Differential chromosomal markers between sympatric karyomorphs of the fi sh Hoplias malabaricus ( Bloch , 1794 ) ( Characiformes : Erythrinidae )

Cytogenetic analyses were performed on Hoplias malabaricus (Bloch, 1794) from the Taquari River, Parana River drainage, revealing two sympatric karyomorphs. One karyomorph was characterized by 2n = 40 m/sm and 2n = 39 m/sm chromosomes for females and males, respectively, and an X1X1X2X2/X1X2Y sex chromosome system. In the second karyomorph, specimens showed 2n = 42 m/sm chromosomes, without sex-related heteromorphism. Both karyomorphs were characterized by a distribution of heterochromatin in the pericentromeric and telomeric regions. In addition to the differences in the diploid numbers and the sex chromosomes, the GC-rich sites and the nucleolar organizer regions also showed clear differences between the karyomorphs. Coupled with the occurrence of unique chromosomal features within each karyomorph, the fact that hybrids have not been identifi ed in the sampled population provides additional support of the existence of a species complex in H. malabaricus.


INTRODUCTION
Chromosomal rearrangements can play a major role in speciation processes, providing reproductive barriers and hence the formation of new biological species (Livingstone, Rieseberg, 2003).This fact has been observed in different biological groups, including house mice, where the accumulation of chromosomal rearrangements has been shown to lead to different "karyotypical races" or karyomorphs, and to a reproductive isolation between populations (Searle, 1998).
In order to provide a more detailed characterization of the diversity and to better understand the karyotypic evolution within this group, in the present work we investigated chromosomal markers in two karyomorphs of this species, collected in both sympatry and syntopy in Taquari River, a tributary of the Tibagi River (Paraná, Brazil), comparing our data with previous work.(Bertollo et al., 1978) and lymphocyte culture (Fenocchio, Bertollo, 1988) and stained with 5% Giemsa in phosphate buffer (pH 6.8)The chromosomes were organized as metacentric (m) and submetacentric (sm) for the preparation of a karyogram.

RESULTS
Two karyomorphs were observed among the examined specimens.In the fi rst case, fourteen females presented a diploid number of 2n=40 m/sm chromosomes (Fig. 2, Aa), and twelve males had a diploid number of 2n=39 m/sm chromosomes, showing the occurrence of a X 1 X 1 X 2 X 2 /X 1 X 2 Y multiple sex chromosome system (Fig. 2, Ba).The second karyotypical form comprised 2n=42 chromosomes (Fig 2,Ca), without chromosomal differentiation between sexes (10 males and 11 females).According to Bertollo et al. (2000), these forms would correspond to karyomorphs D and A, respectively.Heterochromatin blocks were observed at the centromeric regions of all chromosomes and at the telomeric regions of some pairs.In the karyomorph D, the X 1 and X 2 chromosomal pairs, corresponding to pairs 6 and 20, respectively, presented centromeric blocks (Fig. 2

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Comparative Cytogenetics chromosomes of karyomorph D, in both males and females.The X chromosomes presented a larger fl uorescent signal when compared to the others chromosomal pairs (Fig. 3, d, e).This block was more evident in males than females, whereas the Y chromosome presented two GCrich segments in an interstitial position, one on the long arm and the other on the short arm were observed in the 14 th pair (Fig. 2, Cb).Both karyomorphs were characterized by the presence of multiple Ag-NORs (Fig. 3,  a-c).However, the occurrence of bitelomeric NORs, i.e., in both telomeres of the same chromosome, was identifi ed only within karyomorph A (Fig. 3, c).
GC-rich blocks were evident in several

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Comparative Cytogenetics (Fig. 3, e).On the other hand, the karyomorph A presented a single GC-rich block, probably located on the sixth pair (Fig. 3, f).All GCrich segments were negatively stained by DAPI, and AT-rich bands were absent in both karyomorphs (Fig. 3, g, h, i).Eight 18S rDNA cistrons were evident in the karyomorph D, occupying the telomeric regions of three chromosomal pairs in both males and females.Additionally, in females, two interstitial sites on the long arm of a chromosomal pair were detected close to centromeres (Fig. 3, j).In males, this feature was ob-served in a single chromosome, while another chromosome presented interstitial sites on both arms (Fig 3,k).Probably, these chromosomes would correspond to X 1 and Y, respectively.In karyomorph A, six 18S rDNA-bearing chromosomes were detected, comprising two telomeric, two interstitial and two bitelomeric sites (Fig. 3, l).

DISCUSSION
Several cytogenetic studies have been carried out in order to understand the Mogi-Guaçu River (Luiz Antonio-SP) Scavone et al., 1994 Grande River (Conceição das Alagoas-MG) Dergam, 1996 Taquari stream (Londrina-PR)  Born, Bertollo, 2006 Comp.Cytogenet., 2010 4(2) Comparative Cytogenetics remarkable karyotype diversity in the species Hoplias malabaricus (Santos et al., 2009;Rosa et al., 2009a;Blanco et al., 2010a).Such chromosomal diversity is characterized by the occurrence of distinct sex chromosome systems and the identifi cation of at least seven karyomorphs, classifi ed from A to G (Bertollo et al., 2000).The samples of Hoplias malabaricus from the Taquari River presented two sympatric karyotypic forms, characterized as karyomorph A (2n=42) and karyomorph D (2n=39/40).Other examples of sympatric karyomorphs have been reported within H. malabaricus (Table 1).Bertollo et al. (2000) listed nine occurrences throughout Brazil and Argentina.Recent studies have also reported new cases in the Upper Paraná basin (Pazza, Júlio Jr., 2003), in the Tibagi River basin (Rosa, 2006) and in the Parque Estadual do rio Doce (Born, Bertollo, 2006).No possible hybrids have been identifi ed in any of these cases, indicating that such distinct karyotypic forms are likely to be reproductively isolated.Investigations using RAPD markers within a population of H. malabaricus presenting the same sympatric karyomorphs analyzed in the present study, demonstrated a lack of gene fl ow between karyomorphs, reinforcing their putative reproductive isolation (Dergam, 1996).
The general location pattern of pericentromeric and telomeric heterochromatin detected in specimens from the Taquari River is usually found in different populations of H. malabaricus (Lopez, Fenocchio, 1994;Born, Bertollo, 2001;Vicari et al., 2003Vicari et al., , 2006)), although minor differences may also occur among karyomorphs/populations.However, the most contrasting features between both karyomorphs here analyzed refer to the nucleolar organizer regions and location of GC-rich chromosomal sequences.While both males and females from the karyomorph D presented 2-3 telomeric Ag-NORs on long arms of submetacentric chromosomes, specimens from karyomorph A were characterized by the additional occurrence of bitelomeric Ag-NORs.A bitelomeric Ag-NOR pattern was previously reported by Born and Bertollo (2006) in samples of the same karyomorph, found in sympatry with other karyotypical form (karyomorph B: 2n=42, XX-XY), in Parque Estadual do rio Doce.
Data based on FISH results also reinforce the differential location and number of NORs between the two karyomorphs.In fact, eight 18S rDNA-bearing chromosomes were detected in males and females from karyomorph D, and two of them probably correspond to the X 1 and Y chromosomes.According to Bertollo et al. (1997a), the formation of the Y chromosome in males of karyomorph D was related to a centric fusion between one X 1 and one X 2 chromosome.The Y chromosome in the karyomorph D from the Taquari River might have originated by a similar rearrangement.However, once the 18S rDNA sequences are located on both arms of the putative Y chromosome and ribosomal genes are absent in the X 2 chromosome, we hypothesize that a pericentric inversion might have also taken place, moving a portion of the 18S rRNA genes to the short arms of the Y chromosome.
On the other hand, six chromosomes carrying 18S rDNA sites were observed within karyomorph A, two of them in a bitelomeric location, in agreement with the pattern observed in two populations of the same karyomorph in the Tibagi (PR) and Ribeira (SP) rivers (Vicari et al., 2005).Blanco et al. (2010b) showed a differentiation in the number of ribosomal sites in three different populations of H. malabaricus, and differences in the distribution of 5S rDNA and 5SHindIII satellite DNA were also observed.

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Comparative Cytogenetics The GC-rich chromosomal regions also showed a distinct pattern of location between karyomorphs.Males and females of karyomorph D, besides several reduced blocks distributed throughout the karyotype, showed more conspicuous signals related to the putative sex chromosomes, apparently corresponding to C-bands and 18S rDNA sites.However, in karyomorph A, only a single chromosomal pair showed more evident GCrich band, located at centromeric region.
Interpopulation variation has been reported in many groups of neotropical fi sh, and this feature has been commonly correlated with population structure (Galetti Jr. et al., 1994).Species with limited and sedentary populations are more likely to present a greater degree of variation when compared to those with large populations of high mobility.This feature could explain the karyotypical diversity found in H. malabaricus, since it represents a low-vagility species, inhabiting lentic environments.Consequently, the fi xation of chromosomal rearrangements would be easily accomplished, leading to the remarkable karyotypical plasticity observed in different populations of H. malabaricus and favoring the occurrence of distinct karyotypical forms.In sympatric conditions, such differences could be suffi cient to prevent the gene fl ow between different karyomorphs (McCoy, 2003).The fact of the two karyomorphs occur in sympatry and there are no intermediate karyotypic forms, indicates that there must be reproductive isolation, so they are probably different species.
Therefore, our data corroborate the current suggestion that H. malabaricus comprises a species complex of independent evolutionary units (Bertollo et al., 2000).The available information on karyotypic structure and chromosomal markers, along with further morphological and molecular studies, such as mtDNA analysis, are important to provide a more reliable phylogeny of this complex fi sh group.
Fig. 1.Hydrographic map showing lower the Paranapanema river basin (shaded) and the collection site of Hoplias malabaricus (*) in the Taquari River.