Journal of Biology (1990) 37, 105-1 15

Hexaploidy in yellowfish (, Pisces, ) from

L. K. OELLERMANNAND P. H. SICELTON* J.L.B. Smith Institute of Ichthyology, Private Bag 1015, Grahamstown 6140, Republic of

(Received 18 July 1989, Accepted I February 1990)

Five small-scaled yellowfish (large Burbus spp.) from southern Africa are shown to have modal 148 or 150chromosomes. Themajority ofcyprinid species have 2N = 50chromosomes, indicating that the yellowfish karyotype is hexaploid in origin. However, as there is no indication that the species are unisexual or that normal reproduction occurs by any means other than bisexual fertilization, the yellowfish karyotype is considered to have reverted to a diploid condition.

Key words: karyology; yellowfish; Burbus; hexaploidy; southern Africa.

I. INTRODUCTION The name ' yellowfish ' is given to several large-sized Burbus species in southern Africa (Jubb, 1967). The yellowfish fall into two groups on the basis of scale size. The group with smaller scales consists of five recognized species distributed in the system and adjacent drainage systems (Skelton, 1986). The second group, with larger scales, consists of two species, Barbus rnarequensis Smith, 1841 and Barbus codringtonii Boulenger, 1908, distributed in east coastal drainages from the Phongola River to the Zambezi River system. This study concerns the five small-scaled yellowfish species, viz. Barbus cupensis Smith, 1841 from the Olifants River system, Burbus aeneus (Burchell, 1822) and Burbus kimberleyensis Gilchrist & Thompson, 1913 from the Orange River system, Barbus polylepis Boulenger, 1907 from the Limpopo, lncomati and Phongola River systems, and Burbus natalensis Castelnau, 1861 from the rivers of Natal. Yellowfish generally is complicated by the characteristically large degree of intraspecific variation of the species (e.g. Gilchrist & Thompson, 19 13; Barnard, 1943; Groenewald, 1958; Jubb, 1967). Even at present the close similarity between certain species, e.g. B. aeneus and B. cupensis, and B. polylepsis and B. natalensis, and the fact that they are distributed allopatrically raises doubt on their taxonomic status. Yellowfish are popular angling species and most of the species have been successfully bred artificially and raised in captivity. Both B. aeneus and B. natalensis have been translocated beyond their native range in southern Africa (de Moor & Bruton, 1988). Understanding the karyological and genetic structure of these may help to resolve these taxonomic questions. The karyology of fishes has advanced little beyond the description of gross chromosome morphology. A change in a gene sequence causing a phenotypic variation in the species may not necessarily alter the gross shape of the chromo- some. Gold (1980) encountered this problem in the speciose cyprinid *Author to whom correspondence should be addressed. 105 0022-1 I12/90/070105+ 11 $03.00/0 0 1990 The Fisheries Society of the British Isles 106 L. K. OELLERMANN AND P. H. SKELTON

TABLEI. RUSI catalogue number, localities and sources of the small-scaled yellowfish specimens used for karyological studies

Species No. Cat. no. Locality Collector

~ ~ Burbus aeneus 15 28407/8 Kubusie R., L. K. Oellermann Kei R. system, Ciskei B. cupensis 15 28403/4 Olifants R., CDNEC* S.W.Cape B. kimberleyensis 8 28400/1 Le Roux Dam, L. K. Oellermann and Vaal R. Amalinda hatchery CDNEC B. nutalensis 11 28402 MgeniR. Natal Parks Board B. polylepis 4 28406 DorpsR., L. K. Oellermann Olifants-Limpopo R. system

~ *Cape Department of Nature and Environmental Conservation

Notropis: 22 of the species he studied displayed similar gross karyotypes. Although much of fish karyology is aimed at recording karyotypes, a growing number of workers are using karyology as a tool for studying taxonomy, as well as genetics, hybridization, , sex and sex reversal. Taxonomic studies on the small-scaled yellowfish, involving morphometric and meristic characters and osteology, are in progress (Oellermann, 1989). Karyology has been used with varied success in fish taxonomy (e.g. Campos & Hubbs, 1973; Gjedrem et al., 1977; Loudenslager & Thorgaard, 1979; Rukhkyan, 1984; Anemiya & Gold, 1988). There is a fast-growing field of data on cyprinid karyology inter- nationally, but so far no information has been published on African cyprinids. This study initiates the karyology of southern African cyprinids in order to provide (a) karyological descriptions for comparative studies, (b) cytotaxonomic data on the species, and (c) complementary data that could test hypothesized phylogenetic relationships based on anatomical or electrophoretic characters.

11. MATERIALS AND METHODS The small-scaled yellowfish specimens used for this study were mostly sexually immature juveniles, ranging from about 6 months to 2 years old. Specimens were collected in the field by means of seine nets and gill nets, or were provided by Provincial Nature Conservation authorities from hatchery-held stocks. The number of specimens used and their collection data is given in Table 1. All specimens used were preserved and are deposited in the fish collection of the J.L.B. Smith Institute of Ichthyology (RUSI). CHROMOSOME ISOLATION AND ANALYSIS Chromosomes were isolated using a modification of the method described by Kligerman & Bloom (1977) as follows. The specimen is injected with 0.01 ml gg ' body weight of 0.1 % colchicine and placed for 4-6 h in well-aerated water which is 2-5" C above the holding temperature. It is then killed by pithing and whole gill arches removed, teased apart and placed in 0.4% KCl hypotonic solution for 40 min. The gill tissue is fixed in Carnoy solution (I part glacial acetic acid : 3 parts absolute methanol) and then macerated in 50% acetic acid. A suspension of tissue is released from a bulb dropper onto a heated (40" C) glass slide from which it is withdrawn after 30 s. After drying, the slides are stained in fresh 5% Giemsa stain for 1&15 min, rinsed, dried and mounted. HEXAPLOIDY IN BARBUS SPP. 107

Preparations were viewed under a Nikon Optiphot compound light microscope. Photomicrographs were taken through an oil immersion x 100 objective lens. The large number and small size of the yellowfish chromosomes made them difficult to count. The small size also restricted our ability to subdivide the chromosomes into any classes other than bi-armed and uni-armed. Several authors have encountered similar problems (e.g. Schwartz & Maddock, 1986) and have concluded that further subdivision is completely arbitrary. The method used to count the chromosomes was as follows. A transparent sheet was placed over the photograph, and each chromosome was traced onto the sheet, using water-insoluble marker pens. The chromosomes were classed and coloured separately as either bi-armed or uni-armed and then counted. Each photographed chromosome spread was recounted at least three times. The ten most well defined chromosome spreads from each species were used to estimate the Fundamental Number (FN) of the karyotype as follows: FN=Z(n,)+n,, where n,is the number of bi-armed and nz is the number of uni-armed chromosomes. The final karyotype for each species was taken from the best defined chromosome spread [e.g. Fig. l(a)]. The chromosomes were cut from the photograph and paired according to their type and size into a photokaryotype. These karyotypes were traced on a light table and drawn for presentation [Fig. I(b)].

111. RESULTS A representative chromosome spread of B. kimberleyensis is shown in Fig. l(a) and the drawn karyotype for this species in Fig. l(b). The small-scaled yellowfish species can be divided into two groups on the basis of their diploid (2N) chromo- some number. Barbus capensis, B. natalensis and B. polylepis each have 150 chromosomes, while B. aeneus and B. kimberleyensis have 148 chromosomes. Chromosome number varied greatly, mostly due to chromosomal loss from the spreads (Fig. 2). The large number of chromosomes caused great difficulty in spreading the chromosomes sufficiently for counting without losing chromosomes from the spread. Difficulties experienced in obtaining accurate and consistent counts were overcome by applying the counting method described in the ' Materials and Methods ' section. Counts from the slides under the microscope were usually at least 10 chromosomes less than those from the photographs. Mistaken chromo- some identification (e.g. a bi-armed chromosome counted as two uni-armed chromosomes) and chromosome imports from other spreads probably resulted in chromosome counts higher than the modal number. No sexual dimorphism was observed within the small-scaled yellowfish karyo- types. Any major dimorphism in chromosome pairs was probably masked by the multiple chromosomes introduced by polyploidy. The FN for each species was relatively similar, and ranged from 208 in B. cupensis to 196 for B. aeneus (Table 11). The modal chromosome arm numbers (Table 11) differed marginally between the species but must, at this stage, be accepted with caution due to the difficulties in obtaining clear chromosome impressions. The variation in individual counts of bi-armed chromosomes in 10 spreads from which the modal number was derived is given in Table 111.

IV. DISCUSSION The extremely high number of chromosomes is a striking feature for the yellow- fishes. An extensive literature survey on the karyotypes of cyprinids (see NOllaWS 'H 'd (INV NNVNXIB71BO ')I '1 80 I HEXAPLOIDY IN BARBUS SPP. 109

30401

30 -

20 -

10 -

0 I I

Chromosome number 50--I (e

40 t

10 l---4L.090 100 110 120 130 140 150 160 Chromosome number

FIG.2. Percentage spreads with various chromosome numbers for (a) Barbus aeneus (n= 36 spreads), (b) B. capensis (n= 40 spreads), (c) B. kimbedeyensis (n= 33 spreads), (d) B. nafa1ensi.s(n = 28 spreads) and (e) B. po/y/epis (n = 3 I spreads). was duplicated in the cyprinid species which they used as examples of the tetraploid group. Thus, at some stage during their evolution these species experienced a polyploidic event (Mayr et al., 1986) which doubled their chromosome numbers to produce a tetraploid condition. The term ' polyploidic event ' is used to include all possible ways in which polyploidy may have been induced i.e. environmental changes and hybridization. Cyprinid species included in the tetraploid group occur in the genera Acrossochei- lus, Aulopyge, Barbodes, Barbus, Carassioides, , Cyprinus, Neolissocheilus, 110 L. K. OELLERMANN AND P. H. SKELTON

TABLE11. Estimated number of chromosomes, bi-armed chromo- somes, uni-armed chromosomes and Fundamental Number for five small-scaled yellowfish species

Species 2N Bi-armed Uni-armed FN

B. aeneus 148 48 100 196 B. capensis 150 58 92 208 B. kimberleyensis 148 56 92 204 B. natalensis 150 50 100 200 B. polylepis 150 56 94 206

TABLE111. Number of bi-armed chromosomes in 10 spreads for five small-scaled yellowfish species. Abbreviations: AEN, Barbus aeneus; CAP, B. capensis; KIM, B. kimberleyensis; NAT, B. natalensis; POL, B. polylepis. Modal chromosome count of each species is in parentheses

Spread AEN (148) CAP (1 50) KIM (1 48) NAT (1 50) POL (1 50)

1 41 44 46 40 48 2 41 46 52 42 54 3 46 48 52 42 54 4 46 50 54 48 56 5 48 54 55 50 56 6 48 58 56 50 56 7 49 58 56 52 56 8 52 58 56 52 56 9 58 68 57 53 58 10 60 70 58 54 58

Mode 48 58 56 50 56

Percocypris, Pseudobarbus, Schizothoraichthys, Sinocyclocheilus, Spinibarbus and Tor (Mayr et al., 1986; Oellermann, 1989). Tetraploidy can arise from incomplete meiosis I1 during the development of male and female gametes (autotetraploidy). Spermatozoon and ovum would each contain a diploid complement of chromo- somes, and in combination produce a tetraploid zygote. Don & Avtalion (1988) produced viable tetraploid Tilapia by giving the fertilized eggs a temperature shock (a sharp decrease in temperature). Besides the small-scaled yellowfish species studied here, the third cluster of cyprinid chromosome number of around 150 (Fig. 3) includes Barbus marequensis and Varicorhinus nelspruitensis (Oellermann, 1989) and certain varieties of Carassius auratus () and species of the Asian genus Schizothorax (Zan et al., 1986). All these species have the order of three times the chromosome number of a diploid cyprinid and are therefore of hexaploidic origin. In Fig. 4 we attempt to depict the possible ways in which hexaploidy may develop. At least four pathways are possible: the first three involve autopoly- HEXAPLOIDY IN BARBUSSPP. 111

(/7/+------

Diploid chromosome number

FIG.3. The diploid chromosome number ofcyprinid species,determined from the literature (n = 3 15 species).

AUTOPOLY PLOlDY

TETRAPLOID 1 TRTID Triploidic event Tetroploidic event Heroploidic event J. J .1 HEXAPLOID HEXAPLOID HEXAPLOID

ALLOPOLY PLOlDY

DIPLOID

\UNSTABLE TRiPLOiD ()

Spontaneous chromosome.1 doubling

HEXAPLOIDJ. FIG.4. Possible ways of origin of hexaploidy in the small-scaled yellowfish species. ploidy, and the fourth is based on allopolyploidy. The first considers a tetraploid species which experiences a triploidic event, giving rise to a hexaploid genome. This is unlikely to occur, as it would lead to either a sterile population or a species that reproduces through gynogenesis: e.g. Murayama et al. (1986) describe the gynogenesis of a population of triploid Carassius auratus lungsdorji. No unisexual reproductive behaviour has been reported in the yellowfish. Gynogenesis in a naturally occurring population should eventually lead to the of male 112 L. K. OELLERMANN AND P. H. SKELTON triploid individuals. The diploid male line could only survive in the presence of diploid bisexual females. Ecological studies on yellowfish (e.g. Mulder, 1973; Tomasson et al., 1984) and captive (artificial) breeding programmes involving the yellowfish (e.g. Van der Merwe, 1981; Wright & Coke, 1975) show that the five species reproduce bisexually. Zan et al. (1986) reported a population of hexaploid Cyprinus carpio bibelio reproducing gynogenetically, which shows that this path- way has been used at least once. There is, however, a high ratio of hexaploid males present in the population. The sperm has a 50% reduction in DNA content as compared with the male somatic cells and therefore must be undergoing full meiosis. If meiosis redevelops in the female gametes, it may be possible for the hexaploid population to revert to bisexual reproduction. The second possible pathway depicted in Fig. 4 considers a triploid species which experiences chromosome doubling (due to a tetraploidic event) as a means of overcoming sterility due to aneuploidy. The chromosome doubling gives rise to a hexaploid complement of chromosomes. This pathway could be an alternative to gynogenesis for a triploid species. The third possibility involves a single-step increase in ploidy from diploidy to hexaploid. Such a large jump in ploidy level is unlikely. Artificial induction of hexaploidy through environmental manipulation has not been reported in cyprinids. The fourth possible, and perhaps most plausible, way that the yellowfish attained hexaploidy involves a combination of both autopolyploidy and allopoly- ploidy. If a diploid and a tetraploid species hybridized, the product would be an unstable triploid hybrid. Should the hybrid undergo spontaneous chromosome doubling, the result would be a hexaploid. Spontaneous chromosome doubling after hybridization appears to be fairly common in plants (Gibby, 1981) but has not, as yet, been reported in fishes. This possibility seems to be the most likely way for the small-scaled yellowfish to have become hexaploid, because hybridization is common in cyprinids and spontaneous chromosome doubling ensures that is not limited by aneuploidy. Polyploidy has played an important role in evolution, as it provides redundant gene loci (Becaket al., 1966). Redundant gene loci are more likely to escape natural selective pressures conserving active loci, and can therefore more easily accumulate mutations (Wolf et al., 1969). Tetraploids (which originate as diploids) appear gradually to revert to a diploid genome, either by divergence of DNA sequences or by chromosomal rearrangements. Ohno et al. (1967) termed gradual reversion from a quadrivalent to a bivalent state in the autotetraploid genome ' the process of diploidization '. Structural heterogeneity is necessary among the four original homologues (Shaver, 1963), as the preferential separation of one quadrivalent into two bivalents is a prerequisite for functional diversification and diploidization (Ohno, 1970). Diploidization is relatively far advanced in evolutionary polyploid cyprinids (Klose et al., 1969). Hexaploid species have six chromosome sets, effectively tripling the possible combinations of dominant and recessive genes and greatly increasing its heterozy- gosity. This large gene reservoir may explain the plastic nature of the intraspecific yellowfish phenotype, and could also account for their resilience to major pheno- typic changes in the face of diverse habitats. The advantages of polyploidy for cyprinids has been summed up by Uyeno & Smith (1972) as larger size, a longer life, HEXAPLOIDY IN BARBUSSPP. 113 faster growth and greater ecological adaptability than the majority of cyprinids. The yellowfish species and other southern African hexaploids (Barbusmarequensis and Vuricorhinus nelspruitensis) are all relatively large, long-lived and ecologically flexible species (e.g. Tomasson et al., 1984). It is possible that hexaploidy is the basis of the characteristic life-history traits of these species. A combination of Robertsonian translocations and pericentric inversions (the inversion of a length of chromosome, including the centromere, after two breaks occur in the chromosome) can lead to numerous rearrangements in the positions of the centromeres on the chromosomes and in chromosome numbers (Denton, 1973). Such chromosome aberrations may have led to the different karyotypes of the small-scaled yellowfish. The primitive hexaploid karyotype for the yellowfish is probably 150 chromosomes, as this number is characteristic of the majority of the species and can be derived directly from the modal cyprinid diploid number of 50 chromosomes. Only a single centric fusion of one chromosome is necessary to explain the 148 chromosomes of the Orange- species B. aeneus and B. kimberleyensis. The problem of effective sub-classification of the very small yellowfish chromo- somes has already been mentioned. Zan et al. (1986) found it impossible to deter- mine karyotypic differences between the hexaploid Schizothorax species because of the large intraspecific variations in chromosome shape. Further investigation into the proportions of bi-armed chromosomes and differences in FN of the yellowfish is required, as the presented data (Table 11) may be affected by artifacts of chromo- some preparation or misinterpretations due to small size. Therefore, at present , our chromosome data do not allow for reliable interpretation of relationships between the yellowfish species. However, it is reasonable to conclude that hexa- ploidy is sufficiently rare among cyprinids to indicate that it may be a shared derived characteristic (synapomorphy), supporting an hypothesis of monophyletic origin of the southern African yellowfish and their allies.

This study formed part of a M.Sc. thesis submitted to Rhodes University, Grahamstown. Dr G. Coulter provided advice and the work has been improved by the comments of the examiners. Specimens used in this study were provided by Drs A. Bok, K. C. D. Hamman, Mr C. Benade, Mr S. Thorne (all from the Cape Department of Nature and Environmental Conservation), Mr T. Pike (Natal Parks Board) and Mr N. van Loggerenberg (Transvaal Division of Nature Conservation). Mr R. Stobbs and Mr A. Scholtz provided technical assistance. Certain figures were prepared for publication by D. Voorveld. Mr 0. Gon kindly read and improved the manuscript.

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