Research Article |
Corresponding author: Luís Reginaldo Ribeiro Rodrigues ( luisreginaldo.ufpa@hotmail.com ) Academic editor: Grazyna Furgala-Selezniow
© 2018 Ivanny Coelho da Fonseca, Luan Aércio Melo Maciel, Frank Raynner Vasconcelos Ribeiro, Luís Reginaldo Ribeiro Rodrigues.
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:
Fonseca IC, Maciel LAM, Ribeiro FRV, Rodrigues LRR (2018) Karyotypic variation in the long-whiskered catfish Pimelodus blochii Valenciennes, 1840 (Siluriformes, Pimelodidae) from the lower Tapajós, Amazonas and Trombetas Rivers. Comparative Cytogenetics 12(3): 285-298. https://doi.org/10.3897/CompCytogen.v12i3.22590
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The genus Pimelodus LaCépède, 1803 comprises 35 formally recognized species distributed along the major neotropical river basins. Despite conservatism in diploid number with 2n=56, an intense variation of chromosomal morphology (karyotypic formula) has been documented in Pimelodus species. In the present study, we analyzed karyotypes of 20 specimens, identified as Pimelodus blochii Valenciennes, 1840 and collected from the lower courses of the Tapajós, Amazonas and Trombetas Rivers. The karyotypes were characterized by Giemsa conventional staining, C-banding, silver staining (Ag-NOR) and fluorescent in situ hybridization (FISH) with 5S and 18S rDNA probes. The karyotypes showed 2n=56 chromosomes in fish from the Tapajós River. In contrast, fish from the Amazonas and Trombetas Rivers had 2n=58. The nucleolus organizing regions were labeled on the short arm of an acrocentric chromosome as demonstrated by silver staining and FISH. Signals for 18S and 5S rDNA were co-localized on one chromosome pair. Our results demonstrate karyotypic divergence between Tapajós and Amazonas-Trombetas populations of P. blochii, interpreted as supporting the existence of a species complex in this taxon.
Amazon basin, catfish, Pimelodus , rDNA, species complex
The genus Pimelodus LaCépède, 1803 (Siluriformes, Pimelodidae) comprises 35 valid species exclusively distributed in neotropical freshwater drainages. It is commonly recorded in the Amazonas, Orinoco, Araguaia-Tocantins, São Francisco, and Paraná-Paraguay River basins. In the Amazon basin, seven species of Pimelodus have been recorded: Pimelodus albofasciatus Mees, 1974, Pimelodus blochii Valenciennes, 1840, Pimelodus altissimus Eigenmann & Pearson, 1942, Pimelodus jivaro Eigenmann & Pearson, 1942, Pimelodus ornatus Kner, 1858, Pimelodus pictus Steidachner, 1876, and Pimelodus tetramerus Ribeiro & Lucena, 2006 (
Cytogenetic analysis of 32 Pimelodidae family members revealed a conservative karyotypic macrostructure with 2n=56 chromosomes save for a few exceptional karyotypes (2n=50) in Calophysus macropterus Lichtenstein, 1819, Pinirampus pirinampu Spix & Agassiz, 1829 and Luciopimelodus plati Valenciennes, 1835 species (
Diploid chromosome numbers of eleven previously investigated Pimelodus species showed variation from 54 to 58 with eleven distinct karyotype formula (see Table
So far, two distinct karyotypes were reported for two Pimelodus blochii populations; 2n=56 for the Araguaia River, and 2n=58 for the Amazon River (
In the present paper, we investigate the karyotype of Pimelodus blochii from the lower portions of the Tapajós, Amazonas and Trombetas Rivers in order to evaluate their chromosomal features and contribute the debate on the species taxonomy. The karyotypes were characterized by conventional Giemsa staining, C-banding, silver staining (Ag-NOR) and fluorescent in situ hybridization (FISH) technique with 5S and 18S rDNA probes.
Twenty (20) specimens were collected from four localities in the Tapajós, Amazonas and Trombetas Rivers (Table
River | Collection sites | GPS Coordinates (datum WGS84) | n |
---|---|---|---|
Tapajós | Itaituba | 4°16'12.6"S, 55°58'37.1"W | 6 |
Amazonas | Santarém | 2°25'8.0"S, 54°44'28.6"W | 6 |
Chicaia River, Almeirim | 1°38'15.6"S, 52°57'46.2"W | 4 | |
Trombetas | Oriximiná | 1°45'52.2"S, 55°52'18.8"W | 4 |
External morphology and coloration of Pimelodus blochii specimens examined in the present study. A specimen from the Tapajós River (ITB-14, SL=150 mm; W=55 g) B specimen from the lower Amazonas River at Santarém (STXVI-2, SL=145 mm; W=52 g) C specimen from the lower Amazonas River at Almeirim (ALC-2, SL=110 mm; W=9 g) D specimen from the Trombetas River (PO-22, SL=108 mm, W=33 g).
Intra-abdominal colchicine (0.0125%) injection was performed at 0.01 ml/g (
Conventional staining was performed with 5% Giemsa solution (phosphate buffer, pH 6.8). The C-banding protocol from Sumner (1972) was followed with minor changes. The NORs were stained with silver nitrate following the
FISH was used for mapping 18S and 5S rDNA loci (
The PCR products were labeled by nick translation with biotin-14-dATP (BioNick Labeling System kit, Invitrogen/ThermoScientific, Waltham, Massachusetts, USA) and digoxigenin-11-dUTP (DIG-nick translation mix, Roche, Basel, Switzerland). The slides were treated with RNase solution (5 µl RNase 10 mg/mL diluted in 975 µl 2×SSC) for a short period of time. The fixed chromosomes were denatured in 70% formamide (pH 7.0 2×SSC) and heated at 70 °C for 5 min. The hybridization solution mixture was prepared with 20 µl formamide + 8 µl of 50% dextran sulfate + 4 µl of each probe + 4 µl of 20×SSC. The slides were incubated in 2×SSC solution in a humidified and heated (37 °C) chamber overnight.
Post-hybridization washes were performed with 15% formamide at 42 °C for 10 min, three washes in 0.1×SSC at 60 °C for 5 min, and 0.5% Tween20 at room temperature for 5 min. For signal detection, slides were placed in NFDM buffer (20 ml of 20×SSC, pH 7.0 + 5 g of powdered skim milk + 80 ml of distilled water) for 15 min, followed by two washes in 5% Tween20 for 5 min at room temperature.
The hybridized probes were applied in a mixture containing 20 µl anti-digoxigenin-rhodamine (1:200) (Roche, Basel, Switzerland) + 4 µl FITC-Avidin (1:100) (Sigma, St. Louis, Missouri, USA) + 26 µl of C buffer (0.1 M sodium bicarbonate, 0.15 M sodium chloride; pH 7.0) for 60 min. The slides were coated with anti-fading reagent Vectashield H-1000 (Vector Laboratories, Burlingame, California, USA) and chromosomes were counterstained with DAPI (1,2-diamidin-phenyl-indol).
At least 30 metaphases were counted to determine the diploid chromosome number. The best spread metaphase plates were photographed with a CCD camera (Moticam 10 MP) coupled to a Zeiss Axioskop40 microscope for conventional/banding images, and a Nikon Eclipse CI for FISH images. The contrast and brightness were adjusted with ADOBE PHOTOSHOP CS3. The chromosomes were arranged as metacentric (m), submetacentric (sm), subtelocentric (st) and acrocentric (t) following
The diploid chromosome number was observed as 56 in the Tapajós River fish (Itaituba population). On the other hand, 2n=58 chromosomes were recorded from Amazonas (Santarém and Almeirim populations) and Trombetas Rivers (Oriximiná population) samples, with minor variation in the karyotypic formula, as 30m/sm+28a and 26m/sm+32a, respectively (Fig.
Giemsa-stained karyotypes of Pimelodus blochii from the Amazon Basin. A 2n=56 chromosomes (36m/sm + 20a) B 2n=58 chromosomes (30m/sm + 28a) C 2n=58 chromosomes (26m/sm + 32a). Scale bar: 10 μm.
The C-banding results showed small amounts of constitutive heterochromatin in the centromeres. Terminal C-bands were observed in 18 to 24 chromosome pairs from the Tapajós River specimens (Fig.
Metaphases of Pimelodus blochii showing NOR by silver staining (left) and double FISH of 18S rDNA (green) and 5S rDNA (red) (right). Specimens from the Tapajós River (A, E); from the Lower Amazonas River at Santarém (B, F), at Almeirim (D, H); and from the Trombetas River (C, G). Scale bar: 10 μm.
The 5S rDNA probe showed distinct localizations among the samples. Co-localization of 5S and 18S rDNA to a single chromosome pair was detected in the Trombetas and Amazonas Rivers populations (Almeirim population); this syntenic pattern also occurred in the Santarém population but on just one homologous chromosome. The Tapajós River specimens’ karyotypes showed a distinct position for 18S and 5S rDNA (Figs
Variation of 18S rDNA (green signal) and 5S rDNA (red signal) chromosomal sites among Pimelodus blochii populations from Amazon basin. The samples from Amazonas River showed two distinct patterns: one observed in the Almeirim population (A) and other observed in the Santarém population (B).
The karyotype macrostructure of Pimelodus blochii from the Tapajós, Amazonas and Trombetas Rivers are compatible with a previous report (
Compiled data from the literature on karyotypic traits in Pimelodus species. m=metacentric, sm=submetacentric, st=subtelocentric, a=acrocentric, q=long arm, t=terminal, c=centromeric, inter=interstitial, peri=pericentromeric.
Species | 2n | Karyotypic formula | 18S | 5S | References |
---|---|---|---|---|---|
Pimelodus fur | 54 | 32m+8sm+6st+8a | q, t, sm | q inter m, q peri sm | 5 |
P. microstoma | 56 | 22m+22sm+6st+6a | q, t, st | peri sm, q peri st | 1; 2 |
P. argenteus | 56 | 24m+16sm+12st+4a | 3 | ||
P. britskii | 56 | 24m+18sm+8st+6a | q, t, st | p inter sm, q t st | 4 |
P. maculatus | 56 | 32m+12sm+12st | q, t, sm | q inter m, q t sm q peri sm | 5; 6 |
P. absconditus | 56 | 24m+18sm+8st+6a | – | – | 7 |
P. mysteriosus | 56 | 26m+20sm+2st+8a | – | – | 3; 8 |
P. ornatus | 56 | 18m+22sm+6st+10a | – | – | 9; 7 |
P. ortmanni | 56 | 24m+18sm+8st+6a | – | – | 10; 11; 12 |
P. paranaensis | 56 | 22m+22sm+4st+8a | – | – | 13 |
P. blochii | 56/58 | 36m/sm+20st/a; 30m/sm+28a; 26m/sm+32a | q, c, a | q, c, a | 14; 15; 16; 17 |
Despite extensive conservatism in diploid number, variation in karyotypic formula has been frequently detected (
A single NOR (one pair) is the most common pattern observed in the Pimelodus karyotypes (
Our specimens collected from the Chicaia River, a tributary of the lower Amazonas (Almeirim population), have the typical pigmentation for the A variety, whereas the specimens from the Santarém population, collected at the confluence of the Amazonas and Tapajós Rivers, had the B variety pigmentation. Although both populations conserved the diploid number 2n=58 and karyotypic formula (30m/sm+28a), they diverged in their 18S and 5S rDNA locations (Fig.
The populations of Pimelodus blochii from the lower courses of Amazonas, Tapajós and Trombetas rivers presented differentiated karyotypes based on variation in diploid number and chromosome morphology. The specimens collected from the Tapajós River, with 2n=56, are clearly distinguished from the others and may constitute a new, undescribed Pimelodus species.
This research was supported by grants from CAPES/PRO-AMAZÔNIA (Auxpe-3318/2013). The authors are grateful to the native fishermen (Haroldo Leite, Mr Naldo, Mr Nelson, William Vasconcelos, Janison Vasconcelos, Mr Claudenido) for helping us with fieldwork and to the LGBIO/UFOPA staff for help in the laboratory. ICF received Mastership from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). This work is part of the Project “Estudos Citogenéticos e Citogenômicos da Biodiversidade da Amazônia, com implementação de avanços técnicos” (Cytogenetic and cytogenomic studies of Amazonian biodiversity implementing technical advances) coordinated by Dra Cleusa Y. Nagamachi (UFPA).