Cytogenetic analysis in catfi sh species of the genus Peckoltia Miranda Ribeiro , 1912 ( Teleostei : Siluriformes : Loricariidae )

This study describes the karyotypes of three species of the genus Peckoltia (Loricariidae: Ancistrini). Fishes were collected in the Jari (Peckoltia sp. 1 and Peckoltia sp. 2) and Xingu rivers (Peckoltia vittata (Steindachner, 1881)) in the Amazon rainforest. Karyotypes were 2n = 52 for Peckoltia vittata (FN=102: 16 metacentrics (m), 20 submetacentrics (sm), 14 subtelocentrics (st), 2 acrocentrics (a)) and Peckoltia sp. 2 (FN=102: 32 m + sm, 18st, 2a). Peckoltia sp. 1 (FN=102: 44 m + sm, 6st, 2a, 1B) had 2n = 53 due to a B chromosome. The species differ in chromosomal morphology. Nucleolar Organizer Regions (NOR) was identifi ed within a distal region of the long arm of pair 9 in Peckoltia vittata, in pair 10 and in a homologue of pair 25 in Peckoltia sp. 1, as well as in pair 17 and in a homologue of pair 18 in Peckoltia sp. 2. Chromomycin A3 banding agreed with the location of the NORs. C-banding patterns revealed large non-centromeric heterochromatic blocks, probably of a common origin. Our results suggest a higher level of similarity between Peckoltia vittata and Peckoltia sp. 1.

The relatively few cytogenetic studies of the tribe Ancistrini, including our own, have found that the diploid numbers of these fi sh range from 38 to 53, while the FN ranges from 68 to 104.The Table 1 shows this information as compiled by us.The aim of this paper was to study the karyotypes of three species in the genus Peckoltia.To our knowledge, this is the fi rst description of karyotypes in this genus.

MATERIAL AND METHODS
We studied four males and three females of the species Peckoltia vittata (Steindachner, 1908) (Fig. 1, D), which were caught in the Xingu River, in the town of Altamira and Para state (03°12´4˝ N/52°12´41.7˝W).In addition, we studied one female of the taxon Peckoltia sp. 1 (Fig. 2, D) and one female of Peckoltia sp. 2 (Fig. 3, D), which were both caught in the Jari River, located in the town of Monte Dourado and Para state, at Cachoeira Santo Antonio (03°18´14.9˝N/52°03´29.3˝W).Fish specimens were vouchered at the Museu Paraense Emilio Goeldi (MPEG) with reference number MPEG 13427 to Peckoltia vittata, 1 MPEG 3420 to Peckoltia sp. 1 and MPEG 13426 to Peckoltia sp. 2.
Fish were processed using a yeast treatment (Cole, Leavens, 1971).Specimens were injected with 0.5 ml of a 0.025% colchicine solution per 100 g of body weight.After 30 minutes, metaphase chromosomes were prepared using the method described by Bertollo et al. (1978).
For each individual thirty metaphases were examined in order to determine the diploid number (2n), the FN, and to perform the banding analyzed.
For bright fi eld microscopy analysis (Giemsa conventional staining, C-banding, Ag-NOR staining) we used a Zeiss Axiophot 2 microscope, 10x ocular lens and 100x objective lens.The images were captured with an Axiocam Mrm camera, controlled by the Zeiss AxioVision 3.1 software in a PC computer.For metaphases stained with CMA 3 we used the same microscope, camera and software, but illuminated with a HBO-100 Osram Mercury lamp.We used Zeiss fi lters BP 436, FT 460 and LP 470.Representative metaphases were captured digitally, using the Zeiss AxioCam MRm and AxioVision software.

RESULTS
Peckoltia vittata has 2n = 52 chromosomes and a FN of 102 (36 m + sm, 14st, 2a) (Fig. 1,  A). C-banding analysis revealed an interstitial block in the short arm of pair 1 and a large block that spanned most of the short arm of pair 2, the pericentromeric region of pair 11 and most of the long arms of pairs 9, 10, 13 and 21 (Fig. 1, B). NORs were identifi ed in a distal position on the long arm of a large submetacentric chromosome of pair 9 (Fig. 1, A). Size heteromorphism of the NORs was revealed (Fig. 1, A). CMA 3 banding revealed a pair of chromosomes having rich G-C base

Comp. Cytogenet., 2009 3(2)
Comparative Cytogenetics (32 m + sm, 18st, 2a) (Fig. 3, A).C-banding revealed an proximal block in the short arm of pair 1 and in the long arm of pair 2 and in most of the long arm of pairs 9, 10, 17, 18 and 19 (Fig. 3, B).The heterochromatin block on the pair 19 has a size heteromorphism.NORs were identifi ed in the distal region of the long arm of pair 18 and in a homologue of pair 17 (Fig. 3, A).CMA 3 banding was positive in three chromosomes, in agreement with the NOR fi ndings, confi rming that only one of the homologues of pair 17 has a NOR (Fig. 3, C).

DISCUSSION
Our study revealed that Peckoltia has a diploid number of 52, consistent with most other members of the tribe Ancistrini.The number and size of ribosomal sites vary, but all are located in the distal regions of the long arms of specifi c chromosomes.The absence of NORs in one of the homologues may refl ect a genetic deletion or a translocation.Each of the three composition, in agreement with the location of the NORs (Fig. 1, C).
Peckoltia sp. 1 obtained from the Jari river has 2n = 52 chromosomes and a FN of 102 (44 m + sm, 6st, 2a; +1B).In addition, a small supernumerary B chromosome was observed in all metaphases of this specimen (Fig. 2, A).C-banding revealed an interstitial block that spanned the short arm of chromosome pair 1, most of the long arms of pairs 10, 11 and 13, most of the short arm of pair 12 and the long arms (i.e., close to the centromeres) of pairs 15 and 25 (Fig. 2, B).NORs were identifi ed in a distal position on the long arm of pair 10 and in a homologue of pair 25.All NORs were C-band negative (Fig. 2, A).As we found in Peckoltia vittata, the NOR shows a size heteromorphism.CMA 3 banding was positive in three chromosomes, in agreement with the NOR fi ndings, confi rming that only one of the 25 homologues contains a NOR (Fig. 2, C).
Peckoltia sp. 2 obtained from the Jari river has 2n = 52 chromosomes and a FN of 102 Comp.Cytogenet., 2009 3(2) Comparative Cytogenetics species had large heterochromatic blocks, probably of a common origin that covered almost all of the long arms of some submetacentric and subtelocentric chromosomes.The heterochromatic short arm identifi ed in pair 2 of Peckoltia vittata may be a good marker for this species, as it was specifi c to this taxon.The analysis of chromosome morphology and heterochromatin distribution suggested that Peckoltia vittata chromosome pairs 1, 9, 10, 13, 21 and 11 correspond to Peckoltia sp. 1 chromosomes 1, 10, 11, 13, 25 and 15, and Peckoltia sp. 2 chromosomes 1, 18, 9, 10 and 19 (Fig. 4).Thus, the large amount of heterochromatin in Peckoltia probably originated after this genus split from its ancestor.Analysis of constitutive heterochromatin distribution and NOR morphology revealed a higher level of similarity between Peckoltia vittata and Peckoltia sp. 1 than between either of them and Peckoltia sp. 2. This similarity is probably not related to geographical distribution, as Peckoltia sp. 1 and sp. 2 were collected in the Jari river while Peckoltia vittata was collected in the Xingu river.Considering the allopatric relationship among the species from the Xingu and Jari rivers, it is possible that a channel once connected these rivers and allowed the dispersion of species, which were later subjected to vicariance.Alternatively, a large population from one river may have been transferred to the other and then was later subjected to fragmentation via geological movements within in the Amazon.These movements might have created new geographical barriers, which would have limited the gene fl ow in this population.
After conducting an extensive review of Loricariidae karyotypes, Kavalco et al. (2005) suggested that the 2n = 54 karyotype be considered a plesiomorphic condition in this family.Armbruster (2004) published a simplifi ed Loricariidae phylogeny, which is available at http://www.auburn.edu/academic/science_math/res_area/loricariid/fish_key/tree/tree.html.If we put on the phylogeny the diploid number already known for each genus we will fi nd that the 2n = 52 karyotype is found al-

Fig. 1 .
Fig. 1.Karyotype of a Peckoltia vittata, sequentially Giemsa-stained (A) and C-banded (B) chromosomes.The CMA 3 -stained metaphase of the same specimen (C) revealed CMA 3 -positive signals in the end-to-end associated NOR-bearing chromosome pair (arrows).The specimen was obtained from Xingu River (D).

Fig. 2 .
Fig. 2. Karyotype of a Peckoltia sp. 1, sequentially Giemsa-stained (A) and C-banded (B) chromosomes.The CMA 3 -stained metaphase of the same specimen (C) revealed CMA 3 -positive signals in the end-to-end associated NOR-bearing chromosome pair.The specimen was obtained from Jari river (D).

Fig. 3 .
Fig. 3. Karyotype of a Peckoltia sp. 2, sequentially Giemsa-stained (A) and C-banded (B) chromosomes.The CMA 3 -stained metaphase of the same specimen (C) revealed CMA 3 -positive signals in the end-to-end associated NOR-bearing chromosome pair.The specimen was obtained from river Jari (D).

Fig. 4 .
Fig. 4. Possible homologies among species of the Peckoltia genus, as determined via conventional staining, C-banding and NOR staining.