“Split Happens”: a Rare Case of Chromosomal Mosaicism in Characin from an Impacted River Basin

Abstract

Chromosomal polymorphisms have been reported in numerous vertebrates, including fishes. Such variation is usually related to karyoevolutionary trends and speciation but might also be a result of environmental stress. Herein, we provide the first karyotype characterization in populations of a widespread small characin (Hemigrammus marginatus) from an impacted river basin in Northeastern Brazil to infer their chromosome organization and putative genotoxic effects. Therefore, we reported intra and interindividual structural polymorphism and multiple active nucleolar organizer regions in samples from Contas River, a highly polluted environment when compared to less impacted areas. Furthermore, this report represents the first description of chromosomal mosaicism in the family Characidae so far. Besides revealing peculiar karyotypic patterns in relation to populations from other basins, our results suggest that the chromosomal instability in specimens H. marginatus might be associated with the presence of xenobiotics in polluted environments.

Keywords:
Chromosomal polymorphism; environmental stress; pollution

HIGHLIGHTS

First cytogenetic characterization in Hemigrammus marginatus from Northeastern Brazil.

First case of chromosomal mosaicism in Characidae.

Polluted environments are putatively associated with chromosomal instability.

INTRODUCTION

The Contas River Basin stands out as the largest drainage system entirely located in the State of Bahia (Northeastern Brazil), comprising an area of 55,334 km² [¹], and occupying three physiographic regions referred to as Upper, Middle, and Lower Contas [²]. This basin flows through distinct morphoclimatic domains, from Caatinga to Atlantic Forest, being characterized by a high richness of ichthyofauna and remarkable endemicity [³], as revealed by several descriptions of new restricted-range fish species [⁴,⁵].

Nevertheless, human activities have increasingly impacted several spots along Contas River watersheds [¹]. Most of these impacts refer to the use and occupation of land and water resources, including livestock farming, crop irrigation, mining and hydroelectric power plants, besides runoffs of urban and industrial effluents [⁶,⁷]. In this regard, environmental monitoring of anthropogenic effects along this freshwater system has been intensified over the last decades. For instance, chemical analyses of tissues of the frog Leptodactylus ocellatus revealed a positive correlation of the contamination by metals (Mn, Cr, Zn, Ni, Cu, and Fe) when compared to the sediment of Contas River [⁸]. In addition, specimens of L. ocellatus collected in urban areas, especially near the municipality of Jequié, revealed higher concentrations of heavy metals when compared to the samples from rural areas [⁸].

Furthermore, genotoxic assessment studies in native fish species along Contas River reported a remarkable incidence of nuclear abnormalities (NAs), such as micronuclei (MN) and nuclear buds (NBs), in populations from urban regions, as a result of trace metal accumulation and biomagnification [⁹,¹⁰]. MN encompasses broken or entire chromosomes that were lost from the main nucleus during mitosis or the S-phase, but remained encapsulated in their own nuclear envelopes within cells [¹¹]. The origin of NBs is similar to that inferred for MN, but they usually consist of chromosomal fragments attached to the main nucleus as small projections on the nuclear membrane [¹²]. In general, the NAs are highly informative for genotoxic studies because they are bioindicators of DNA damage, being associated with the accumulation of heavy metals and other pollutants in fish cells [¹³]. In fact, some xenobiotics (e.g., pesticides) trigger clastogenic effects, thus leading to DNA instability and chromosomal abnormalities [¹⁴-¹⁵]. Therefore, highly polluted freshwater environments might affect cell homeostasis and, particularly, division processes [¹⁴].

Cytogenetic analyses have proved to be helpful in understanding the potential effects of polluted environments on genome architecture since chromosomal rearrangements are main drivers of reproductive isolation and speciation, as commonly reported in fishes [¹⁶,¹⁷]. A notable example of the association between chromosomal variability and environmental pollutants was described in Hoplerythrinus unitaeniatus (Erythrinidae) [¹⁸]. Natural populations of this fish species from isolated and human-modified aquatic ecosystems in Prata River (state of Minas Gerais, Southeastern Brazil) presented an intricate pattern of intra and interindividual chromosomal variation [¹⁸]. Another case of chromosomal mosaicism was also identified in Trichomycterus paolence, in which the numerical variation among karyotypes of a single specimen was putatively associated with somatic nondisjunction and consequential post-zygotic aneuploidies [¹⁹]. These examples of intra-individual chromosomal instability may be potentially favored by the accumulation of pollutants in freshwater habitats.

Considering the lack of cytogenetic studies in fish populations from several groups along Northeastern Brazil basins and the role of these analyses in inferring environmental stress, we report the first cytogenetic characterization of Hemigrammus marginatus from distinct localities in Contas River Basin. Considering the environmental impacts of some sites, we tested the potential effects of pollution on the cell homeostasis of animals from polluted habitats.

MATERIAL AND METHODS

Study area

The specimens of Hemigrammus marginatus were collected at two sites (localities 1 and 2) along the Middle Contas River sub-basin (Figure 1A). The collection site 1 refers to the main channel of Contas River in the ecotone between Caatinga and Atlantic Forest, downstream the Pedras Dam and upstream Jequié (-13.870806, -40.186673). The collection site 2 is approximately 30 km apart from locality 1 and refers to the Preto do Criciúma River, a tributary of Contas River. The latter flows from a rural area along the Atlantic Forest towards Jitaúna (-13.999085, -39.927951).

Figure 1
Map of the Contas River basin (A), highlighting the collection sites (1 - main channel of Contas River in Jequié, and 2 - Preto do Criciúma River) of the characin Hemigrammus marginatus (B) along the Contas River basin.

According to the Resolution n. 127 9/2020 by the State Water Resources Council (CONERH) [²⁰], the water quality of both sampled localities diverges substantially from each other [²¹]. Therefore, locality 1 is categorized as “Class 4”, with a remarkable presence of thermotolerant coliforms due to the massive release of untreated sewage. On the other hand, the locality 2 meets the standards established for “Class 1”, characterizing a good-quality water source and suitable for human use after simple treatment.

Furthermore, the land cover and land use around these sites are also significantly differentiated. To illustrate these differences, we defined an area of 5-km around each collection site from the MapBiomas Collection 8 [²²] to evaluate the proportion of land cover and land use categories in each region (Figure 2). Remarkably, half of the area around location 1 is occupied by pastures and agriculture (52%) while the other half comprises natural areas (46%) (Figure 2A). Conversely, location 2 is surrounded mainly by Atlantic Forest fragments (63%), followed by pastures (26%) and a few crops (9.4%) (Figure 2B). Satellite images of both collection sites obtained using the software Google Earth® were also informative to reveal the deep changes in natural landscape close to locality 1 when compared to locality 2 (Figure 2C, D).

Figure 2
Maps of land cover/land use maps (area radius = 5 km) and satellite images (Google Earth®, Sep 19^th, 2023) of collection sites in Contas River - locality 1 (A, C, D), and Preto do Criciúma River - locality 2 (B, E). The colors represent land use and cover categories according to MapBiomas, Collection 8 [²²] (green = natural areas; light brown = pastures; red = urban and highly modified areas; pink = agricultural fields). The central black dot represents the collection sites

Cytogenetic analysis

Thirty-seven specimens of Hemigrammus marginatus were collected at the two sites in the Contas River Basin, as follows: 20 (14 females and 6 males) from Contas River (location 1) and 17 (10 females and 7 males) specimens from Preto do Criciúma River (location 2). The specimens were captured under permit by SISBIO (n. 31360-1) using cast and gill nets.

Afterwards, all individuals were inoculated with fungal antigens and kept in fish tanks for 12 to 24 h to stimulate cell division [²³]. Next, they were euthanized in iced water [²⁴], and portions of the anterior kidney were removed to obtain the mitotic chromosomes at metaphase stage [²⁵].

The constitutive heterochromatin was detected by C-banding [²⁶] while the active nucleolar organizer regions (Ag-NORs) were visualized by silver nitrate staining [²⁷]. The GC- and AT-rich sites were analyzed by base-specific fluorochromes using chromomycin A₃ (CMA₃), distamycin A (DA), and 4′,6-diamidino-2-phenylindole (DAPI), respectively [²⁸].

About 10 metaphases per individual were analyzed using an epifluorescence photomicroscope (Olympus BX-51) and the images were acquired using the software Image Pro-Plus v. 6.2. (Media Cybernetics). The chromosomes were classified based on their arm ratio [²⁹] and the pairs were organized into karyotypes by decreasing size order within each morphological class (m - metacentric; sm - submetacentric; a - acrocentric). The software Easy Idio 1.0 [³⁰] was used to measure the chromosomes as well as to construct the ideograms.

RESULTS

All specimens of H. marginatus shared a modal diploid number (2n) of 50 chromosomes (Figure 3A). Overall, we observed a standard karyotype organized into 12m+36sm+2a chromosomes in both populations with a fundamental arm number (FN) equal to 98. As frequently reported in characins with 2n=50, a large first metacentric pair was commonly observed, while no heteromorphic sex chromosomes were detected.

On the other hand, we identified intra and interindividual chromosomal variation in two specimens from location 1, hereafter referred to as specimens A and B. Specimen A exhibited three distinct karyomorphs, characterizing the occurrence of chromosomal mosaicism. We designated them as karyomorphs I, II, and III. The karyomorph I was similar to the standard karyotype described above (Figure 3A) while karyomorph II exhibited a size heteromorphism between homologous from the pair 1 (Figure 3B). The karyomorph III diverged from the standard karyotype as a result of structural differences between chromosomes 1a and 1b, resulting in a karyotype organized into 11m+37sm+2a (Figure 3C). In addition, all cells from specimen B presented two acrocentric pairs, thus resulting in a karyotype organized into 12m+34sm+4a (FN= 96), being named as karyomorph IV (Figure 3D). Ideograms of karyomorphs II, III, and IV are shown in Figure 4 to provide a comparative analysis of the major differences in chromosome macrostructure.

Figure 2
Giemsa-stained karyotypes of Hemigrammus marginatus. karyomorph I (standard) (A); karyomorph II showing heteromorphism in the size of chromosomes of pair 1 (B); karyomorph III showing a morphological difference between chromosomes 1a and 1b (C); karyomorph IV (specimen “B”), distinguished by the presence of two acrocentric pairs (D).

Figure 3
Ideograms representing the distinct karyomorphs in populations of Hemigrammus marginatus from Contas River basin. The karyomorph I was commonly observed in specimens from both sampled collection sites (A) while the others were restricted to specimens from the main channel in Contas River (locality I). The karyomorphs II (B) and III (C) show the size and structural heteromorphism in pair 1, respectively (specimen “A”), while the ideogram of karyomorph IV (specimen “B”) is differentiated by the presence of two acrocentric pairs (24 and 25) (D).

In turn, the distribution of constitutive heterochromatin was similar between both populations (Figures 5A, 5B), although heterochromatic blocks were less conspicuous in specimens from location 2, especially in pairs 6, 8, and 17 (Figures 5A, 5B). In addition, the C-banding revealed conspicuous heterochromatic blocks on the short arms of pair 2 and near the centromeres of pair 8. Less stained C-bands were observed at pericentromeric regions of pairs 6, 9, 10, 11, 12, 17, 22, and 23, centromeric regions of pairs 1, 3, 4, 14, 20, and 21, and terminal regions of pairs 3, 5, 9, and 25.

A multiple Ag-NOR system was detected in specimens from location 1, including a major active NOR at the interstitial region on short arms of pair 2, as well as additional sites on the long arms in one homologous of pair 8 and short arms in one homologous from pair 13 (Figure 5C). On the other hand, the specimens from location 2 exhibited single active Ag-NORs on the short arms of pair 2, coinciding with the major cluster observed in samples from location 1 (Figure 5D). The GC-rich sites (CMA₃ ⁺/DAPI^- signals) were interspersed with Ag-NORs from pair 2 in all specimens from both collection sites (Figures 5C, 5D).

Figure 4
C-banded (A, B) and silver-nitrate stained (C, D) karyotypes of Hemigrammus marginatus from Contas (A, C) and Preto do Criciúma (B, D) rivers. The major Ag-NOR chromosomes bearing GC-rich signals are shown inbox.

DISCUSSION

A modal diploid number of 2n=50, no heteromorphic sex chromosome system, and the presence of a large first metacentric pair, as observed in populations of H. marginatus from the present study, have been previously reported in samples of the same species from Upper Paraná River basin in southern Brazil [³¹]. However, the specimens from the Contas and Paraná watersheds were differentiated by their karyotype formula (standard karyotype of 12m+36sm+2a in the former versus 10m+34sm+6a in the latter) [³¹]. These results indicate independent karyoevolutionary pathways between both lineages, probably driven by their high degree of geographical isolation, thus leading to distinct chromosomal rearrangements, particularly pericentric inversions.

Besides the differences in chromosome macrostructure commonly observed in fish populations and species from distinct basins, cases of polymorphic C-bands have already been reported in several characins [³¹,³²]. In fact, the heterochromatin pattern of H. marginatus from the Contas River basin differs from those reported for other populations [³¹]. Again, these findings reinforce the unique evolutionary histories of each lineage, providing a preliminary evidence of cryptic diversity. On the other hand, no significant differences in C-banding were observed among samples from each locality in the Contas River basin, suggesting that heterochromatin regions were not involved in the process of karyotype diversification among nearby populations. Since Preto do Criciúma and Contas rivers are 30 km apart from each other and belong to the same watershed, it was expected that samples from both localities share a common biogeographical history and short periods of divergence, if any, thus hindering conspicuous differences in chromosome structure [³³].

On the other hand, the specimens from Contas River presented multiple Ag-NORs, including a major active rDNA cluster co-located with GC-rich segments (Figure 5C) equivalent to the single NOR-bearing pair observed in the population from Preto do Criciúma River (Figure 5D). Even though this is a common feature in fishes [³⁴], it should be pointed out that silver nitrate staining only reveals ribosomal gene sequences that were actively expressed in the preceding interphase since Ag ions present high affinities with ribosomal proteins [³⁵]. However, even though CMA₃ ⁺signals and NORs are usually colocated in fish chromosomes, additional Ag-NORs might lack GC-rich sites, as reported in several species characterized by multiple NOR systems [³⁶], including the samples from the present study. Therefore, the polymorphism in Ag-NORs when the samples of Contas and Preto do Criciúma rivers are compared to each other would reflect a differential expression pattern of ribosomal sequences between both populations (Figures 5C, 5D).

Such polymorphic nature of Ag-NORs has been widely reported in representatives of the family Characidae, ranging from single NORs in genera like Moenkhausia [³¹], Bryconamericus [³⁷], Piabina [³⁸], and Tetragonopterus [³⁹] to multiple NOR systems in many taxa, including cases of intraspecific variation [⁴⁰]. As a matter of fact, the multiple NOR system herein observed in the population of H. marginatus from Contas River contrasts with the single NORs in specimens from Preto do Criciúma River as well as with the pattern previously reported in populations of the same taxon in Upper Paraná River basin [³¹].

In addition, cases of chromosomal polymorphism have been occasionally detected in Characiformes fishes, such as Hoplerythrinus unitaeniatus [¹⁸] and Serrapinnus heterodon [⁴¹]. In the present study, we describe the first case of both chromosomal polymorphism and mosaicism in the genus Hemigrammus related to inter- and intraindividual variation in karyotype structure, respectively. This unusual condition was observed in specimens of H. marginatus from Contas River, a highly polluted environment [¹,²,⁶,⁷]. Environmental pollutants and other xenobiotics are recognizably responsible for disorders in DNA replication thus triggering chromosomal instability (CIN) and aneuploidies [⁴²]. Putatively, the local presence of chromosome mosaicism observed in specimens from a polluted habitat such as Contas River could be correlated with the potential effects of environmental stress on cell homeostasis.

In particular, the intraindividual chromosome differences (karyomorphs II and III) in specimen A of H. marginatus should result from structural chromosomal rearrangements. In karyomorph II (Figure 3B), duplication-deletion events via unequal crossing-over involving the terminal portion of homologous from pair 1 might explain the heteromorphic size condition, even though artifactual effects related to the degree of chromatin compartmentalization should not be disregarded [⁴³]. Likewise, we infer that the differentiated morphology in homologous of the first m pair in karyomorph III (Figure 3C) is an outcome of a heterozygous pericentric inversion. In this scenario, chromosome 1b would be a variant form derived from karyomorph I.

On the other hand, the karyomorph IV differs from the standard karyotype (karyomorph I) by the presence of an additional acrocentric pair in spite of the maintenance of 2n=50. Moreover, the acrocentric chromosomes from pair 24 in karyomorph IV are similar in size to the sm pairs 19 and 20 in karyomorph I. Therefore, the extra acrocentric chromosomes in the karyomorph IV (Figure 3D) should have evolved from a pericentric inversion involving one of the smallest sm pairs observed in the standard karyotype (Figure 3A).

Unfortunately, the understanding of the processes directly responsible for chromosomal abnormalities in wild animals from impacted environments remains largely unknown. Nonetheless, highly polluted environments, such as the Contas River channel, associated with errors in somatic segregation could be potential causal factors for this phenomenon. According to reports from regulatory agencies [¹,²,⁷], several human activities have been affecting the water quality in Contas River. It should also be emphasized that such environmental disturbances along this river are not a recent event, since historical and cultural records have shown increased and conspicuous levels of anthropogenic intervention in Contas River over time [⁶,⁴⁴,⁴⁵].

In spite of the limited evidence of the cause-effect relationships between xenobiotics and cell homeostasis in non-model organisms, cytologic analyses in fishes have been widely used as bioindicators to assess the genotoxic effects of water contaminants in aquatic biota [⁴⁶-⁴⁹]. For instance, the clastogenic activity of xenobiotics on the DNA of fishes has proved to affect chromosomal stability, as evidenced by the increasing of Rex 1 retrotransposon copies and heterochromatic blocks in specimens of Colossoma macropomum (Characiformes, Serrasalmidae) exposed to copper sulfate [⁵⁰]. Comparably, previous ecotoxicological analyses on the aquatic biota from the Contas River along the same collection site of H. marginatus showed that cumulative amounts of trace metal pollutants in fishes were directly related to nuclear abnormalities in native fish species [⁹,¹⁰].

Several molecular mechanisms have been described to explain such chromosomal alterations, mostly related to errors in DNA replication, repair, and recombination processes. Furthermore, some genomic regions are more susceptible to rearrangements, such as repetitive DNA sequences [⁵¹-⁵²]. For example, ribosomal cistrons and hAT elements are clustered with microsatellite motifs near chromosomal fusion points in Rineloricaria latirostris (Siluriformes: Loricariidae) [⁵³] while 5S rRNA pseudogenes were found to be hotspots for chromosomal rearrangements in this species [⁵⁴]. As a matter of fact, rDNA regions such as NORs have been regarded as unstable genomic regions prone to double-strand DNA breaks, transposon invasions, and chromosomal rearrangements in a plethora of groups [⁵⁵-⁵⁷]. In addition, an increased number of active ribosomal cistrons might represent an adaptive and positively selected trait to stressful environmental conditions [⁵⁸,⁵⁹]. In this sense, previous reports on aquatic organisms have shown that specimens from highly polluted environments tend to accumulate active NORs [⁶⁰]. Our results corroborate this hypothesis since the specimens of H. marginatus from a highly polluted site in Contas River showed multiple Ag-NORs whereas those from Preto do Criciúma River (less impacted site) shared a single NOR-bearing pair. Thus, the presence of additional active NOR sites associated with a highly polluted environment could explain to some degree the emergence of chromosomal rearrangements in the population of H. marginatus from the Contas River.

Moreover, additional evidence for the role of environmental contamination as a potential driver of chromosomal polymorphism and mosaicism in H. marginatus from polluted habitats is provided by the interpopulation comparative cytogenetic analysis from the present study. Differently from the main channel of Contas River, the landscape surrounding Preto do Criciúma River is clearly less impacted by human use, besides presenting abundant riparian vegetation, whereas the former is characterized by degraded areas driven by urbanization activities and untreated sewage effluent (Figure 2). Moreover, livestock farming and monoculture crops have been intensified in areas close to the Contas River [⁶,⁶¹], which represent potential sources of pesticide runoffs to aquatic habitats [⁶²,⁶³]. As a result, no cases of chromosomal variation were detected in the population of H. marginatus from Preto do Criciúma River even though a similar number of specimens was cytogenetically analyzed when compared to the population from Contas River.

CONCLUSION

The inter and intraindividual chromosomal variability in H. marginatus from a highly impacted river might be associated with the differential levels of pollution in aquatic environments, thus serving as a potential bioindicator of water quality and risk assessment to both local biota and humans. Furthermore, the comparative analyses of cytogenetic data in Hemigrammus marginatus reinforce that this putatively widespread taxon should actually represent a species complex, what remains to be further investigated. Furthermore, based on the comparative analyses of cytogenetic data in Hemigrammus marginatus suggests that this putatively widespread taxon in Brazilian hydrographic basins might encompass cryptic forms what remains to be further investigated.

Acknowledgments

The authors are grateful for the permit granted by the Committee of Ethics in Experimentation with Animals from the State University of Southwestern Bahia (CEUA/UESB 32/2013).

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• Funding:

  This research was financially supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (financial code 001).

Edited by

Editor-in-Chief:

Paulo Vitor Farago

Associate Editor:

Marcelo Ricardo Vicari

Publication Dates

• Publication in this collection
  08 Nov 2024
• Date of issue
  2024

History

• Received
  14 Feb 2024
• Accepted
  14 Aug 2024