Chromosome Research 8: 603^613, 2000.
# 2000 Kluwer Academic Publishers. Printed in the Netherlands
603
A biodiversity approach in the neotropical Erythrinidae ¢sh, Hoplias
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 Genëtica e Evoluc°a¬o, Universidade Federal de Sa¬o Carlos, C.P. 676, 13565-905,
Sa¬o Carlos, SP, Brazil; Tel: (016) 260.8309; Fax: 55 16 261.2081; E-mail: [email protected];
2
Departamento de Cieªncias Morfobiolögicas, Universidade do Rio Grande, Rio Grande, RS, Brazil;
3
Departamento de Biologia Animal, Universidade Federal de Vic°osa, Vic°osa, MG, Brazil; 4 Departamento
de Genëtica, Universidad Nacional de Misiones, Posadas, Argentina
Received 20 May 2000; received in revised form and accepted for publication by M. Schmid 10 July 2000
Key words: cytotaxonomy, geographic distribution, Hoplias malabaricus ¢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, Uruguay and Surinam. Seven general cytotypes are clearly identi¢ed
on the basis of their diploid number (2n ˆ 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
The Erythrinidae family comprises some
neotropical ¢shes with a wide distribution in South
America (Britski et al. 1986). Within this taxon,
Hoplias malabaricus is the most widespread
species. Although usually considered as a single
biological species, the taxonomy of this group is
poorly understood (Oyakawa 1990). Growing evi-
dence has pointed to the karyotypic diversity of
H. malabaricus, showing interpopulational differences in the diploid number and chromosome
morphology, as well as in sex chromosome systems
(Bertollo et al. 1979, 1983, Ferreira et al. 1989,
Dergam & Bertollo 1990, Scavone et al. 1994,
Lopes & Fenocchio 1994, Bertollo et al. 1997a,
1997b, Lopes et al. 1998, Bertollo & Mestriner
1998, Born & Bertollo 2000).
604
Specimens with a putative hybrid karyotype
have not been found when distinct chromosomal
forms (cytotypes) are sympatric. Such is the case
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
form (Lopes et al. 1998). Similar situations are
also observed in some Brazilian localities (Scavone
et al. 1994, Bertollo et al. 1997a).
In this paper, we provide an overview of the
karyological diversity in Hoplias malabaricus,
with the description of a new cytotype from the
Amazon basin and the comparative analysis of
the several known cytotypes, their geographic
distributions and sympatric regions, compiled
from our studies with this ¢sh group over the last
two decades. The available karyotypic data for
Hoplias malabaricus have led to the hypothesis
that this ¢sh represents a species complex (Bertollo
et al. 1986, Dergam & Bertollo 1990, Bertollo et al.
1997a, Lopes et al. 1998); this is reinforced by the
present study.
Materials and methods
Data are available from thirty-six distinct
localities, thirty-two in Brazil, two in Argentina,
one in Uruguay and one in Surinam (Table 1, Figure 1). Samples sizes are also given in Table 1.
Karyological analyses were performed from
cephalic kidney cells, with either of the following
protocols: short-term culture cells (Fenocchio et
al. 1991), Hank's saline treatment (Foresti et al.
1993) or the conventional air-drying method
(Bertollo et al. 1978). In the latter case, specimens
were previously treated with 0.05% colchicine solution (1 ml/100 g body weight), 50^60 minutes
before sacri¢ce. Some specimens were also ¢rst
stimulated with a yeast solution as a mitogenic
(Lee & Elder 1980). Meiotic preparations were
basically obtained by the method of Kligerman
& Bloom (1977), according to the description in
Bertollo & Mestriner (1998).
The diploid number was determined for each
specimen studied. The homologous pairs were
arranged in decreasing order of size in the
karyotype, and partial idiograms were drawn to
depict some relevant aspects of the karyotypes.
The chromosomes were classi¢ed as metacentrics,
L. A. C. Bertollo et al.
submetacentrics, subtelocentrics, and acrocentrics
according to their morphology and arm ratios
(Levan et al. 1964).
Results and discussion
Based on their macrostructure, we are able to
determine seven basic karyotypic con¢gurations
referred to hereafter as cytotypes (Figures 2 &
3). Each of these cytotypes shows unique combinations of chromosome numbers and/or
morphologies; some of their most remarkable
aspects are summarized here. The distribution
of the cytotypes is based on the available data
up till now.
Cytotype A
Cytotype A presents 2n ˆ 42 meta- and submetacentric chromosomes in both sexes (Figures
2A & 3A), without an apparent sex chromosome
system. This cytotype shows a wide distribution,
from northern to southern Brazil, Uruguay
(Dergan, unpublished), and northern Argentina
(Figure 1).
Cytotype B
Cytotype B also shows 2n ˆ 42 chromosomes both
in males and females, the general karyotypic structure being similar to cytotype A. However, this
cytotype can be differentiated by an exclusive
XX/XY sex chromosome system: females present
two subtelocentric X chromosomes (pair 6); in
the male karyotype, only one of this chromosome
is identi¢ed, together with the Y chromosome,
probably the smallest submetacentric in the complement (Figures 2B and 3B). The X chromosome
also carries ribosomal cistrons and can be
polymorphic in size (Born & Bertollo 2000). This
cytotype has a geographic distribution restricted
to a lake system in the Vale do Rio Doce, Minas
Gerais State ^ Brazil (Figure 1).
Cytotype C
Cytotype C is characterized by 2n ˆ 40 meta- and
submetacentric chromosomes, both in males and
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.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
A
A
A
A
A
A
A
A
A
A
A
A
A
2
1
2
3
3
2
6
9
7
1
12
21
6
2
2
2, 11
2
9
2, 14
10
12
2
2
2
3, 4
15
14. Parque Florestal do Rio Doce (MG) ^ lagoons: rio Doce
B
11
12, 16
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
Manaus (AM) ^ rio Negro; igarapë Mindu
Tucuru|¨ (PA) ^ rio Tocantins
Porto Velho (RO) ^ rio Madeira
Aripuana¬ (MT) ^ rio Aripuana¬
Cuiabä (MT) ^ lagoons: rio Cuiabä
Aragarc°as (GO) ^ lagoons: rio Araguaia
Goiäs Velho (GO)
Corumbä (MS) ^ rio Paraguai
Miranda (MS) ^ lagoons: rio Miranda
Misiones ^ Argentina ^ r|¨ o Paranä
Corrientes ^ Argentina ^ r|¨ o Aguapey, r|¨ o Riachuelo
C
C
C
C
C
C
C
C
C
C
C
15
7
6
1
14
7
2
8
10
18
4
1,
1
1,
1
1,
2
1
1,
1,
3
4,
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
Itirapina (SP) ^ Lobo reservoir: ribeira¬o do Lobo
Sa¬o Carlos (SP) ^ UFSCar reservoir: ribeira¬o Monjolinho
Pirassununga (SP) ^ rio Mogi-Guac°u
Ipeüna (SP) ^ rio Passa-Cinco
Piracicacaba (SP) ^ rio Piracicaba
Novo Horizonte (SP) ^ rio Treªs Pontes
Mirassolaªndia (SP) ^ ribeira¬o Barra Grande
Reserva Ecolögica Jata|¨ (SP) ^ lagoons: rio Mogi-Guac°u
Conceic°a¬o das Alagoas (MG) ^ Volta Grande reservoir: rio Grande
Londrina (PR) ^ ribeira¬o Treªs Bocas
D
D
D
D
D
D
D
D
D
D
40
42
9
9
7
3
3
8
4
7
2, 6
2, 7, 8
2, 8
9
9
9
2
9
10
2
36. Porto Trombetas (PA) ^ rio Trombetas
E
1
2
37.
38.
39.
40.
41.
42.
F
F
F
F
F
F
6
2
3
4
4
4
1
1
1
1, 2
1
1, 7
G
G
G
1
3
15
2
2
2, 6
Manaus (AM) ^ igarapë Mindü
Poconë (MT) ^ lagoons: rio Bento Gomes
Araguaiana (MT) ^ cörrego Dois de Agosto
Treªs Marias (MG) ^ rio Sa¬o Francisco
Reserva Ecolögica do Jata|¨ (SP) ^ lagoons: rio Mogi-Guac°u
S. J. do Marinheiro (SP) ^ Aègua Vermelha reservoir: rio Grande
Conceic°a¬o das Alagoas (MG) ^ Volta Grande reservoir: rio Grande
Juquiä (SP) ^ rio Juquiä
Itatinga (SP) ^ Jurumirim reservoir: rio Paranapanema
Poc°o Preto (SC) ^ rio Iguac°u
Gua|¨ ba (RS) ^ rio Gua|¨ ba
Corrientes ^ Argentina ^ r|¨ o Aguapey
Tacuarembö ^ Uruguai ^ r|¨ o Negro
Paramaribo ^ Surinam
Tucurui (PA) ^ rio Tocantins
Sa¬o Luiz (MA)
Natal (RN) ^ lagoa Redonda: N|¨ zia Floresta
Recife (PE)
Treªs Marias (MG) ^ rio Sa¬o Francisco
43. Porto Trombetas (PA) ^ rio Trombetas
44. Porto Velho (RO) ^ rio Madeira
45. Aripuana¬ (MT) ^ rio Aripuana¬
2
2
2
2
2
5
n ˆ Number of specimens studied; Brazilian States in brackets: AM: Amazonas; GO: Goiäs; MA: Maranha¬o; MG: Minas Gerais; MS:
Mato Grosso do Sul; MT: Mato Grosso; PA: Parä; PE: Pernambuco; PR: Paranä; RN: Rio Grande do Norte; RO: Rondoªnia; RS:
Rio Grande do Sul; SC: Santa Catarina; SP: Sa¬o Paulo.
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
differentiation (Figures 2C and 3C). This cytotype
is also widespread, occuring from northern Brazil
to northeastern Argentina (Figure 1).
607
in one locality, Proto Trombetas (northern Brazil,
rio Trombetas, Parä State ^ see Figure 1).
Cytotype F
Cytotype D
Cytotype D shows 2n ˆ 40 chromosomes in
females, with a reduction to 2n ˆ 39 in males,
all of them meta- and submetacentrics (Figures
2D and 3D). This differentiation is due to a unique
multiple sex chromosome system for this cytotype,
with X1 X1 X2 X2 females and X1 X2 Y males
(Bertollo et al. 1983, 1997b). The Y chromosome
is one of the largest in the complement, while
the X1 and the putative X2 are similar to chromosomes number 6 and 20, respectively. During male
meiosis, eighteen bivalents and a typical trivalent
can be seen, the latter formed by the Y, X1 and
X2 chromosomes which can present heterosynapsis during pachytene (Bertollo & Mestriner
1998). This is not a widely distributed cytotype,
apparently being limited to the Upper Paranä
hydrographic basin (Figure 1).
Though the diploid number of the cytotypes A
and B is higher than cytotypes C and D, these four
cytotypes show karyotypes with a similar general
appearance, the ¢rst four chromosomal pairs
being larger than the remaining ones which are
gradually reduced in size (Figures 2A^D & 3A^D).
Cytotype E
Cytotype E represents a newly discovered
chromosomal form, with a diploid number equal
to cytotype A (2n ˆ 42), and mostly biarmed
chromosomes (meta- and submetacentrics).
However, an unique combination of characters
of this cytotype is the relatively large size of the
¢rst chromosome pair, and the morphology of pair
6, an acrocentric chromosome of rare occurrence
in Hoplias malabaricus (Figures 2E and 3E).
Female karyotypes are still unknown. For the present, this karyotypic form has been observed only
Cytotype F, like cytotype C is characterized by
2n ˆ 40 meta- and submetacentric chromosomes,
without differentiation between males and
females. Its distinctive feature is the presence of
a large-sized metacentric pair, the number 1 in
the karyotype, which constitutes also the largest
chromosome known for Hoplias malabaricus.
The C-banding pattern clearly shows that these
characters do not result from heterochromatin
accumulation (Bertollo et al. 1997a). The second
pair of homologs is somewhat larger than the third
and fourth ones, and is similar to pair no. 1 of
cytotype E. The remaining chromosomal pairs
typically show a gradual reduction in size (Figures
2F & 3F). This cytotype occurs from Surinam to
southeastern Brazil, with a preferential distribution in the oriental part of the continent (Figure
1).
Cytotype G
Cytotype G presents 2n ˆ 40 chromosomes in
females, with an increase to 2n ˆ 41 in the males
(Figures 2G & 3G). This heteromorphism results
from a multiple sex chromosome system, with
females XX and males XY1 Y2 . The X chromosome is a metacentric, the largest in the complement, while the Ys (an acrocentric and a
submetacentric) are medium sized. During male
meiosis, nineteen bivalents and a typical trivalent
can be seen, the latter corresponding to the X,
Y1 and Y2 chromosomes (Bertollo et al. 1983).
Details of the synaptic behavior of these chromosomes are still unknown. The presence of the
largest metacentric and the acrocentric chromosomes is shared with cytotypes F and E. The size
of the chromosome pairs follows the general pattern described above for cytotype F. The
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
appears to be restricted to a few Amazonian sites
(Figure 1).
Although these seven cytotypes are diagnosed
by gross differences in chromosome morphology
and/or diploid numbers, minor karyotypic differences concerning the centromere location (median
to submedian positions) can be found among
populations of widespread cytotypes. Though,
in some cases, this should represent a technical
matter, in some others, it is a real change, as occurs
among distinct populations of the cytotype A
(2n ˆ 42) which are being comparatively studied
(Born, in preparation).
Based on their general macrokaryotypic
features, we can recognize two major
chromosomal groups in Hoplias malabaricus:
one composed of the cytotypes A, B, C and D
(cluster I), and another by the cytotypes E, F
and G (cluster II).
Despite the differences in the diploid numbers,
within cluster I cytotypes A and B present an overall karyotypic structure that agrees with cytotypes
C and D (Figures 2A^D & 3A^D) and so these
four karyotypic forms appear to show a close evolutionary relationship. Cytotype D (2n ˆ 40
females/2n ˆ 39 males) seems to be derived from
a karyotype like cytotype C (2n ˆ 40
females/2n ˆ 40), a translocation giving rise to
a multiple X1 X2 Y sex chromosome system
(Bertollo et al. 1997a, 1997b). In a similar way,
the cytotype B, with a differentiated XX/XY
sex chromosome system, can represent a derivative
form from a karyotype like cytotype A with no
heteromorphic sex chromosomes.
Within cluster II, cytotypes F and G are similar
concerning the morphology and size of the ¢rst
big metacentric and the next ¢ve pairs of
homologues (Figures 2F, G and 3F, G).
Additionally, the presence of an unusual
acrocentric, the Y1 chromosome in the male
cytotype G, suggests an af¢nity with cytotype E
which also has a similar acrocentric member (pair
6, Figures 2E & 3E). If we assume the presence
of a multiple sex chromosome system as a good
taxonomic marker, cytotype G (female XX/male
XY1 Y2 ) seems to be derived from a karyotype like
cytotype E, where the acrocentric chromosome 6
would be related to karyotypic rearrangements
giving rise to the big X metacentric chromosome.
The same rearrangement could also originate
611
the big metacentric pair 1 in cytotype F, but
now in a homozygous form among males and
females. Additionally, chromosomal pairs 1, 2
and 3 in cytotype E are comparable in shape
and size to homologs 2, 3 and 4 in both cytotypes
F and G, respectively (Figures 2E^G & 3E^G).
Thus, the remaining chromosome 4 in cytotype
E could be a good candidate for the
rearrangements related to the acrocentric chromosome 6, considered above. Indeed, the Y1 and Y2
chromosomes in the male cytotype G are
morphologically equivalent to chromosomes 6
and 4 in cytotype E, respectively, representing
the two chromosomes without a homolog after
the heterozygous rearrangement in males.
Chromosome banding methods would be important to con¢rm these propositions. Indeed, such
procedures were effective in clarifying some
speci¢c problems, such as the rearrangements
related to the X1 X2 Y sex chromosome system differentiation in cytotype D (Bertollo et al. 1997b).
However, a general comparative banding pattern
analysis among the distinct cytotypes is not
practically viable at the moment, considering
the real dif¢culty in obtaining informative multiple
bands on ¢sh chromosomes, as well as new
samples for this study in view of the geographical
distance of several collection sites (Figure 1).
Meanwhile, these studies remain as future objectives to be reached.
For the moment, our clustering suggests that the
overall karyotypic structure, multiple sex chromosome systems, and chromosomes with unusual
morphology would be more informative for
phylogenetic relatedness among the chromosomal
forms than the diploid number similarity. This
is also supported by mtDNA data (Dergam 1996).
Although molecular data strongly support a
monophyly for a set of populations with 2n ˆ 42
chromosomes (cytotype A) from south and
southeast Brazil, other allopatric populations of
this same cytotype show a closer phylogenetic
relatedness to 2n ˆ 40 populations of cytotype C
(Dergam op. cit.).
Sympatry of cytotypes is also indicated in Figure 1. This is the ¢rst report for some localities
of the Amazon basin: (1) igarapë do Mindü:
Manaus-AM (cytotypes A and C), (2) rio
Trombetas: Proto Trombetas-PA (cytotypes E
and G), (3) rio Madeira: Porto Velho-RO
612
(cytotypes C and G), and (4) rio Aripuana¬:
Aripuana¬-MT (cytotypes C and G). Others have
already been described (Scavone et al. 1994,
Dergam 1996, Bertollo et al. 1997a, Lopes et al.
1998). Despite sympatry, no evidence of gene £ow
between cytotypes has been reported until now.
Speci¢cally for cytotypes A and C, and for
cytotypes A and D, RAPD-PCR genomic markers
are also congruent with lack of gene £ow (Dergam
1996), providing additional evidence for cytotypes
as distinct evolutionary units.
Although chromosomal rearrangements can
play an important role in evolution, the major
problem is to identify this role (Sites & Moritz
1987). In spite of this question, the present status
of H. malabaricus re£ects the ¢xation of several
chromosomal changes and a diversi¢cation within
this ¢sh group. So, our cytogenetic studies support
the view that the nominal taxon Hoplias
malabaricus is composed of several independent
biological units, characterized by unique ¢xed
cytogenetic characters, a fact that attests the
urgent need for a thorough taxonomic revision
of this `species complex'. Recent molecular data
(RAPD-PCR genomic markers) also corroborate
this view (Dergam et al. 1998). This diversity is
not surprising, taking into account that H.
malabaricus is a group with a wide geographic distribution. Thus, faced with this distribution, new
karyotypic forms or even different subgroups
within the general cytotypes yet described cannot
be ruled out. Even though the history of this
species is somewhat confused among the
Erythrinidae ¢sh, it is its proper type locality
(Oyakawa 1990). According to the original
description of Block, in 1794, probable type
localities would be Malabar or Tranquebar, but
these are coastal Indian sites (Oyakawa op. cit.);
however, circumstancial evidence points to
Surinam as the most likely origin of the type
(Dergam 1996). Thus, given the problematical
situation of this taxon, we cannot be sure that
cytotype F from Surinam (Tijgerkreek, 58 km west
of Paramaribo) is the presumed one of the type
locality, although it is a candidate for this.
L. A. C. Bertollo et al.
Acknowledgements
This work was supported by Conselho Nacional de
Desenvolvimento Cient|¨ ¢co e Tecnolögico
(CNPq). We are grateful to the Brazilian Embassy
and to the Agriculture and Fishery Ministry in
Surinam, and to Drs. E. Feldberg, H. Gurgel, J.
A. Pereira, P. M. Galetti Jr, P. C. Venere, Y. Sato,
J. I. R. Porto, A. Schwarzbold, L. R. Malabarba,
L. Giuliano-Caetano, J. C. Garavelo, W. Garutti
and A. D. Carvalho for their help in supplying ¢sh.
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