Priscila Polaquini de Macedo Leite1 , Francisco de Menezes Cavalcante Sassi1, Manoela Maria Ferreira Marinho2, Mauro Nirchio3, Renata Luiza Rosa de Moraes, Gustavo Akira Toma1, Luiz Antonio Carlos Bertollo1 and Marcelo de Bello Cioffi1
Lebiasinidae is a well-supported monophyletic taxon containing seven genera and 75 valid species distributed in two subfamilies, Lebiasininae and Pyrrhulininae (Weitzman, Weitzman, 2003; Netto-Ferreira, Marinho, 2013; Fricke et al., 2021). Lebiasininae encompasses three genera, Lebiasina Valenciennes, 1847 (26 sp.), Piabucina Valenciennes, 1850 (1 sp.) and Derhamia Géry & Zarske, 2002 (1 sp.), and Pyrrhulininae other four ones, Nannostomus Günther, 1872 (21 sp.), Pyrrhulina Valenciennes, 1846 (19 sp.), Copella Myers, 1956 (6 sp.), and Copeina Fowler, 1906 (2 sp.) (Netto-Ferreira, Marinho, 2013; Fricke et al., 2021). All lebiasinids are found in freshwater and are endemic to Central and South Americas, except in Chile’s hydrographic basins. They are characterized by the absence of adipose fin, small anal fins, and an elongated body, which varies greatly in size, from 1.6 cm in Nannostomus to 20 cm in Lebiasina (Weitzman, Weitzman, 2003; Netto-Ferreira, 2010).
The first morphological investigations reported that Lebiasinidae would be related to Erythrinidae, Ctenoluciidae, Serrasalmidae, and Hepsetidae (Ortí, Meyer, 1997; Buckup, 1998; Oyakawa, 1998). However, molecular data suggested that many morphological synapomorphies among the abovementioned groups could be convergent evolutionary traits associated with their predatory lifestyle but positioning Lebiasinidae as a sister group to Ctenoluciidae (Oliveira et al., 2011). Lately, additional data from ultraconserved elements (UCEs) also corroborated their phylogenetic relatedness (Arcila et al., 2017; Betancur‐R. et al., 2019; Melo et al., 2022).
Thus far, only three Lebiasina species were found to occur in Brazilian waters at Serra do Cachimbo (PA): Lebiasina marilynae Netto-Ferreira, 2012, L. melanoguttata Netto-Ferreira, 2012, and L. minuta Netto-Ferreira, 2012, with a fourth additional species, Lebiasina yepezi Netto-Ferreira, Oyakawa, Zuanon & Nolasco, 2011, found in the Brazil-Venezuela border (Netto-Ferreira et al., 2011; Netto-Ferreira, 2012). Cytogenetic analyses pointed that the diploid number (2n = 36) is conserved for L. bimaculata Valenciennes, 1847,and L. melanoguttata, the only two species for which chromosomal data are known (Sassi et al., 2019). Significantly, this number is also conserved in all Ctenoluciidae representatives (de Souza e Sousa et al., 2017; Souza et al., 2021). In fact, two general trends appear to occur within the Lebiasinidae family: i) species with 2n = 36 bi-armed chromosomes, as in Lebiasina (Sassi et al., 2019) and some Nannostomus species (Sember et al., 2020), and ii) with higher diploid numbers, with mostly mono-armed chromosomes, as in Pyrrhulina (de Moraes et al., 2017, 2019, 2021), Copeina (Toma et al., 2019) and Nannostomus species (Sember et al., 2020). Up to now, Pyrrhulina semifasciata Steindachner, 1876 is the only species in the family that displays a morphologically differentiated sex chromosome system, of the X1X1X2X2/X1X2Y type (de Moraes et al., 2019).
The present study aimed to extend the knowledge on the trends and underlying mechanisms of karyotype differentiation in Lebiasinidae. Our main goal was to characterize the chromosomal patterns of the species Lebiasina minuta and highlight the contrasting evolutionary pathways inside the genus Lebiasina. Besides, we will test the hypothesis that a karyotype composed of 36 exclusively bi-armed chromosomes is also shared by other Lebiasina species, representing thus, a probable synapomorphy for this genus. For this, we applied conventional (Giemsa staining, C-banding) and molecular (mapping of repetitive DNA markers, comparative genomic hybridization (CGH), and whole chromosome painting (WCP)) in three Lebiasina species, one of them (L. minuta) now analyzed for the first time. This study is included in a series focusing on the cytogenetics and cytogenomics of Lebiasinidae fishes.
Material and methods
Sampling, chromosomes obtainment, and C-banding. Samples of Lebiasina minuta were collected at Serra do Cachimbo, Xingu River basin (Fig. 1; Tab. 1).The samples of L. bimaculata and L. melanoguttata were the same used by Sassi et al. (2019). The specimens were properly identified by morphological and meristic criteria by Dr. Manoela M. F. Marinho, an expert on Lebiasinidae taxonomy and were deposited in the fish collection of the Museu de Zoologia da Universidade de São Paulo (voucher number MZUSP 126519). The map was designed using the software QGIS Desktop 3.18 and Adobe CC Photoshop 2020.
FIGURE 1| Distribution of Lebiasina species with available cytogenetic data, highlighting the Brazilian state of Pará (orange) and Ecuadorian (purple) territories A. 1. L. bimaculata,2. L. melanoguttata (Sassi et al., 2019), and 3. L. minuta (this study). B. Highlights the position of A in South America, and C. indicates that, although close, species 2 and 3 does not share an overlapped distribution.
TABLE 1 | Sample sites, geographic coordinates, sampling number (N), diploid number (2n) and distribution of ribosomal DNA sequences on chromosomes of Lebiasina species.
Arenillas river lakes, El Oro – Ecuador
Sassi et al. (2019)
Serra do Cachimbo, Cachoeira da Serra – PA, Brazil
Pairs 1 and 13
Pairs 1, 2, 3, 7 and 9
Sassi et al. (2019)
PCH Três de Maio, Cachoeira da Serra – PA, Brazil
Pairs 1 and 13
Mitotic chromosomes were obtained from anterior kidney cells employing the classical air-drying method (Bertollo et al., 2015). Chromosomes were stained with 10% Giemsa diluted in Sorensen phosphate buffer (pH 6.8) and the constitutive heterochromatin regions were evidenced through the C-banding protocol (Sumner, 1972).
Probe obtainment and FISH-based experiments. Both 18S and 5S ribosomal DNA sequences (rDNAs) were cytogenetically mapped by FISH, using Hoplias malabaricus Bloch, 1794 (Characiformes, Erythrinidae) genome-isolated sequences. The 18S rDNA probe corresponds to a 1,400 base pairs (bp) segment of the respective gene (Cioffi et al., 2009), and the 5S rDNA probe includes 120 bp of the respective gene plus 200pb of non-transcribed spacers – NTS (Pendás et al., 1994). Both probes were labeled using a Nick-translation kit (Jena Bioscience, Jena, Germany), the 5S rDNA being labeled with Atto550-dUTP (red color) and 18S rDNA with Atto448-dUTP (green color). Three microsatellite sequences – (GA)15, (CA)15, (CGG)10 – that showed accumulation in other Lebiasinidae species previously analyzed (de Moraes et al., 2017, 2019; Sassi et al., 2019; Toma et al., 2019), were directly labeled during their synthesis (Kubat et al., 2008) with Cy-3 (Sigma-Aldrich, Darmstadt, Germany),and mapped on chromosomes, in addition to the telomeric sequence (TTAGGG)n using the Telomere PNA FISH Kit/FITC (DAKO, Glostrup, Denmark).
Microdissected first chromosome pair of Lebiasina bimaculata was used for whole chromosome painting (WCP) (Sassi et al., 2019) and labeled with Spectrum-Orange dUTP fluorophore (Vysis Inc, EUA). Two sets of comparative genome hybridizations (CGH) were also designed. The first one aimed to compare the genomic content of all analyzed Lebiasina species. For that, the male genomic DNAs (gDNAs) of L. bimaculata, L. melanoguttata, and L. minuta, were extracted from liver tissues (Sambrook, Russell, 2001), labeled by Nick-translation with Atto425-dUTP (light blue), Atto488-dUTP (green), and Atto550-dUTP (red) (Jena Biosciences, Jena, Germany), respectively, and co-hybridized against the male chromosomal background of L. minuta, using C0t-1 DNA (i.e., part of genomic DNA enriched for highly repetitive sequences) as a blocker of excessed shared repetitive sequences (Zwick et al., 1997). The final hybridization mixture was composed of 500ng of L. minuta gDNA, 500ng of the each compared gDNAs, and 25μl of unlabeled C0t-1 DNA, in a hybridization buffer containing 50% of formamide, 2× SSC, 10% SDS, 10% dextran sulfate and Denhardt´s buffer (pH = 7.0). The second experiment focused on intraspecific variations between males and females of L. minuta. Male and female-derived gDNA were also obtained by the standard phenol:chloroform:isoamyl alcohol protocol (Sambrook, Russell, 2001), and labeled with Atto550-dUTP (red), and Atto488-dUTP (green), respectively. A male metaphase preparation was used to co-hybridize both genomes. The final hybridization mix was composed of 500ng of male-derived gDNA, plus 500ng of female-derived gDNA and 15µg of unlabelled male-derived C0t-1 DNA. The chosen ratio of probes versus the C0t-1 DNA amount was based on previous data of our research group (Sassi et al., 2020). In all FISH-base experiments, chromosomes were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) and slides mounted with an antifade solution (VECTASHIELD, Vector Laboratories, Burlingame, CA, USA). All the hybridizations procedures followed the high-stringency protocol described in Yano et al. (2017).
Optical analyses and image processing. Metaphase plates were captured at a photomicroscope Olympus BX50 with CoolSNAP and images processed by the software ImagePro Plus 4.1. Chromosomes were classified according to their arms ratio (q/p), following Levan et al. (1964). Karyotypes were assembled with Adobe Photoshop CC 2020 software after the analysis of at least 30 metaphases for each sex to confirm the 2n number, karyotype structure and FISH results.
Conventional data and repetitive DNA mapping. Both males and females of L. minuta have 2n = 36 meta- and submetacentric chromosomes without heteromorphic sex chromosomes (Fig. 2A). C-positive heterochromatin occurs at the pericentromeric regions in all chromosomes (Fig. 2B). The “double-FISH” procedure showed a syntenic condition for both 5S and 18S rDNA sequences in the long (q) arms of the first chromosomal pair of males and females (Fig. 2C) with the 18S rDNA located on the terminal region, whereas 5S rDNA site is found in the pericentromeric region. Additional 5S rDNA sites are also found in the short (p) arms of the 13th chromosomal pair (Fig. 2C).
FIGURE 2| Male and female karyotypes of Lebiasina minuta after A. Giemsa staining, B. C-banding, and C. “double-FISH” with 5S (red) and 18S (green) rDNA probes. Scale bar = 5 µm.
Microsatellites (CA)15 and (GA)15 marks are mainly found in almost all chromosomes of L.minuta (Figs. 3A–B), while (CGG)10 marks occur only in the q terminal region of the first chromosome pair (Fig. 3C). Telomeric sequences (TTAGGG)n were only identified in their standard terminal regions in all chromosomes (Fig. 3D), without interstitial telomeric sites (ITS).
FIGURE 3| Metaphase chromosomes of Lebiasina minuta hybridized with microsatellite probes (A, B and C) and telomeric probes (D), using red signals. Scale bar = 5 µm.
Whole chromosome painting (WCP). The whole chromosome painting (WCP) with a derived probe from the first chromosome pair of L. bimaculata completely hybridized the first chromosome pair of L. minuta, with small-scattered signals in other chromosomes (Fig. 4).
FIGURE 4| Whole chromosome painting (WCP) highlighting the first chromosome pair of Lebiasina minuta completely hybridized with the probe from the first chromosome pair of L. bimaculata.
Intra- and interspecific comparative genomic hybridization (CGH). The genomic probes of L. bimaculata and L. melanoguttata successfully hybridized with the chromosomes of L. minuta. Notably, the Brazilian species L. melanoguttata and L. minuta share more repetitive sequences in their genomes than with the Ecuadorian species L. bimaculata, especially at the pericentromeric regions (Figs. 5A–D). The intraspecific comparison did not identify any sex-specific region, thus discarding the occurrence of distinguishable sex chromosomes (Figs. 5E–H).
FIGURE 5| First Row: Mitotic chromosome spreads of Lebiasina minuta males after CGH— interspecific comparisons (A–D). Male-derived genomic probe of L. minuta (A); L. melanoguttata (B); L. bimaculata (C) hybridized against male metaphase plates of L. minuta (D). Second Row: Mitotic chromosome spreads of Lebiasina minuta males after CGH— intraspecific comparisons (E–H). DAPI image (E); Male-derived genomic probe of L. minuta (F); Female-derived genomic probe of L. minuta (G) hybridized against male metaphase plates of L. minuta (H). The common genomic regions of both compared karyomorphs are depicted in yellow. Scale bar = 5 µm.
General chromosomal conservation. The current results show that the presence of 2n = 36 biarmed chromosomes is a general conserved characteristic in all the Lebiasina species analyzed. Such feature is also found in some other lebiasinids, such as Nannostomus eques Steindachner, 1876(Sember et al., 2020), but notably in all representatives of the Ctenoluciidae family, which is represented by the genera Boulengerella Eigenmann, 1903(Souza et al., 2021)and Ctenolucius Gill, 1861 (Souza et al., 2021). Such chromosomal relationship between the Lebiasinidae and Ctenoluciidae families, taken as sister groups (Arcila et al., 2017; Betancur‐R. et al., 2019), indicates that 2n = 36 biarmed chromosomes is a plesiomorphic condition within lebiasinids (Sassi et al., 2020). Thus, while Lebiasininae has retained the 2n = 36 biarmed chromosomes throughout its evolutionary history, the subfamily Pyrrhulininae followed a very different pathway, its species differing by presenting larger diploid numbers and mainly acrocentric chromosomes. Therefore, our results further support the evolutionary differentiation within the Lebiasinidae family by introducing new data on L. minuta.
Another shared characteristic refers to the first pair of chromosomes among the Lebiasina species, which can be easily differentiated, as it is the largest metacentric of the karyotype. Besides its morphological conservation, all three species also share a general genomic composition for this chromosome pair, as evidenced by the WCP experiments (Fig. 4). Indeed, this occurrence does not only refer to L. bimaculata and L. minuta as shown in the current study, but also to L. bimaculata and L. melanoguttata (Sassi et al., 2019). Therefore, this chromosome stands out as a possible useful marker for further investigations regarding the evolutionary process in the genus Lebiasina, as well as in the Lebiasinidae as a whole.
Chromosomal and genomic evolutionary differentiation. Despite the general conservation of the diploid number and chromosomal morphology, some particular features highlight the evolutionary differentiation that was fixed by each of the Lebiasina species, both at the chromosomal and genomic levels. This can be firstly easily verified concerning the distribution pattern of the constitutive heterochromatin (Fig. 2). It was previously shown that L. bimaculata and L. melanoguttata present centromeric and telomeric sites in several chromosome pairs, in addition to an exclusive set of noticeable interstitial bands in L. melanoguttata (Sassi et al., 2019). In turn, L. minuta differs from its sister species by having only pericentromeric sites, both in male and female karyotypes. It is known that heterochromatin has an important role in maintaining the chromosome structure and, consequently, it is usually associated with some specific regions, such as centromeres and telomeres. However, different distribution patterns are often found, even among closely related species as in Lebiasina, indicating probable additional roles played by the heterochromatin throughout the evolutionary pathways of the different species. In fact, the heterochromatic regions are composed by various types of repetitive sequences (Charlesworth et al., 1994; Kidwell, 2002) and, therefore, act as hotspots for chromosomal repatterning processes, driving an intragenomic dynamism during the evolutionary process of many fish species (Cioffi, Bertollo, 2012).
In fact, the distribution of repetitive DNAs represents a powerful tool in exploring the genome dynamics in fishes (Cioffi, Bertollo, 2012), which can be observed regarding the distribution of the rDNA sites among the Lebiasina species (Fig. 6). Concerning the 5S rDNA, all species share a site on the first chromosome pair, although in a different position and condition in L. bimaculata, including an additional site on chromosome 13 in L. melanoguttata and L. minuta. In turn, the 18S rDNA has a very different distribution among species. A single site is found in L. bimaculata and L. minuta, on chromosome pairs three and one, respectively, while L. melanoguttata has several sites distributed in the karyotype, including a bi-telomeric one in pair two (Fig. 6). Thus, the 18S rDNA presents a greater evolutionary dynamism among species compared to that noticed for the 5S. However, regardless of their numerical dispersion, it is notable that all 18S sites occupy a terminal position on chromosomes, which can be considered as a symplesiomorphic trait for lebiasinids, since Nannostomus, Pyrrhulina, Lebiasina, Copeina, and the representatives of the sister family Ctenoluciidae, also share such feature (Sember et al., 2020). In the same way, the syntenic condition for both 18S and 5S rDNAs in the first chromosomal pair of L. minuta and L. melanoguttata also occurs in other lebiasinids karyotypes, such as Pyrrhulina australis Eigenmann & Kennedy, 1903, Pyrrhulina aff. australis, P. brevis Steindachner, 1876 and Pyrrhulina cf. laeta (Cope, 1872) (de Moraes et al., 2017, 2019, 2021). This fact represents an exception among fishes, since a non-syntenic organization for both rDNA classes has been originally assumed to be the plesiomorphic condition for this group (Amemiya, Gold, 1988; Gornung, 2013). Such a syntenic condition can create recombination hotspots in association with heterochromatin (Gornung, 2013; Sochorová et al., 2018), facilitating intrachromosomal rearrangements, as observed in mice and humans (Cazaux et al., 2011; Tchurikov et al., 2021).
FIGURE 6 | Representative idiograms of L. bimaculata (A); L. melanoguttata (B) and L. minuta (C) highlighting the distribution of the 18S (green) and 5S (red) rDNA sequences; (CGG)n microsatellite (blue) and C-positive heterochromatin (black): Data for L. bimaculata and L. melanoguttata are from Sassi et al. (2019).
Although sharing the same 2n, CGH experiments (Fig. 5) and the microsatellite distribution (Fig. 3) suggest an advanced stage of sequence divergence among the Lebiasina species under study. Such internal reorganization in chromosomes is likely to be related to less identified rearrangements in fish karyotypes and has a marked role in the karyotype evolution of several animal groups (Matsuoka et al., 2004; Barby et al., 2019). Our results indicate that repetitive sequences have divergent patterns of distribution and accumulation, probably fostering the chromosomal differentiation and biodiversity, thus highlighting the differential paths taken by the evolutionary process when comparing the genome organization of the trans-Andean species, L. bimaculata, with the two exclusive Brazilian species, L. minuta and L. melanoguttata.
In spite of the difficulties in obtaining good chromosomal preparations for small-sized fish, the cytogenetics of the Lebiasinidae family has experienced considerable progress in recent years, both on conventional and molecular procedures. Our current data on L. minuta support that 2n = 36 two-armed chromosomes is a plesiomorphic condition for the genus Lebiasina, reinforcing its proximity to Ctenoluciidae species as a sister group. However, despite the maintenance of a general karyotypic macrostructure, these species highlight differential evolutionary features regarding the distribution of repetitive elements of the genome, indicating their dynamic in their genomic differentiation pathways. It is also notable that the distribution and amplification of repetitive DNA classes across the chromosomes followed independent evolutionary trajectories among the Lebiasina species. The two exclusively Brazilian species, L. minuta and L. melanoguttata, are more related to each other, sharing a more genomic closeness than with the trans-Andean species, L. bimaculata. In fact, allopatry is often considered the most common source of speciation among Neotropical fishes (Seehausen, Wagner, 2014) and here may have contributed to the biodiversity of such Lebiasinidae fishes.
This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq – 302449/2018–3 to MBC); Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP – 2020/11772–8 to MBC, 2020/03046–5 to PPML, 2020/02681–9 to FMCS). This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brasil (CAPES – Finance code 001).
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Priscila Polaquini de Macedo Leite1 , Francisco de Menezes Cavalcante Sassi1, Manoela Maria Ferreira Marinho2, Mauro Nirchio3, Renata Luiza Rosa de Moraes, Gustavo Akira Toma1, Luiz Antonio Carlos Bertollo1 and Marcelo de Bello Cioffi1
 Laboratório de Citogenética de Peixes, Departamento de Genética e Evolução, Universidade Federal de São Carlos (UFSCar), Rod. Washington Luiz, s/n, 13565-905 São Carlos, SP, Brazil. (PPML) email@example.com (corresponding author), (FMCS) firstname.lastname@example.org, (RLRM) email@example.com, (GAT) firstname.lastname@example.org, (LACB) email@example.com, (MBC) firstname.lastname@example.org.
Priscila Polaquini de Macedo Leite: Formal analysis, Methodology, Validation, Visualization, Writing-original draft.
Francisco de Menezes Cavalcante Sassi: Data curation, Formal analysis, Investigation, Methodology, Software, Writing-review and editing.
Manoela Maria Ferreira Marinho: Formal analysis, Investigation, Validation, Writing-review and editing.
Mauro Nirchio: Formal analysis, Validation, Writing-review and editing.
Renata Luiza Rosa de Moraes: Investigation, Methodology, Writing-review and editing.
Gustavo Akira Toma: Formal analysis, Methodology, Visualization, Writing-review and editing.
Luiz Antonio Carlos Bertollo: Investigation, Methodology, Validation, Visualization, Writing-review and editing.
Samples were collected under licenses 48628-2 and A96FF09 issued by the Brazilian bureaus of environmental control ICMBio/SISBio and SISGEN, respectively. All procedures followed ethical, and anesthesia conducts according to the Committee of Ethics in Animal Use and Experimentation of the Universidade Federal de São Carlos (Process number CEUA 1853260315).
The authors declare no competing interests.
How to cite this article
Leite PPM, Sassi FMC, Marinho MMF, Nirchio M, Moraes RLR, Toma GA, Bertollo LAC, Cioffi MB. Tracking the evolutionary pathways among Brazilian Lebiasina species (Teleostei: Lebiasinidae): a chromosomal and genomic comparative investigation. Neotrop Ichthyol. 2022; 20(1):e210153. https://doi.org/10.1590/1982-0224-2021-0153
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Accepted January 11, 2022 by Claudio Oliveira
Submitted October 22, 2021
Epub March 21, 2022