kidney cells as described by Moreira-Filho and. Bertollo. (1991b) using. (A) the direct method ..... Supernumerary B chromosomes restricted to males in Astyanax.
_??_1995 The Japan Mendel Society
Cytogenetic
Cytologia 60: 273-281 , 1995
Diversity in the Astyanax
scabripinnis
(Pisces, Characidae) Complex II. Different Cytotypes Living in Sympatry 1. L. Souza, O. Departamento
Moreira-Filho
de Genetica
and
L. A. C. Bertollo
e Evolucao, Universidade
Federal de Sao Carlos ,
Caixa Postal 676, 13565-905, Sao Carlos, SP, Brazil Accepted
June
21, 1995
The large complex of hydrographic basins including the La Plata-Uruguay-Parana - Paraguay rivers and extending from North-Northeast Argentina to East Bolivia and South and Center-West Brazil represents one of the most important regions in South America in terms of ichthyofauna. Like other basins on the continent, this complex harbors fish groups of difficult taxonomic positioning (Gery 1968). Among them is the subfamily Tetragonopterinae which, according to Britski (1972), represents the largest number of species among characids.
Cytogenetic investigation of neotropical fish have demonstrated that some Characidae subfamilies present a predominantly conservative karyotype evolution in terms of diploid number and macrokaryotype structure, as is the case for Salmininae (Marco 1986), Acestror hynchinae and Cynopotaminae (Falcao 1983, Falcao and Bertollo 1985), Bryconinae (Vascon et al. 1984, Santos et al. 1985, Julio et al. 1990, Margarido et al. 1994), Characidiinae (Miyazawa and Galetti Jr. 1994), Triportheinae (Falcao et al. 1985) and Glandulocaudinae (Guimaraes et al. 1993). However, other characid subfamilies have demonstrated high variability in terms of diploid number and karyotype formula, generally also presenting considerable taxonomic complexity, as is the case for the subfamily Serrasalminae (Arefjev 1990a, Cestari and Galetti Jr. 1992a,b), Cheirodontinae (Arefjev 1990b, Wasko and Galetti Jr. 1994), and Tetragonopterinae (Portela et al. 1988, Arefjev 1990b). Among Tetragonopterinae, Astyanax is the largest unit from a taxonomic viewpoint and is dominant in the continental waters of the neotropical region (Gery 1977). This genus presents wide differences in karyotype constitution among species, with a variation in diploid number ranging from 2n=36 in A. schubarti (Foresti et al. 1977, Morelli et al. 1983) to 2n=50 in A. fasciatus mexicanus (Kirby et al. 1977), A. bimaculatus (Jim and Toledo 1975, Foresti et al. 1977, Morelli et al. 1983), A. taeniatus (Rocon-Stange et al. 1986), A. eigenmaniorum (Fauaz et al. 1994), and in most species belonging to the A. scabripinnis complex (Souza and Moreira-Filho 1995). Variations in diploid number are also detected in the same nominal species, as is the case for the A. fasciatus (Morelli et al. 1983, Justi 1993) and A. scabripinnis (Moreira-Filho 1989, Moreira-Filho and Bertollo 1991a, Maistro 1991, Souza and Moreira - Filho 1995), these differences being usually detected among allopatrically distributed popula tions. A. scabripinnis was considered to be a species complex by Moreira-Filho and Bertollo (1991a) on the basis of the karyotypic and morphometric differences detected among popula tions from different basins or even from the same hydrographic basin. These investigators pointed out that these populations are isolated from one another by larger drainage systems, in agreement with Caramaschi (1986). Thus, the allopatric form of distribution of this complex is due to its limitation to the headwaters of small streams. The objective of the present investigation was to carry out a population study on this
274
I. L. Souza, O. Moreira-Filho
and L. A. C. Bertollo
Cytologia
60
species from a karyotype point of view, with efforts to obtain the largest possible number of specimens yielding good results using individuals of both sexes and of different sizes (different ages) and different methods of chromosome preparation. Material A.
scabripinnis
Itirapina,
State
were
obtained
using
(A)
solution
in
hypodermal
a
facilitate
cell
0.0125%
colchicine
a Pasteur
pipette
dropped and Levan
kidney
cells
as
or tissue
glass
routinely moist
Moreira-Fulho
an is
no
added
left for
in an cell
to
a
this
suspension,
oven
heated
(1994)
and
and to
is
and for
chromosome
the
M
with
the
aspiration
preparation
observation.
were
0.075
suspension;
approximately
70•Ž types
of
according identified
as
to
cell
according
of
a
to
4
glass to
drops
stirred material
the
of with
is then
suspensions
procedure to
i) KCl
expiration
3
The
the
follows:
aid
and
is carefully
Fixed
culture
hypotonic
iii)
30min.
(1991b)
short-term
technique
5.0ml
of
preparations Bertollo
(B)
completed
cell
municipality
and
(1978),
movements
approximately
chromosome
al.
in
homogeneous
at 36-37•Ž
fixation
slides
gentle
basin,
Chromosome
preparation
fragmented
fragmentation
using
River
Moreira-Filho
et
chromosome and
obtain
to
(Tiete
by
Bertollo
alternative
ii)
brook
cytogenetically.
described
by
removed
needle,
and are
and
(C)
cuvette;
with
separation
onto
et al.
cephalic
employed
methods
Galo
analyzed
or
small
Canta
were
method
syringe
the
Brazil)
1991),
organ
from
Paulo,
direct
et al.
desired
processed
Sao
from the
(Fenocchio the
specimens of
and
were of
criterion
Souza of
(1964).
Results
A total of 64 specimens were submitted to cytogenetic analysis; of these, 21 females and 20 males presented satisfactory results for chromosome counts, C banding and karyotype mount ing. The specimens submitted to the alternative method of chromosome preparation were mainly small ones or specimens with precarious survival conditions that would not have resisted colchicine treatment in vivo. Despite these drawbacks, more than 70% of these individuals presented good results for chromosome analysis and banding, thus demonstrating that in vitro colchicine application directly to the hypotonic solution is very efficient and can be used routinely in fish cytogenetics laboratories.
A diploid number of 2n=50 was detected in 20 females and 20 males (Table 1a) with a karyotype consisting of 2 pairs of metacentric chromosomes (M), 13 pairs of submetacentrics (SM), 2 pairs of subtelocentrics (ST) and 8 pairs of acrocentrics (A), and with a fundamental number of FN=84 (Fig. 1a). However, one female and one male from the same collection presented 2n=48 (Table 1b), with three metacentric (M) pairs, 11 submetacentric (SM) pairs, 4 subtelocentric (ST) pairs and 6 acrocentric (A) pairs (Fig. 1b), with the same number of arms (FN=84) as the 2n=50 karyotype mentioned earlier. We defined the 2n=50 karyotypes as cytotype I and the 2n=48 karyotypes as cytotype II. Fig. 2a shows constitutive heterochromatin distribution among the chromosomes of cytotype I, with small pericentromeric blocks in all chromosomes in the complement; weak labeling in the telomeric region of the long arm of pair 1; C-positive bands occupying the entire short arm of pairs 16, 17; terminal bands on the long arm of chromosome pairs 16, 18, 19, 20, 21 and 22; weak labeling in the interstital region of the long arm of pair 18. The C+ bands observed in cytotype II were distributed into small blocks in the pericentromeric region of most chromosomes; on the short arm of pairs 6 and 18; in interstitial and terminal regions on the long arm of pairs 15, 19, 20, 22 and 23, and in the terminal region of the long arm of pair 21 (Fig. 2b).
1995
Cytogenetic
Table
1.
Frequency
Diversity in the Astyanax scabripinnis
distributions
of diploid chromosome
Canta Galo brook-Tiete
Method: A=Bertollo
et al. 1978; B=Fenocchio
basin, Itirapina,
Complex II .
counts in A . scabripinnis SP, Brazil
et al. 1991; C=present
paper
275
from
276
I. L. Souza, O. Moreira-Filho
and L. A. C. Bertollo
Cytologia
60
Fig. 1. Karyotypes of A. scabripinnis collected from the Canta Galo brook, a cytotype I (2n= 50), and b cytotype II (2n=48). Fig. 3 presents
the idiograms
of the cytotypes
detected
in the present
study.
Discussion
The cytogenetic studies on A. scabripinnis were initiated by Moreira-Filho et al. (1978), and the karyotypes of a significant number of populations have been described thus far. Interpopulation differences have been demonstrated in terms of diploid number, karyotype structure, pattern of constitutive heterochromatin distribution and Ag-NORs position, reinfor cing the hypothesis that these populations are isolated from one another by larger water basins.
1995
Cytogenetic
Diversity in the Astyanax scabripinnis
Complex II .
Fig. 2. Metaphase chromosomes of A. scabripinnis from the Canta Galo brook submitted to C banding. a cytotype I (2n=50) and b cytotype II (2n=48).
277
278
I. L. Souza, O. Moreira-Filho
and L. A. C. Bertollo
Cytologia
60
Fig. 3. a and b refer to the idiograms of A. scabripinnis from the Canta galo brook. a cytotype I (2n=50) and b cytotype II (2n=48). The darker spots represent the more clearly visible regions of C+ bands.
Thus, by being restricted to the headwaters of small streams, different populations can develop its own evolutionary course and the various chromosome rearrangements observed have demonstrated the process of differentiation between them. However, two allopatrically distributed populations were also found in the same stream, having quite divergent cytotypes, without a contact zone detected thus far (Souza and Moreira-Filho 1995). In the present study, the numerical and structural chromosome differences among two cytotypes living in syntopy and sympatry in the Canta Galo brook, but with the same number of chromosomal arms, suggest that various rearrangements, such as those of the Robertsonian type, and other structural ones, took place in this cytotypic diversification. This case, seems to indicate a situation differing from those previously described. Thus, the low frequency of cytotype II in relation to cytotype I suggests that this collection site represents a parapatric zone in which the presence of such divergent types may result from a secondary contact between populations initially belonging since no signs of hybridization
to different streams and possibly have been observed.
already
reproductively
isolated,
Although Moreira-Filho and Bertollo (1991a) have detected populations with 2n=46 and 2n=48, most A. scabripinnis specimens studied thus far present 2n=50 (Souza and Moreira - Filho 1955). A total of three populations revealed a diploid number of 2n=48 (Moreira-Filho and Bertollo 1991a, Maistro 1991 and present paper) and, even though they are located in streams from different basins, their karyotypes are similar, suggesting a disruptive distribution by vicariance. In contrast to Salmininae, Bryconinae, Characidiinae and some other characids, A. scabripinnis has withstood several structural chromosome alterations such as inversions, trans locations, increases or losses of heterochromatin, as well as Robertsonian rearrangements. Furthermore, some populations have a high percentage of individuals carrying B macrochro mosomes (Salvador and Moreira-Filho 1992, Maistro et al. 1992, Neo and Moreira-Filho 1994, Vicente 1994), and one of them in particular showed incidence of B microchromosomes (Rocon-Stange and Almeida-Toledo 1993). Triploid specimens with the presence of two B macrochromosomes were also detected (Fauaz et al. 1994).
A. scabripinnis presents high phenotypic plasticity (Caramaschi 1986), and some studies of its biology (Barbieri 1992a,b, c) have shown that this species complex has low food require ments, a predominance of females in the population and an early reproductive age, this last characteristic being in agreement with data reported by Araujo (1983). Their allopatric distribution in small populations, reproductive potential, high phenotypic plasticity and other
1995
Cytogenetic
Diversity in the Astyanax scabripinnis Complex II.
279
peculiar biological attributes must have facilitated the fixation of chromosome alterations so contributing to their wide diversity. These biological peculiarities, allied to geomorphologic events, probably led to the present populational distribution of this fish complex.
Summary
Cytogenetic studies were conducted in Astyanax scabripinnis (Pisces, Characidae, Tetrago nopterinae) species complex, from the Canta Galo brook (Tiete River basin, State of Sao Paulo, Brazil). Two sympatric cytotypes differing in diploid number and karyotype structure were detected. Almost all specimens presented 2n=50, with a karyotype consisting of 4M+26SM +4ST+16A and a fundamental number FN=84. However, two specimens in this sample-a female and a male-collected syntopically with that cytotype were found to be 2n=48 (6M+ 22SM+8ST+12A) and to have the same number of chromosome arms (FN=84). The two cytotypes showed some differences in constitutive heterochromatin distribution. The probable events implicated in this divergence and their significance are discussed, together with aspects of the evolutionary process of this species complex.
Acknowledgments
The authors are grateful to Dr. Pedro M. Galetti Jr. for valuable suggestions, and to Adriana Medaglia and Alois Copriva for technical assistance. This research was supported by The Federal University of Sao Carlos (UFSCar) and by CNPq (proc. 80.1348/88-9) and CAPES. References
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