and BERREBIP., 1993. - Karyotypes, dishbution of centromdc heterochro- matin and po¡ymorphiSmur of NORs in Barbus nalis lis /rom roufhem France and easfem Sioorrkia: preliminary resuBr. Cahiers Ethol., 131 195.198. N J.F., 1990. Two diploid and ferroplaid, - lineages, RISHIS. and ADARASHDEEP KAURTHIND, 1992. mchromatin and NOR localization on the heichthycs. Cyprinidae). Aquat. Living Resour., 3: chromosomes oj Puntius sophore (Ham.) (Cy In: t#erspcctivcs in cytology and gene. ticsr, ed. by G.K. Manna and S.C. Roy, 7: if four cyprinid fishes from Thailand. Bull. Natnl. Sci. In: SKELTONP.H., TWEDDLED.and JACKSONP.B.N., cyprinids in Africa. <