Research Article |
Corresponding author: Diego Javier Grassi ( diegograssi@scripps.edu ) Academic editor: Grazyna Furgala-Selezniow
© 2017 Diego Javier Grassi, Ana Cláudia Swarça, Jorge Abdala Dergam, Maria Cristina Pastori, Alberto Sergio Fenocchio.
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:
|
Hoplias malabaricus (Bloch, 1794), a predatory freshwater fish with a wide distribution throughout South America, represents a species complex with seven well characterized karyomorphs at the cytogenetic level. Although this species has been extensively studied in several Brazilian basins, data are still scarce for hydrographic systems from other South American countries. This study aims to characterize cytogenetically the Hoplias malabaricus populations from the Argentinean Central Region, close to the southernmost distribution of this species complex. A total of 32 specimens from the Ctalamochita River, a tributary of Lower Paraná Basin located in the province of Córdoba, were analyzed using cytogenetic techniques (Giemsa staining, C- and Ag-NOR banding and fluorescent in situ hybridization with 18S rDNA). All the specimens showed diploid number 2n=42, chromosomic formula 22m + 20sm and absence of sexual chromosomes. Thus, the analyzed populations belong to the karyomorph named A. These populations showed a remarkable degree of divergence in their cytogenetic traits such as karyotypic formula, C-banding, NORs and 18S rDNA patterns for Hoplias malabaricus from other populations bearing the same karyomorph in the Middle and Upper Paraná Basin. These findings are consistent with molecular data from a recent study (where specimens collected in the present work were included), which indicate a closer phylogenetic relationship of Hoplias malabaricus populations from the Ctalamochita River with those from the Uruguay basin and the coastal regions of South Brazil than with populations from the Middle and Upper Paraná Basin. Overall, these pieces of evidence highlight the distinctive features of Hoplias malabaricus from the Ctalamochita River, and also reveal a complex history of dispersion of these populations. The present work is the first to provide cytogenetic information and include some phylogeographic aspects of Hoplias malabaricus populations living in close proximity to the southernmost extreme of its distribution area. Therefore, this study expands significantly upon the previously known geographical coverage for karyomorph A and contributes to a better understanding of the karyotypic diversification within this species complex.
Biogeography, cytogenetics, Hoplias malabaricus , karyomorph A
Hoplias malabaricus (Bloch, 1794) is a predatory freshwater fish that belongs to the order Characiformes and family Erythrinidae. It is a widespread species inhabiting 44 out of 52 South American ecoregions proposed by
Classically, Hoplias malabaricus specimens have been recognized by a few morphological features, such as convergence of dentary bone toward the symphysis, resulting in a ‘‘V’’ shape pattern and tooth plates in the tongue. Although Hoplias malabaricus has been considered as a single biological species in reference to its morphological traits, every sampled population shows a bimodal pattern of variation: they are either 2n=42 or 2n=40 with slight modifications. Several cytogenetic studies indicate that Hoplias malabaricus represents a species complex, with seven well characterized karyomorphs (or karyotypic variants, also known as cytotypes) nominated with letters A to G, which differ with regards to their diploid numbers, chromosome morphology and the presence of sex chromosome systems (
Frequently, cytogenetic evidence is also supported by, and consistent with, molecular data obtained from analysis of nuclear and mitochondrial DNA sequences (nuDNA and mtDNA), which reveal a marked divergence and genetic structuration between karyomorphs, suggesting reproductive isolation between the karyomorphs. Additionally, combination of cytogenetic and molecular approaches together with geological information (i.e., estimate data of ancient episodes such as stream piracy between rivers, orogenic activity, etc., involved in vicariance events) has been particularly useful in the study of dispersal events and phylogenetic relationships (
All the aforementioned evidence strongly indicates the existence of a complex of cryptic species grouped into the typical Hoplias malabaricus morphotype (
It is noteworthy to mention that, although the lack of detailed morphometric information has led to group different species, well defined at the genetic level, in a single one on the basis of a common morphology, some karyomorphs of H. malabaricus seem to exhibit subtle morphological differences. For example, in a recent study focused on analysis of morphometric parameters (
Despite its wide geographic distribution, most of karyotypic and molecular research in H. malabaricus populations has been carried out in Brazil and the few studies conducted in Argentina have been restricted to Mesopotamic region (
The Pampa Plain is a vast region characterized by gentle slopes, occasionally interrupted by low geomorphological reliefs. The hydrographic systems developed in this area form both endorheic and exorheic basins. Among the latter, the Ctalamochita River (also known as Tercero River) is one of the most important hydrographical systems. This river, located in the province of Córdoba, is a tributary of the Lower Paraná River Basin. Along the Ctalamochita River, there are five reservoirs (artificial lakes), the two most important of them called Embalse Río Tercero (built in 1936) and Piedras Moras (built in 1978). The fish assemblage in the Ctalamochita River is characterized by the presence of members of the families Characidae, Pimelodidae and Erythrinidae, among others. Therefore, H. malabaricus is an indigenous species (
The aim of the present study was characterizing at the cytogenetic level Hoplias malabaricus populations from headwater of the Ctalamochita River using standard and fluorescent in situ hybridization techniques.
Thirty two specimens of Hoplias malabaricus consisting of 8 males, 10 females and 14 specimens of undetermined sex were collected in the headwaters of the Ctalamochita River, Córdoba Province, Argentina (Fig.
- Embalse Río Tercero reservoir and Rio Grande stream
# 1 (32°13'S, 64°32'W): 4 specimens (2 males, 2 females)
# 2 (32°13'S, 64°25'W): 14 specimens (14 juvenile stages, undetermined sex)
- Piedras Moras reservoir and Soconcho stream
# 3 (32°11'S, 64°19'W): 3 specimens (1 male, 2 females)
# 4 (32°11'S, 64°18'W): 11 specimens (5 males, 6 females)
Specimens (Fig.
a Ctalamochita River location in Province of Córdoba, Argentina (left and middle images). Asterisk (*) indicate location of the Colorado River in Argentina (in red), the limit of distribution of Hoplias malabaricus and most of Neotropical fishes. Distribution of sampling sites is indicated along headwater basin (right image, black dots) b Representative specimen of Hoplias malabaricus caught in the Ctalamochita River and analyzed in the present study (right picture). All specimens showed the typical morphological feature identifying Hoplias malabaricus, the V-shaped gular region (left picture). Bar = 10 cm.
Mitotic chromosome preparations were obtained according to the technique described by
Nucleolus organizer regions (Ag-NORs) were visualized with silver staining following
Fluorescent in situ hybridization (FISH) experiments were performed using biotinylated 18S rDNA probes (1700 pb long fragments) obtained from the nuclear DNA of the fish Oreochromis niloticus (Linnaeus, 1758) labeled with biotin-14-dATP by nick translation (Gibco cat N°18247-015), according to the manufacturer’s instructions. The hybridization technique, post-hybridization washes and visualization were carried out following
The preparations were analyzed with an Olympus BX50 microscope, and the best metaphases were captured with a SONY camera, model Exware HAD coupled to the microscope. The FISH slides were observed and the images acquired with a Leica DM 4500 microscope equipped with a DFC 300F9 camera and Leica IM50 4.0 software.
More than thirty metaphases from each specimen were analyzed and the best of them were used to make karyotypes. The chromosomes were arranged in groups classified according to their arm ratios as metacentrics and submetacentrics (
Tissue samples from several specimens were collected for further molecular analysis (study of sequences of mitochondrial and nuclear DNA) and were deposited in the Laboratory of Molecular Systematic Beagle, Universidade Federal of Viçosa, Minas Gerais, Brazil.
The entire collection was split in two groups, which were deposited at the Museum of Zoology, Universidad Nacional of Córdoba, Argentina (specimens numbered from 1 to 18) and at Fish Cytogenetics Laboratory, Universidad Nacional of Misiones, Argentina (specimens numbered from 19 to 32).
All the specimens of Hoplias malabaricus collected from the headwater region of the Ctalamochita River exhibited a diploid number of 2n=42 chromosomes. The karyotype was composed of 22m + 20sm, with a NF=84 (Fig.
a Male Hoplias malabaricus karyotype with conventional Giemsa staining (karyomorph A) b Metaphase corresponding to karyotype showed in (a) c Male meiotic metaphase showing formation of 21 bivalents d Female Hoplias malabaricus karyotype (karyomorph A) e C-banding karyotype exhibiting centromeric staining in most of chromosomes and telomeric signal in pairs number 14 and 20 f Ag-NOR banding karyotype displays telomeric signal in chromosome pair 20 gFISH with 18S rDNA probes labeling two chromosomic pairs, numbers 12 and 20 (boxed). Bar = 5 µm.
Analysis of meiotic preparations allowed the identification of 21 bivalents without atypical pairing among them, supporting lack of chromosomal differentiation between sexes (Fig.
Patterns of heterochromatin distribution revealed by C-banding were mostly associated with centromeric regions of all chromosomes of the complement, as well as in the telomeric region of some pairs (Fig.
Nucleolus Organizer Regions identified by impregnation by silver nitrate (AgNORs) were located in telomeric position on the long arm of a small chromosome pair number 20 (Fig.
These data coincided with results from FISH technique with 18S rDNA probes, which revealed two positive submetacentric pairs: pair number 12, clearly identified due to its size and exhibiting probe signal in its pericentromeric position, and pair number 20, showing probe signal in its long arm (Fig.
It is important to note that, concerning all chromosome markers and macrostructural features analyzed in this study, no significant differences were found between populations of Embalse Río Tercero and Piedras Moras reservoir. Thus, it seems these populations are relatively homogeneous (Fig.
Idiogram referring to the Hoplias malabaricus populations (karyomorph A) from the Ctalamochita River, highlighting the chromosome markers. Black: C-positive heterochromatin; red: 18S rDNA sites; yellow: nucleolar organizer regions (NORs). Putative pericentromeric NOR in chromosome pair 12 was also included.
Hoplias malabaricus populations from the headwater of the Ctalamochita River (Córdoba Province, Argentina) were characterized at the cytogenetic level, providing the first data for this hydrographic system and also for the Argentinean Central Region. These populations exhibited karyotypes with 2n=42 chromosomes composed of 11 metacentric and 10 submetacentric pairs, without apparent heteromorphic sex chromosomes. These results qualify them as belonging to karyomorph A previously described by
Despite sharing some chromosomal similarities with other karyomorph A-bearing populations occurring in the Upper and Middle regions of Paraná Basin, cytogenetic traits of the Ctalamochita population such as karyotypic formula, C-banding, NORs and 18S rDNA patterns were significantly divergent (Figure
Fixation of chromosome rearrangements within major karyomorphs is not completely surprising taking into consideration some biological aspects of Hoplias malabaricus, as its wide distribution in most of South America basins, sedentary habits and adaptations to live in small and isolated populations. These features would favor a random fixation of chromosome rearrangements and, thus, certain degree of intra-karyomorph variation would be expected (
With respect to distinctive cytogenetic features of these populations, it is important to highlight relevant traits for submetacentric chromosome pairs 12 and 20. In both cases the presence of heterochromatic bands coincident with 18S rDNA sites was demonstrated; in chromosome pair 12 located in pericentromeric position and in chromosome pair 20 in the long arm. With regards to Ag-NORs only chromosome pair 20 had a clear, positive staining for silver nitrate impregnation, showing a conspicuous positive NOR in telomeric position of the long arm. As was mentioned previously, in some cases (approximately 5%) weak NOR-positive staining was observed in pericentromeric position of submetacentric pair number 12, but it was not possible conclude if this variability was caused by limited accessibility of silver nitrate to highly compacted heterochromatic regions or by the occurrence of structural Ag-NOR polymorphisms (
Overlapping of C- and Ag-NOR banding suggests the presence of heterochromatic blocks interspersed with ribosomal cistrons in the same region, as previously reported for several chromosomic pairs in karyomorph A (
These high levels of chromosomal differentiation within the 2n=42 A karyomorphs proposed by
Although the spread of karyomorph A populations through interaction between coastal drainages and dispersal into continental basins has been previously studied and demonstrated only for restricted Brazilian coastal regions (
Additionally, fluctuations of sea level during glacial-interglacial cycles and temporary connections between adjacent basins would also have had an important role in spreading karyomorph A Hoplias malabaricus populations through coastal regions (
While glacial – interglacial cycles may have promoted dispersal of H. malabaricus coastal populations, at the same time they would also have had profound effects on the Pampa Plain ecosystems. The dominant climate characteristics of the Pampa Plain region during these glacial periods were: cold with frequent snowing and windy conditions, with scarce precipitations and frequent decreases of basin water flow. Thus, cycles of expansion of steppe-like climate over the Pampa Plain region occurred during glacial epochs. Constant propagule pressure from the Brazilian region favored exchange and turnover of species in these regions after glacial periods, and this fauna advance over Pampa Plain region would have taken place approximately 15 times during the last one million years (
The chronological frame of these events fits with the estimated dispersal time of the haplogroup including Hoplias malabaricus populations from Ctalamochita and Uruguay Rivers and its tributaries (
In light of the previously mentioned data, it is evident that the current geographic distribution pattern of Hoplias malabaricus in the Ctalamochita River, and probably most of the ichthyofauna in the Pampa Plain region, is a result of the intricate combination of climatic and geomorphological factors. Overall, cytogenetic and molecular evidence presented in this study highlights the distinctive chromosomal features of Hoplias malabaricus from the Ctalamochita River, and also reveals a complex history of dispersal of these populations.
Taking into consideration the new findings described previously, the present work is the first to provide cytogenetic information and also including some phylogeographic aspects of Hoplias malabaricus populations living in close proximity to the southernmost extreme of its distribution area, in Argentinean Central Region. Therefore, this study expands significantly upon the previously known geographical coverage for karyomorph A and contributes to a better understanding of the karyotypic diversification within this species complex.
To Dr. Gustavo Haro (Universidad de Córdoba), for his invaluable knowledge about ichthyofauna of rivers of Córdoba and save part of the collection used in this study in the Museum of Zoology, Universidad Nacional of Córdoba, Argentina. Mr. Mario Ledesma (Estación de Hidrobiología y Piscicultura. Entidad Binacional Yacyretá. Misiones, Argentina) for his advices about technical issues and processing of images.