Research Article |
Corresponding author: Alber Sousa Campos ( albercampos07@gmail.com ) Academic editor: Petr Rab
© 2020 Alber Sousa Campos, Ramon Marin Favarato, Eliana Feldberg.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Campos AS, Favarato RM, Feldberg E (2020) Interspecific cytogenetic relationships in three Acestrohynchus species (Acestrohynchinae, Characiformes) reveal the existence of possible cryptic species. Comparative Cytogenetics 14(1): 27-42. https://doi.org/10.3897/CompCytogen.v14i1.33483
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The karyotypes and chromosomal characteristics of three Acestrorhynchus Eigenmann et Kennedy, 1903 species were examined using conventional and molecular protocols. These species had invariably a diploid chromosome number 2n = 50. Acestrorhynchus falcatus (Block, 1794) and Acestrorhynchus falcirostris (Cuvier, 1819) had the karyotype composed of 16 metacentric (m) + 28 submetacentric (sm) + 6 subtelocentric (st) chromosomes while Acestrorhynchus microlepis (Schomburgk, 1841) had the karyotype composed of 14m+30sm+6st elements. In this species, differences of the conventional and molecular markers between the populations of Catalão Lake (AM) and of Apeu Stream (PA) were found. Thus the individuals of Pará (Apeu) were named Acestrorhynchus prope microlepis. The distribution of the constitutive heterochromatin blocks was species-specific, with C-positive bands in the centromeric and telomeric regions of a number of different chromosomes, as well as in interstitial sites and completely heterochromatic arms. The phenotypes of nucleolus organizer region (NOR) were simple, i. e. in a terminal position on the p arm of pair No. 23 except in A. microlepis, in which it was located on the q arm. Fluorescence in situ hybridization (FISH) revealed 18S rDNA sites on one chromosome pair in karyotype of A. falcirostris and A. prope microlepis (pair No. 23) and three pairs (Nos. 12, 23, 24) in A. falcatus and (Nos. 8, 23, 24) in A. microlepis; 5S rDNA sites were detected in one chromosome pair in all three species. The mapping of the telomeric sequences revealed terminal sequences in all the chromosomes, as well as the presence of interstitial telomeric sequences (ITSs) in a number of chromosome pairs. The cytogenetic data recorded in the present study indicate that A. prope microlepis may be an unnamed species.
Repetitive DNA, fish cytotaxonomy, FISH, ITS
The family Acestrorhynchidae is a group of South American fishes, for which contradictory views on its phylogenetic position within Characiformes are debated. Based on the analysis of morphological data, for example,
The genus Acestrorhynchus includes piscivorous fishes with an elongated body and snout, conical teeth and robust canines with a characteristic arrangement in the maxilla, together with a number of other diagnostic traits (
The Acestrorhynchus species can be distinguished on the basis of their coloration patterns and can be allocated to three groups: (i) Acestrorhynchus lacustris group (
The cytogenetic data available for Acestrorhynchus indicate a conserved 2n = 50 in all species (
The present study examined the karyotypes and chromosomal characteristics of three Amazonian Acestrorhynchus species using both conventional and molecular cytogenetic protocols. Our results were compared with the existing data attempting to better understand the chromosomal differentiation of the genus and the rearrangements involved in this process.
The present study analyzed the cytogenetic characteristics of Acestrorhynchus falcatus (Block, 1794), Acestrorhynchus falcirostris (Cuvier, 1819), and Acestrorhynchus microlepis, (Schomburgk, 1841) where the latter species had variation in the chromosome complement of representatives from different collecting localities (Fig.
Map of the Brazilian Amazonia region, showing the individual collection localities. 1 Acestrorhynchus falcirostris – Balbina reservoir on the Uatumã River, Amazonas state 2 Acestrorhynchus falcirostris and Acestrorhynchus microlepis – Catalão Lake, at the confluence of the Negro and Solimões rivers, Amazonas state 3 Acestrorhynchus falcatus, Acestrorhycnhus falcirostris, and Acestrorhynchus prope microlepis – Apeu Stream, basin of the Guamá River, Pará.
The Acestrorhynchus species included in the present study, collecting localities, and the number of individuals analyzed. ♂ = male; ♀ = female.
Species | Sampling locations | Hydrographic Basin | Coordinates | Number of analized animals | Vouchers |
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A. falcatus | Apeu Stream, Pará, Brazil | Guamá River | 1°23'20.4"S, 47°59'07.5"W | 8♂ 2♀ |
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A. falcirostris | Catalão Lake, Amazonas, Brazil | Solimões River | 3°09'20.4"S, 59°54'47.1"W | 1♂ 7♀ |
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Balbina UHE, Amazonas, Brazil | Uatumã River | 1°55'07.6"S, 59°29'19.7"W | 1♂ 2♀ |
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Apeu Stream, Pará, Brazil | Guamá River | 1°23'20.4"S, 47°59'07.5"W | 3♂ 1♀ |
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A. microlepis | Catalão Lake, Amazonas, Brazil | Solimões River | 3°09'20.4"S, 59°54'47.1"W | 1♂ 2♀ |
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A. cf. microlepis | Apeu Stream, Pará, Brazil | Guamá River | 1°23'20.4"S, 47°59'07.5"W | 4♂ 2♀ |
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The chromosomal preparations were obtained following the protocols of
The 5S and 18S ribosomal DNA probes were obtained from the genomic DNA of A. falcirostris, which was extracted using the Wizard Genomic DNA Purification kit. The rDNA probes were amplified by polymerase chain reaction (PCR), using the primers 18Sf (50-CCG CTG TGG TGA CTC TTG AT-30), and 18Sr (50 - 31 CCG AGGACC TCA CTA AAC CA- 30) (
The PCR products were verified in 1.5% agarose gel, and quantified in NanoVue Plus (GE Healthcare). The 18S rDNA gene was marked with digoxigenin-11-dUTP (Dig Nick Translation mix, Roche), while the 5S rDNA gene and telomeric sequences were marked with biotin-14-dATP (Biotin Nick Translation mix, Roche), following the manufacturer´s instructions. The hybridization signals were detected using anti digoxigenin-rhodamine (Roche Applied Science) for the 18S rDNA probe, and streptavidin (Sigma-Aldrich) for the 5S rDNA probes and telomeric sequences. Fluorescence in situ hybridization (FISH) was based on the protocol of
The chromosomes of about 30 metaphases per individual were analyzed and the images were captured using an Olympus BX51 epifluorescence microscope, and processed using Image Pro Plus 4.1 software (Media Cybernetics, Silver Spring, MD, USA). The chromosomes were classified according to
All Acestrorhynchus falcatus, A. falcirostris, A. microlepis, and A. prope microlepis individuals possessed invariably 2n = 50 and a fundamental number (FN) 100. Their karyotypes were very similar to each other and composed of 16m+28sm+6st in A. falcirostris and A. falcatus, while 14m+30sm+6st in A. microlepis and A. prope microlepis (Fig.
Karyotypes of the species under study arranged from chromsomes stained conventionally with Giemsa, C-banded, and after Ag-NOR impregnation: a–c A. falcirostris d–f A. falcatus g–i A. prope microlepis j–l A. microlepis. Scale bar: 10 µm.
The NORs were located in a distal position on the p arms of pair No. 23 in all the species, except for A. microlepis, in which the NORs were located on the q arms of pair No. 23 (Fig.
The positive 18S rDNA sites corresponded to the NOR signals in A. falcirostris and A. prope microlepis, at pair No. 23 (Fig.
Karyotypes of the species under study, arranged from chromosomes showing “double” FISH with 18S rDNA (red) and 5S (green) probes a, c, g and FISH with (TTAAGG)n probe, in green b, d, f, h A. falcirostris (a, b), A. falcatus (c, d), A. prope microlepis (e, f), A. microlepis (g, h). Scale bar: 10 µm.
The blocks of constitutive heterochromatin were distributed in centromeric and telomeric regions in karyotypes of all species, though with unique features found in each species, as follows:
A. falcatus: heterochromatin in centromeric and telomeric blocks in pairs Nos. 2, 4, 6, 8, 10, 15, 16, 20, and 22, and in centromeric blocks only in pairs Nos. 1, 3, 5, 7, 9, 11, 13, 14, 17, 19, and 21, while pairs Nos. 12, 23, 24, and 25 have entirely heterochromatic p arms, and pair No. 18 had no clear heterochromatic signal (Fig.
A. falcirostris: heterochromatin in centromeric and telomeric blocks in pairs No. 1, 3, 4, 5, 7, 8, 9, 10, 12, 14, 15, and 18, in telomeric blocks only in pairs Nos. 2, 6, 17, 19, 20, 21, and 22 and in pericentromeric blocks only in pairs Nos. 11, 16, 23, and 24. Pairs Nos. 13 and 25 have centromeric blocks and terminal blocks on the q arms. In pair No. 23, a differential accumulation of heterochromatin was observed in the p arms, with blocks adjacent to the NOR (Fig.
A. prope microlepis: heterochromatin in centromeric and pericentromeric regions. Pair No. 4 also had telomeric signals, while in pairs Nos. 2, 8, 17, 18, 20, 23, 24, and 25, there is a block in a more interstitial position. Pair No. 19 displayed size heteromorphism of a heterochromatin block, observed after both Giemsa staining and C-banding (Fig.
A. microlepis: heterochromatin found primarily in centromeric regions, with some proximal signals, but in a pattern distinct from that observed in A. prope microlepis, in terms of the location and position of the heterochromatin on some chromosome pairs (Fig.
The mapping of the 5S rDNA gene revealed a pericentromeric signal in only one pair in each species (pair No. 17 in A. falcirostris, pair No. 3 in A. falcatus, A. microlepis prope microlepis) (Fig.
Telomeric sequences were detected in the terminal regions of all chromosomes, as expected, but also with additional interstitial telomeric sequences (ITSs) in a number of chromosome pairs in all species under study, displaying species-specific patterns in terms of their localization (Fig.
All species analyzed in the present study have invariably 2n = 50 chromosomes, with no morphologically distinguishable sex chromosomes. There is a considerable variation, however, in the karyotype structures and the FN values (
Cytogenetic data available for the representatives of the genus Acestrorynchus. (2n = diploid chromosome number, FN = Fundamental Number, NOR = Nucleolus Organizer Region, ITS = Interstitial Telomeric Sequence, m = metacentric, sm = submetacentric, st = subtelocentric, a = acrocentric chromosomes p = short arm, q = long arm).
Groups | Species | 2n | FN | NOR | Karyotype formulae | N° and location 18S rDNA | N° and location 5S rDNA | N° of pairs ITS | References |
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lacustris | A. altus | 50 | 94 | 2 pairs | 8m+22sm+14st+6a | - | - | - |
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A. falcatus | 50 | 100 | 1 pair | 16m+28sm+6st | 3 pairs; (p) | 1 pair (3); pericentromeric | 6 pairs | Present study | |
A. lacustris | 50 | 98 | 1 pair | 12m+32sm+4st+2a | - | - | - |
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A. lacustris | 50 | 98 | - | 8m+34sm+6st+2a | - | - | - |
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A. pantaneiro | 50 | 86 | 1pair | 36 m-sm+14st-a | - | - | - |
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microlepis | A. cf. microlepis | 50 | 100 | 1pair | 14m+30sm+6st | 1 pair; (p) | 1 pair (3); pericentromeric | 18 pairs | Present study |
A. microlepis | 50 | 100 | 1pair | 14m+30sm+6st | 2 pairs; (q) and 1 pair bitelomeric | 1 pair (3); pericentromeric | 19 pairs | Present study | |
nasutus | A. falcirostris | 50 | 100 | 1 pair | 16m+28sm+6st | 1 pair; (p) | 1 pair (17); pericentromeric | 10 pairs | Present study |
Based on the analysis of morphological characters,
A similar scenario is found in the Erythrinoidea (Ctenoluciidae, Hepsetidae, Lebiasinidae and Erythrinidae), a fish groups that are also closely-related to the Acestrorhynchidae (
The comparison of the different markers provides valuable insights into the chromosomal differentiation of Acestrorhynchus. In karyotypes of all species, the blocks of constitutive heterochromatin are located primarily in centromeric or telomeric regions, although large heterochromatic blocks are associated with the NORs, as seen as in most species of teleost fish of different families of different orders such as Anguilliformes, Siluriformes, Characiformes, among others (
The telomeric sequence was a particularly valuable cytogenetic marker, with a species-specific configuration in the four studied taxa, due to the large number of ITSs distributed in different pairs. In fishes, as in other vertebrates, the pericentromeric ITSs are found within or adjacent to the constitutive heterochromatin (
In Acestrorhynchus species all six ITS types have been observed. Larger sequences were observed in association with the blocks of constitutive heterochromatin in some chromosome pairs as revealed by the C-banding, although a number of the observed ITSs were not associated in any way with the heterochromatin (Fig.
Other types of ITS, not associated with the heterochromatin would have arisen through terminal translocations, the insertion of telomeric repetitions during the repair of breaks in double-strand DNA, or by the duplication or transposition of genes (
In the specific case of A. microlepis, remarkable differences were found between the individuals collected at the two localities (Catalão Lake and Apeu Stream, respectively), both in the location of the NORs and the number and location of the 18S rDNA sites. Thus the individuals of Pará (Apeu) were provisionally named A. prope microlepis. These chromosomal differences may reflect the presence of an unnamed species, that is, a past speciation event, which would have been caused by the geographic distance between the two populations. This distance would have minimized gene flow, isolating the populations, and permitting the fixation of specific rearrangements. A probable rearrangement was a pericentric inversion involving the NOR carrier pair, since NOR in three species was on the short arm and A. microlepis was on the same pair, but located on the long arm.
Another possible type of arrangement is the translocation of major ribosomal 18S sites, which were present in four other sites, in addition to the NORs. This movement may have been facilitated by transposable elements (TEs) associated with the heterochromatin, which has great potential to cause chromosomal rearrangements, as well as through ectopic recombination that can generate intrachromosomal recombination between copies of the same family of transposable elements, arranged in opposite positions (