Chromosome Research 8: 603^613, 2000. 603 # 2000 Kluwer Academic Publishers. Printed in the Netherlands

A biodiversity approach in the neotropical ¢sh, malabaricus. Karyotypic survey, geographic distribution of cytotypes and cytotaxonomic considerations

Luiz A. C. Bertollo1, Guassenir Gonc°alves Born2, Jorge A. Dergam3, Alberto Sergio Fenocchio4 & Orlando Moreira-Filho1 1 Departamento de Gene¨ tica e Evoluc°a¬o, Universidade Federal de Sa¬o Carlos, C.P. 676, 13565-905, Sa¬o Carlos, SP, ; Tel: (016) 260.8309; Fax: 55 16 261.2081; E-mail: [email protected]; 2 Departamento de Cieª ncias Morfobiolo¨ gicas, Universidade do Rio Grande, Rio Grande, RS, Brazil; 3 Departamento de Biologia , Universidade Federal de Vic°osa, Vic°osa, MG, Brazil; 4 Departamento de Gene¨ tica, Universidad Nacional de Misiones, Posadas,

Received 20 May 2000; received in revised form and accepted for publication by M. Schmid 10 July 2000

Key words: cytotaxonomy, geographic distribution, ¢sh, karyotypic diversity, sympatric cytotypes

Abstract

Hoplias malabaricus, a widely distributed neotropical freshwater ¢sh, shows a conspicuous karyotypic diversi¢cation. An overview of this diversity is presented here comprising several Brazilian populations, and some others from Argentina, and Surinam. Seven general cytotypes are clearly identi¢ed on the basis of their diploid number (2nˆ 39 to 2n ˆ 42), chromosomal morphology and sex chromosome systems, which can be clustered into two major karyotypic groups. This clustering suggests that karyotype structure would be more informative than the diploid number regarding cytotype relationships in this ¢sh group. While some cytotypes show a wide geographical distribution, some others appear to be endemic to speci¢c hydrographic basins. Sympatric cytotypes can occur without detection of hybrid forms; this situation points to a lack of gene £ow, a fact that is also reinforced by studies with genomic markers. The karyotypic data support the view that the nominal taxon H. malabaricus corresponds to a species complex comprising distinct evolutionary units, each with well-established chromosomal differences.

Introduction dence has pointed to the karyotypic diversity of H. malabaricus, showing interpopulational differ- The Erythrinidae family comprises some ences in the diploid number and chromosome neotropical ¢shes with a wide distribution in South morphology, as well as in sex chromosome systems America (Britski et al. 1986). Within this taxon, (Bertollo et al. 1979, 1983, Ferreira et al. 1989, Hoplias malabaricus is the most widespread Dergam & Bertollo 1990, Scavone et al. 1994, species. Although usually considered as a single Lopes & Fenocchio 1994, Bertollo et al. 1997a, biological species, the of this group is 1997b, Lopes et al. 1998, Bertollo & Mestriner poorly understood (Oyakawa 1990). Growing evi- 1998, Born & Bertollo 2000). 604 L. A. C. Bertollo et al.

Specimens with a putative hybrid karyotype submetacentrics, subtelocentrics, and acrocentrics have not been found when distinct chromosomal according to their morphology and arm ratios forms (cytotypes) are sympatric. Such is the case (Levan et al. 1964). in the rio Aguapey (northeastern Argentina) where specimens with 2n ˆ 40 and 2n ˆ 42 chromosomes are found together, without a 2n ˆ 41 intermediary Results and discussion form (Lopes et al. 1998). Similar situations are also observed in some Brazilian localities (Scavone Based on their macrostructure, we are able to et al. 1994, Bertollo et al. 1997a). determine seven basic karyotypic con¢gurations In this paper, we provide an overview of the referred to hereafter as cytotypes (Figures 2 & karyological diversity in Hoplias malabaricus, 3). Each of these cytotypes shows unique com- with the description of a new cytotype from the binations of chromosome numbers and/or Amazon basin and the comparative analysis of morphologies; some of their most remarkable the several known cytotypes, their geographic aspects are summarized here. The distribution distributions and sympatric regions, compiled of the cytotypes is based on the available data from our studies with this ¢sh group over the last up till now. two decades. The available karyotypic data for Hoplias malabaricus have led to the hypothesis Cytotype A that this ¢sh represents a species complex (Bertollo et al. 1986, Dergam & Bertollo 1990, Bertollo et al. Cytotype A presents 2n ˆ 42 meta- and sub- 1997a, Lopes et al. 1998); this is reinforced by the metacentric chromosomes in both sexes (Figures present study. 2A & 3A), without an apparent sex chromosome system. This cytotype shows a wide distribution, from northern to southern Brazil, Uruguay Materials and methods (Dergan, unpublished), and northern Argentina (Figure 1). Data are available from thirty-six distinct localities, thirty-two in Brazil, two in Argentina, Cytotype B one in Uruguay and one in Surinam (Table 1, Fig- ure 1). Samples sizes are also given in Table 1. Cytotype B also shows 2n ˆ 42 chromosomes both Karyological analyses were performed from in males and females, the general karyotypic struc- cephalic kidney cells, with either of the following ture being similar to cytotype A. However, this protocols: short-term culture cells (Fenocchio et cytotype can be differentiated by an exclusive al. 1991), Hank's saline treatment (Foresti et al. XX/XY sex chromosome system: females present 1993) or the conventional air-drying method two subtelocentric X chromosomes (pair 6); in (Bertollo et al. 1978). In the latter case, specimens the male karyotype, only one of this chromosome were previously treated with 0.05% colchicine sol- is identi¢ed, together with the Y chromosome, ution (1 ml/100 g body weight), 50^60 minutes probably the smallest submetacentric in the comp- before sacri¢ce. Some specimens were also ¢rst lement (Figures 2B and 3B). The X chromosome stimulated with a yeast solution as a mitogenic also carries ribosomal cistrons and can be (Lee & Elder 1980). Meiotic preparations were polymorphic in size (Born & Bertollo 2000). This basically obtained by the method of Kligerman cytotype has a geographic distribution restricted & Bloom (1977), according to the description in to a lake system in the Vale do Rio Doce, Minas Bertollo & Mestriner (1998). Gerais State ^ Brazil (Figure 1). The diploid number was determined for each specimen studied. The homologous pairs were Cytotype C arranged in decreasing order of size in the karyotype, and partial idiograms were drawn to Cytotype C is characterized by 2n ˆ 40 meta- and depict some relevant aspects of the karyotypes. submetacentric chromosomes, both in males and The chromosomes were classi¢ed as metacentrics, females, without an apparent sex chromosome Chromosomal diversity in Hoplias ¢sh 605

Table 1. Collection sites of Hoplias malabaricus, with the respective cytotypes and sample sizes. Locality Cytotype n References

1. Manaus (AM) ^ igarape¨ Mindu¨A22 2. Pocone¨ (MT) ^ lagoons: rio Bento Gomes A 1 2 3. Araguaiana (MT) ^ co¨ rrego Dois de Agosto A 2 2, 11 4. Treª sMarias(MG)^rioSa¬ o Francisco A 3 2 5. Reserva Ecolo¨ gica do Jata|¨ (SP) ^ lagoons: rio Mogi-Guac°u A 3 9 6. S. J. do Marinheiro (SP) ^ Aè gua Vermelha reservoir: rio Grande A 2 2, 14 7. Conceic°a¬ o das Alagoas (MG) ^ Volta Grande reservoir: rio Grande A 6 10 8. Juquia¨ (SP) ^ rio Juquia¨ A912 9. Itatinga (SP) ^ Jurumirim reservoir: rio Paranapanema A 7 2 10. Poc°o Preto (SC) ^ rio Iguac°u A 1 2 11. Gua|¨ba (RS) ^ rio Gua|¨ba A 12 2 12. Corrientes ^ Argentina ^ r|¨oAguapey A 21 3,4 13. Tacuarembo¨ ^Uruguai^r|¨o Negro A 6 15

14. Parque Florestal do Rio Doce (MG) ^ lagoons: rio Doce B 11 12, 16 15. Manaus (AM) ^ rio Negro; igarape¨ Mindu C 15 1, 2 16. Tucuru|¨ (PA) ^ rio Tocantins C 7 1 17. Porto Velho (RO) ^ rio Madeira C 6 1, 2 18. Aripuana¬ (MT) ^ rio Aripuana¬ C11 19. Cuiaba¨ (MT) ^ lagoons: rio Cuiaba¨ C141,2 20. Aragarc°as (GO) ^ lagoons: rio Araguaia C 7 2 21. Goia¨ s Velho (GO) C 2 1 22. Corumba¨ (MS) ^ rio Paraguai C 8 1, 2 23. Miranda (MS) ^ lagoons: rio Miranda C 10 1, 2 24. Misiones ^ Argentina ^ r|¨oParana¨ C183 25. Corrientes ^ Argentina ^ r|¨o Aguapey, r|¨o Riachuelo C 4 4, 5

26. Itirapina (SP) ^ Lobo reservoir: ribeira¬ o do Lobo D 40 2, 6 27. Sa¬ o Carlos (SP) ^ UFSCar reservoir: ribeira¬ o Monjolinho D 42 2, 7, 8 28. Pirassununga (SP) ^ rio Mogi-Guac°u D 9 2, 8 29. Ipeu¨ na (SP) ^ rio Passa-Cinco D 9 9 30. Piracicacaba (SP) ^ rio Piracicaba D 7 9 31. Novo Horizonte (SP) ^ rio Treª sPontes D 3 9 32. Mirassolaª ndia (SP) ^ ribeira¬ o Barra Grande D 3 2 33. Reserva Ecolo¨ gica Jata|¨ (SP) ^ lagoons: rio Mogi-Guac°u D 8 9 34. Conceic°a¬ o das Alagoas (MG) ^ Volta Grande reservoir: rio Grande D 4 10 35. Londrina (PR) ^ ribeira¬ oTreª sBocas D 7 2 36. Porto Trombetas (PA) ^ rio Trombetas E 1 2 37. Paramaribo ^ Surinam F 6 1 38. Tucurui (PA) ^ rio Tocantins F 2 1 39. Sa¬ oLuiz(MA) F 3 1 40. Natal (RN) ^ lagoa Redonda: N|¨zia Floresta F 4 1, 2 41. Recife (PE) F 4 1 42. Treª s Marias (MG) ^ rio Sa¬ o Francisco F 4 1, 7 43. Porto Trombetas (PA) ^ rio Trombetas G 1 2 44. Porto Velho (RO) ^ rio Madeira G 3 2 45. Aripuana¬ (MT) ^ rio Aripuana¬ G152,6 n ˆ Number of specimens studied; Brazilian States in brackets: AM: Amazonas; GO: Goia¨ s; MA: Maranha¬ o; MG: Minas Gerais; MS: Mato Grosso do Sul; MT: Mato Grosso; PA: Para¨ ; PE: Pernambuco; PR: Parana¨ ; RN: Rio Grande do Norte; RO: Rondoª nia; RS: Rio Grande do Sul; SC: Santa Catarina; SP: Sa¬ oPaulo. References: 1. Bertollo et al. (1997a); 2. Present paper; 3. Lopes & Fenocchio (1994); 4. Lopes et al. (1998); 5. Jorge (1995); 6. Bertollo et al. (1983); 7. Dergam & Bertollo (1990); 8. Bertollo et al. (1997b); 9. Scavone et al. (1994); 10. Dergam (1996); 11. Born (unpublished); 12. Bertollo et al. (1979); 13. Ferreira et al. (1989); 14. Cavallini & Bertollo (unpublished); 15. Dergam (unpublished); 16. Born & Bertollo (2000). 606 L. A. C. Bertollo et al. Chromosomal diversity in Hoplias ¢sh 607 differentiation (Figures 2C and 3C). This cytotype in one locality, Proto Trombetas (northern Brazil, is also widespread, occuring from northern Brazil rio Trombetas, Para¨ State ^ see Figure 1). to northeastern Argentina (Figure 1). Cytotype F Cytotype D Cytotype F, like cytotype C is characterized by 2n ˆ 40 meta- and submetacentric chromosomes, Cytotype D shows 2n ˆ 40 chromosomes in without differentiation between males and females, with a reduction to 2n ˆ 39 in males, females. Its distinctive feature is the presence of all of them meta- and submetacentrics (Figures a large-sized metacentric pair, the number 1 in 2D and 3D). This differentiation is due to a unique the karyotype, which constitutes also the largest multiple sex chromosome system for this cytotype, chromosome known for Hoplias malabaricus. with X X X X females and X X Ymales 1 1 2 2 1 2 The C-banding pattern clearly shows that these (Bertollo et al. 1983, 1997b). The Y chromosome characters do not result from heterochromatin is one of the largest in the complement, while accumulation (Bertollo et al. 1997a). The second the X and the putative X are similar to chromo- 1 2 pair of homologs is somewhat larger than the third somes number 6 and 20, respectively. During male and fourth ones, and is similar to pair no. 1 of meiosis, eighteen bivalents and a typical trivalent cytotype E. The remaining chromosomal pairs can be seen, the latter formed by the Y, X and 1 typically show a gradual reduction in size (Figures X chromosomes which can present hetero- 2 2F & 3F). This cytotype occurs from Surinam to synapsis during pachytene (Bertollo & Mestriner southeastern Brazil, with a preferential distri- 1998). This is not a widely distributed cytotype, bution in the oriental part of the continent (Figure apparently being limited to the Upper Parana¨ 1). hydrographic basin (Figure 1). Though the diploid number of the cytotypes A and B is higher than cytotypes C and D, these four Cytotype G cytotypes show karyotypes with a similar general appearance, the ¢rst four chromosomal pairs Cytotype G presents 2n ˆ 40 chromosomes in being larger than the remaining ones which are females, with an increase to 2n ˆ 41 in the males gradually reduced in size (Figures 2A^D & 3A^D). (Figures 2G & 3G). This heteromorphism results from a multiple sex chromosome system, with females XX and males XY1Y2 . The X chromo- Cytotype E some is a metacentric, the largest in the com- plement, while the Ys (an acrocentric and a Cytotype E represents a newly discovered submetacentric) are medium sized. During male chromosomal form, with a diploid number equal meiosis, nineteen bivalents and a typical trivalent to cytotype A (2n ˆ 42), and mostly biarmed can be seen, the latter corresponding to the X, chromosomes (meta- and submetacentrics). Y1 and Y2 chromosomes (Bertollo et al. 1983). However, an unique combination of characters Details of the synaptic behavior of these chromo- of this cytotype is the relatively large size of the somes are still unknown. The presence of the ¢rst chromosome pair, and the morphology of pair largest metacentric and the acrocentric chromo- 6, an acrocentric chromosome of rare occurrence somes is shared with cytotypes F and E. The size in Hoplias malabaricus (Figures 2E and 3E). of the chromosome pairs follows the general pat- Female karyotypes are still unknown. For the pre- tern described above for cytotype F. The sent, this karyotypic form has been observed only geographic distribution of this chromosomal form

Figure 1. Opposite. Distribution of Hoplias malabaricus cytotypes A (solid squares), B (open triangle), C (open circles), D (solid circles), E (open square), F (solid stars) and G (solid triangles) comprising 32 distinct regions in Brazil, 2 in Argentina, 1 in Uruguay and 1 in Surinam. The sampled regions in the Brazilian Sa¬ o Paulo (SP) state are shown in the detail. Sympatry among distinct cytotypes is indicated by the large open circles. 608 L. A. C. Bertollo et al.

Figure 2.(A^D) Conventional Giemsa-stained karyotypes of Hoplias malabaricus: cytotype A (female/male 2n ˆ 42, without heteromorphic sex chromosomes), cytotype B (female/male 2n ˆ 42, with an XX/XY sex chromosome system), cytotype C (female/male 2n ˆ 40, without heteromorphic sex chromosomes) and cytotype D (female 2n ˆ 40/male 2nˆ 39, with an

X1X1X2X2/X1X2Y multiple sex chromosome system). Bars ˆ 5 mm. Chromosomal diversity in Hoplias ¢sh 609

Figure 2.(E^G) Conventional Giemsa-stained karyotypes of Hoplias malabaricus: cytotype E (male 2n ˆ 42, with an unusual acrocentric pair 6), cytotype F (female/male 2n ˆ 40, without heteromorphic sex chromomomes and with a large metacentric pair

1), and cytotype G (female 2n ˆ 40/male 2n ˆ 41, with an XX/XY1Y2 multiple sex chromosome system). Bars ˆ 5 mm. 610 L. A. C. Bertollo et al.

Figure 3. Partial idiograms of the Hoplias malabaricus cytotypes A^G, showing some of their most remarkable characteristics. Chromosomal diversity in Hoplias ¢sh 611 appearstoberestrictedtoafewAmazoniansites the big metacentric pair 1 in cytotype F, but (Figure 1). now in a homozygous form among males and Although these seven cytotypes are diagnosed females. Additionally, chromosomal pairs 1, 2 by gross differences in chromosome morphology and 3 in cytotype E are comparable in shape and/or diploid numbers, minor karyotypic differ- and size to homologs 2, 3 and 4 in both cytotypes ences concerning the centromere location (median F and G, respectively (Figures 2E^G & 3E^G). to submedian positions) can be found among Thus, the remaining chromosome 4 in cytotype populations of widespread cytotypes. Though, E could be a good candidate for the in some cases, this should represent a technical rearrangements related to the acrocentric chromo- matter, in some others, it is a real change, as occurs some 6, considered above. Indeed, the Y1 and Y2 among distinct populations of the cytotype A chromosomes in the male cytotype G are (2n ˆ 42) which are being comparatively studied morphologically equivalent to chromosomes 6 (Born, in preparation). and 4 in cytotype E, respectively, representing Based on their general macrokaryotypic the two chromosomes without a homolog after features, we can recognize two major the heterozygous rearrangement in males. chromosomal groups in Hoplias malabaricus: Chromosome banding methods would be import- one composed of the cytotypes A, B, C and D ant to con¢rm these propositions. Indeed, such (cluster I), and another by the cytotypes E, F procedures were effective in clarifying some and G (cluster II). speci¢c problems, such as the rearrangements Despite the differences in the diploid numbers, related to the X1X2Y sex chromosome system dif- within cluster I cytotypes A and B present an over- ferentiation in cytotype D (Bertollo et al. 1997b). all karyotypic structure that agrees with cytotypes However, a general comparative banding pattern CandD(Figures2A^D&3A^D)andsothese analysis among the distinct cytotypes is not four karyotypic forms appear to show a close evol- practically viable at the moment, considering utionary relationship. Cytotype D (2n ˆ 40 the real dif¢culty in obtaining informative multiple females/2n ˆ 39 males) seems to be derived from bands on ¢sh chromosomes, as well as new a karyotype like cytotype C (2n ˆ 40 samples for this study in view of the geographical females/2n ˆ 40), a translocation giving rise to distance of several collection sites (Figure 1). amultipleX1X2Y sex chromosome system Meanwhile, these studies remain as future objec- (Bertollo et al. 1997a, 1997b). In a similar way, tives to be reached. the cytotype B, with a differentiated XX/XY For the moment, our clustering suggests that the sex chromosome system, can represent a derivative overall karyotypic structure, multiple sex chromo- form from a karyotype like cytotype A with no some systems, and chromosomes with unusual heteromorphic sex chromosomes. morphology would be more informative for Within cluster II, cytotypes F and G are similar phylogenetic relatedness among the chromosomal concerning the morphology and size of the ¢rst forms than the diploid number similarity. This big metacentric and the next ¢ve pairs of is also supported by mtDNA data (Dergam 1996). homologues (Figures 2F, G and 3F, G). Although molecular data strongly support a Additionally, the presence of an unusual monophyly for a set of populations with 2n ˆ 42 acrocentric, the Y1 chromosome in the male chromosomes (cytotype A) from south and cytotype G, suggests an af¢nity with cytotype E southeast Brazil, other allopatric populations of which also has a similar acrocentric member (pair this same cytotype show a closer phylogenetic 6, Figures 2E & 3E). If we assume the presence relatedness to 2n ˆ 40 populations of cytotype C of a multiple sex chromosome system as a good (Dergam op. cit.). taxonomic marker, cytotype G (female XX/male Sympatry of cytotypes is also indicated in Fig- XY1Y2) seems to be derived from a karyotype like ure 1. This is the ¢rst report for some localities cytotype E, where the acrocentric chromosome 6 of the Amazon basin: (1) igarape¨ do Mindu¨: would be related to karyotypic rearrangements Manaus-AM (cytotypes A and C), (2) rio giving rise to the big X metacentric chromosome. Trombetas: Proto Trombetas-PA (cytotypes E The same rearrangement could also originate and G), (3) rio Madeira: Porto Velho-RO 612 L. A. C. Bertollo et al.

(cytotypes C and G), and (4) rio Aripuana¬ : Acknowledgements Aripuana¬ -MT (cytotypes C and G). Others have already been described (Scavone et al. 1994, This work was supported by Conselho Nacional de Dergam 1996, Bertollo et al. 1997a, Lopes et al. Desenvolvimento Cient|¨ ¢co e Tecnolo¨ gico 1998). Despite sympatry, no evidence of gene £ow (CNPq). We are grateful to the Brazilian Embassy between cytotypes has been reported until now. and to the Agriculture and Fishery Ministry in Speci¢cally for cytotypes A and C, and for Surinam, and to Drs. E. Feldberg, H. Gurgel, J. cytotypes A and D, RAPD-PCR genomic markers A. Pereira, P. M. Galetti Jr, P. C. Venere, Y. Sato, are also congruent with lack of gene £ow (Dergam J. I. R. Porto, A. Schwarzbold, L. R. Malabarba, 1996), providing additional evidence for cytotypes L. Giuliano-Caetano, J. C. Garavelo, W. Garutti as distinct evolutionary units. and A. D. Carvalho for their help in supplying ¢sh. Although chromosomal rearrangements can play an important role in evolution, the major problem is to identify this role (Sites & Moritz References 1987). In spite of this question, the present status of H. malabaricus re£ects the ¢xation of several Bertollo LAC, Mestriner CA (1998) The X1X2Y sex chromo- chromosomal changes and a diversi¢cation within some system in the ¢sh Hoplias malabaricus (Pisces, this ¢sh group. So, our cytogenetic studies support Erythrinidae). II. Meiotic analyses. Chromosome Res 6: 141^147. the view that the nominal taxon Hoplias Bertollo LAC, Takahashi CS, Moreira-Filho O (1978) malabaricus is composed of several independent Cytotaxonomic considerations on (Pisces, biological units, characterized by unique ¢xed Erythrinidae). Brazil J Genet 1: 103^120. cytogenetic characters, a fact that attests the Bertollo LAC, Takahashi CS, Moreira-Filho O (1979) urgent need for a thorough taxonomic revision Karyotypic studies of two allopatric populations of the Hoplias (Pisces, Erythrinidae). Brazil J Genet 2: 17^37. of this `species complex'. Recent molecular data Bertollo LAC, Takahashi CS, Moreira-Filho O (1983) Multiple (RAPD-PCR genomic markers) also corroborate sex chromosomes in the genus Hoplias (Pisces, Erythrinidae). this view (Dergam et al. 1998). This diversity is Cytologia 48: 1^12. not surprising, taking into account that H. Bertollo LAC, Moreira-Filho O, Galetti Jr PM (1986) malabaricus isagroupwithawidegeographicdis- Cytogenetics and taxonomy: considerations based on chromosome studies of freshwater ¢sh. JFishBiol28: tribution. Thus, faced with this distribution, new 153^159. karyotypic forms or even different subgroups Bertollo LAC, Moreira-Filho O, Fontes MS (1997a) within the general cytotypes yet described cannot Karyotypic diversity and distribution in Hoplias malabaricus be ruled out. Even though the history of this (Pisces, Erythrinidae): Cytotypes with 2n ˆ 40 chromosomes. species is somewhat confused among the Brazil J Genet 20: 237^242. Bertollo LAC, Fontes MS, Fenocchio AS, Cano J (1997b) The Erythrinidae ¢sh, it is its proper type locality X1X2Y sex chromosome system in the ¢sh Hoplias (Oyakawa 1990). According to the original malabaricus. I. G-, C- and chromosome replication banding. description of Block, in 1794, probable type Chromosome Res 5: 493^499. localities would be Malabar or Tranquebar, but Born GG, Bertollo LAC (2000) An XX/XY sex chromosome these are coastal Indian sites (Oyakawa op. cit.); system in a ¢sh species, Hoplias malabaricus,witha polymorphic NOR-bearing X chromosome. Chromosome however, circumstancial evidence points to Res 8: 111^118. Surinam as the most likely origin of the type Britski HA, Sato Y, Rosa ABS (1986) Manual de Identi¢cac°a¬o (Dergam 1996). Thus, given the problematical de Peixes da Regia¬ o de Treª sMarias, 2nd edn. Bras|¨ lia: situation of this taxon, we cannot be sure that Codevasf. cytotype F from Surinam (Tijgerkreek, 58 km west Dergam JA (1996) Phylogeography and character congruence within the Hoplias malabaricus Bloch, 1794 (Erythrinidae, of Paramaribo) is the presumed one of the type , Ostariophysi) species complex. PhD thesis. locality, although it is a candidate for this. Colorado State University, USA. Dergam JA, Bertollo LAC (1990) Karyotypic diversi¢cation in Hoplias malabaricus (Osteichthyes, Erythrinidae) of the Sa¬ o Francisco and Alto Parana¨ basins, Brazil. Brazil J Genet 13: 755^766. Dergam JAè , Suzuki HI, Shibatta OA et al. (1998) Molecular biogeography of the Neotropical ¢sh Hoplias malabaricus Chromosomal diversity in Hoplias ¢sh 613

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