-- --I.--. al. 1991). These tw 1: (i.e. about 150 mm SL and m. . Vol. 48, n. 3.4: , barbels (SKELTON et

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1 -- --I.--. CARYOLOGIA ---I_-.-. Vol. 48, n. 3.4: , PETR Karyotypes of three <(small>> Barbus species (Cyprinidae) from Republic of Guinea (Western Africa) with a review on karyology of African small Barbus -{ RAB, ANNIE MACHORDOM *, ANABEL PERDICES and JEAN-FRANCOIS GUEGAN ** Institute of Animal Physiology and Genetics, Department of Genetics, Laboratory of Fish Genetics, Acndemy of Sciences of Czech Republic, Libechod Czech Republic; Consejo Superior de Investigaciones Cientificns, Museo Naciond de Ciencias N$rurales, Laborarory Biodiversitas, Madrid, Spain; ** Antenne ORSTOM. Laboratoire d Ichtfiyologie Génirale ct Appliquée, Muséum National d Histoire Naturelle, Paris Cedex 05, Fri&. I SUMMARY - Karyotyp up of asmalln African from the Republic of me (Zn) and chromo- 96, for 8 ablober 2~ 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 two distinct groups: ctsmall (about 230 s L. -. barbels (SKELTON et t al. 1991). These tw 1: (i.e. about 150 mm SL and m us Barbus involve in fact dargen (about 70 species) r especially in adult size cal (e.g. VERVOORT 1980; RAB 1981; V COLLARES-PEREIRA and MADEIRA 1990, LUBTSOV and KRYSANOV 1993; RAB et al. AN et aì 1995) investiga-..

2 300. KAU, MAClIOKDOM, PERDICES 2nd CUECAN tions demonstrated that only chose species wich evolutionary tetraploid (2s = 100) and hexaploid (2n = Ievels,,are assignable to the genus Barbus sensu stricto (and/or to che broader cntegbry of barbin lineage), while chose possessing a diploid chromosome numbqr (2~ = f 50) belong CO distinct lineages of cyprinine cyprinids (sensu HOW,ES 1991) such as Puntitrs and related genera (MAGTOON and ARAI 1989, 1990; ARAI and MAGTOON 1991; 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 BOUlenger, 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. KARI'L MATERIALS AND METHODS i The specimens used in this srudy represenr a parr of large sample during a joint French-Spaniard expedition in Republic of Guinea in All specimens karyotyped 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 MNCN) from the Mongo river (Upper Little Scarcies basin) ar Marela, 1 specimen (female1 of Barbus abbbes (No ) from the Kaba River (Upper Little Scarcies basin) at Kouloundala, and 1 specimen (sex unknown) of Barbus macrops (No ) from the Samou river (Konkouré basin) at Débélé. Chromosome preparations were made directly in field conditions according to the method described in DOUSSALI DE'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.- -

3 .? TABLE 1 - Comparison of current and previously published data on the chromosome number (2n). haploid karyotype characteristics and chromosome arm number (N F 1 of species of mnalln African Barbus and related genera < 2n Haploid No. and sex Species karyotype N.F. of material Locality Reference - examined Barbus uiuiparur 24' not reported - not Natal, aquarium stock POST 1965 Weber Barbus bariiiordes Boulenger, 1914 ** 48 16m+8sm 96 3 uv. Angola, aquarium stock RAß 1981 Barbus hoiotaenia Boulenger, m+l3sm 100 I& 29 Zaire, aquarium stock RAB 1981 Barbus anema Boulenger lm,sm+4a 92 3? Alvero R., Ethiopia KRYSANOV & GOLUIITSOV 1993 Barbus kenlenit 50 17m, sn+8a 84 17? L. Abaya, Harr R., KRYSANOV & Co~uursov 1993 Peter, 1868 Silc R.. Ethiopia ßurbur paludinorus 50 23m. sm + 2a 96 IS? Bulbula R, Ethiopia KRYSANOV & GoLuursov 1993 hers, 1852 KRYSANOV & GOt.UWlSt)V 1903 KRYSANOV & Go1 UII rwv 19') 3 Hare R, Kulfo R, KKYSANOV & GOI 1111 I WV 1993 Mongo R I Guinea this report Barbus bigomei Levequi, Teugels 48 9m+15sm 98 I.!, 2cf Kaba K.. Guinea this report. and Thys vin den Audenarde Barbus macrops ßoulenger, I9 I I 50 7m, t 14sm t 4st-a 92 I? Samour R., Guinea this report Caecobarbrir gecrtrii ßoulenger, I92 I.. ~ 50 6m. + 14sm x 39 cavc near Thysville. Zaire VERVOOKT 1980 Explanations. III. nlciaccntric. sm. submetacentric. il. acroceniric cliromosumcs; * only haploid chromosome nunlber rc1,orlcd; *' thc il:rinc is il syiiuiiymc of 8arhnr /urdolt~trrs Gunthcr, _.._.e-i CI O.-I m 5 : r F o 5 B R \r o i I i i i i I _ I..,...

4 I,..I 302 RAB, MACHOHDOM, PERDlCES an CUECAN.. '. organizer regions (NORs) were analyzed by *e colloidal silver nitrate method of HOWELL an BLACK (1980). Chromosomes wes classified according to LEVAN et al. (1964). -v....' i r, 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 ',... 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 conrraction 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 quality, the karyotype features of these three barbels may however permit to discuss about the composition of their chromosomal sets. This is more especially 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 chromosomes in the karyorypes of all three species was distinctly larger, and it can be Fig Karyotypes of Barbus bigomei (A), B. ablaber (BI and B. macrops (C); 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.

5 RAB, MACHORDOM, PERDICES and GUECAN -+ ' &e colloidal silver nitrate method of Les classified according to LEVAN ef al...)+ A. s 2..?....I.i: ne number 2n = 48. The karyotype )airs of submetacentric chromosomes, d telomerically in one middle-sized KAHYOTYI'ES OF TtIItEE ccsmhl1.n HAKIIU?: 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 conand 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 romosomal sets. This is more especialid chance to analyze a limited number the first pair of metacentric chromoies was distinctly larger, and it can be 1- blnbcs (B) and B. macropr (CI: karyogram of E. e plate displayed in the inset. m. metacentric, sm itric chromosomes. Scale bars equal 5 gm. Y " e. 3-

6 .".., ~.^.. t......:, '... :,.''(.... :!..~.,,,. :..,...,.... -_.-..-_ RAD, MAC!{OKDOh!, I'EKDICES and GUECAN E) -. - I'., Fig. 2 - The metaphase plares (A. G)&d inrerphase nuclci (E) of Burbur bigomei (A, B), B. ablobes.c. D! snd E. macrop (E) stained sequcntiallv wich Giemsa 1.4, C) and silver (B, D) or nonsequeniia]ly -:h ilver ie1. The NOR bearing chromosome pairs in ßarbus bigomei [A, B) and B. &,ber (c, D) are iramed and also enlarged in the insers. &&phase nuclei of B. mucrops display IWO positivc signals..i,.

7 KAU, MACI{OKDOfvl, I'EKDICES and 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 location of NORs is, therefore, the their karyotypes more precisely. We paired NORs. This could be the case barbels and such an information can s on the number and '... hexaploid African Barbus (work in by means of checking the number classified into this ((catch-all) polyploid genus Barbus sensu KRYSANOV (1993) clearly stated: (( Barbus with this group is not new ( karyological data seem to be mos determination of ploidy level ng very simple silver staining pensive and can be performed diploid status of three new II)) African barbels usually phylogenetically to the true Asian genus Punlius (and/or r to chis African group of up. GoLußsro\j and idea to relate small African of in LÉVÊQUE and DACET 1984). ence supporting chis hypothe- nrerphasc nuclci (E) of Barbus bigom?i (A. BI, 11. ablaber with Gicmsa L48 C) and silver (B, D) or nonsequ e pairs in Rubus bigomei (A. BI and B. abhbes (C, -, ".. )hasr nuclci of ß. macrops display two positive sifinals. 92 except for B. kerslenii where NF er remarkable contrast concerns the distribution of the two d ome numbers 2n = 48 and 50. The formula 2n = 48 was foun f Puntius which is about 9% of the total number of karyotyped ((Barbusa representing almost 24% of ka cates that the 2n = 48 formula might be _...,...

8

9 itnll, MACIIORDOM, IJPIWICES wid CUECAN eculations about relationships between Asian Punkius from a cytocaxonomical dative and, undoubtedly, we do need :y prinids. and Guinea) and CNSHB (Guinea) for support in iks are due to R. Bigorne and B. Hugueny for their. Diop for his scientific assistance on the field, C. lg field protocol of chromosome preparations, and ome suspensions. This study was supported by an.cch Republic No (P.R.). and a PICASSO ireign Of[iCKS (A.M., A.P., ]..F.C.). N J.F., Two lineages, diploid and ferroplaid, heichthycs. Cyprinidae). Aquat. Living Resour., 3: if four cyprinid fishes from Thailand. Bull. Natnl. Sci. rbus ne~lec~us problem and a review of cerfain Nilotic nics. Cyprinidae). Bull. British Mus. Natur. Hist. ;., AGNESE J.F., GUEGAN J.F. and MAcHOnDoI\I A., Barbus (Osleichthyes, Cyprinidae): on the coding o/ LesOur.. 3: 313'323. O. - Cyloraxonomic studies in Iberian cyprinids. Ill. rbirr Cuvier , with some reconsiderations oh Ihr ilogia. 43: , Une technique rapide d'analyse chromosomr. arcrrqirr. Cybium, 9: Frequency analyses 01 active NORs ln nuclei o/.ppi. Genet., 85: I. - Karyological study o/ some cyprinid species /rom irge and small Barbus of Africa. J. Fish Biol., 42: 445- KARYOTYPES OF THREE (<SMALL>> BARBUS 307 LÉVÊQUE C., PhUCY D. and TEIJGELS G.G., Fairve des poissons d'eaux douces cl saumdtrer de 1'Alrique de 1'0uesf. Ed. by C. Lévtque, D. Paugy, and G.G. Tcugels, vol. 1, Paris, ORSTOM and Tervuren, MRAC. MAGTOON W. and ARAI R., Karyotypes 01 /ive puntjrrs species an one Cyclocheilichfys species IPisces, Cyprinidne) /rom Thailand. Bull. Natnl. Sci. MUS., 15: , Karyotypes of three cyprinid fishes, Osteochilus hasselti, O. hasselti, and Labcobarbus lineatus, /rom Thailand. Jpn. J. Ichthyol.. 36: OELLERMAN L.K. and SKELTON P.H., Hexaploidy in ye/locufirh species (Barbus, Pisces. Cyprinidae) from southem Africa. J. Fish Biol., 37: POST A., Vergleichende Untersuchungen der Ch~mosomenzahl bei 5usswasser.Teleosleen. Z Syst. Evol.. Forsch., 3: RAB P., Karyotypes O/ two African barbels Barbus barilojdes and Barbus boiotaenia. Folia Zool.. 30: RAB P. and ROTH P., Chromosome studies in.european leuciscine fishes (Pisces, Cyprinidne). Karyotypes of Rutilus pigus virgo an R. rutilus. Folia Zwl., 38: RAB P., OZOUF.COSTAZ C. and BERREBI P., Karyotypes, dishbution of centromdc heterochromatin and po ymorphismur of NORs in Barbus nalis lis /rom roufhem France and easfem Sioorrkia: preliminary resubr. Cahiers Ethol., RISHI S. and ADARASH DEEP KAUR THIND, mchromatin and NOR localization on the chromosomes oj Puntius sophore (Ham.) (Cy In: t#erspcctivcs in cytology and gene. ticsr, ed. by G.K. Manna and S.C. Roy, 7: SKELTON P.H., TWEDDLE D. and JACKSON P.B.N., cyprinids in Africa. In: <<Cyprinid fishes. Systematics, biology and exploitation. I. Winfield and J. Nelson, pp Chapman & Hall. VASIL'EV V.P., Euolufronnry karyologv o, Nauka Press, Moscow (in Russian) VenvooxT A., Karyotype o/ Caecobarbus er Veleosfer Cypnnidae) Nucleus, Yu X., ZHOU T., LI K., LI Y. and ZHOU M., 1987 karyosysfemaftcs o/ cypnnrd frshes and a summary o/ fish chromosome studies in China. Yu X., ZHOU T, LI Y., LI K. and ZHOU M 198 oromes 0,' Chrnese /reshwatn/isher 179 pp., Science Press (in Chinese). ed 27 July 1994, accepted 18 June 1995 mnio I New evidence of hexaploidy in large D some considerations on the origin of hexaploidy. J. Fish rolled silver staining of nucleolus organizer regions with a