A new karyotype and a molecular phylogeny of Lebiasinidae (Teleostei: Characiformes) shed light on the chromosomal evolution of the family

Renata Luiza Rosa de Moraes^1,2, Fernando Henrique Santos de Souza^1,2, Geize Aparecida Deon^1,2, Manoela Maria Ferreira Marinho³, Marcelo de Bello Cioffi¹ and Francisco de Menezes Cavalcante Sassi^1,4

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Associate Editor: Priscila Camelier

Section Editor: Izeni Farias

Editor-in-chief: Carla Pavanelli

Abstract​

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Introduction​

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Research on non-model species generally presents several challenges when compared to model ones. Genomic resources, such as whole-genome scaffold data or well-annotated reference genomes, are usually scarce, or even unavailable for these species (Ekblom, Galindo, 2011; Russel et al., 2017). Cytogenetic techniques are a very powerful tool in the study of non-model organisms since they characterize the chromosomal morphologies, ploidy, and position of specific genomic markers without prior information (Cioffi et al., 2012). As a result, it can aid in clarifying taxonomic issues and improving comprehension of the relationships between species or populations (Potter, Deakin, 2018). For example, cytogenetics has contributed to finding cryptic diversity and even species complexes in fishes (e.g., Moreira-Filho, Bertollo, 1991; Bertollo et al., 2000; Kavalco et al., 2009; Castro et al., 2015; Ferreira et al., 2017; Nirchio et al., 2018; Pazza et al., 2018; Rocha-Reis et al., 2018, among others). Other approaches, such as the use of mitochondrial markers like 16S rRNA, cytochrome oxidase I (COI), and the Control Region (CR), are widely applied to understand how different groups of organisms evolved and to study population genetics (e.g., Imoto et al., 2013; Bronstein et al., 2018). However, mitochondrial DNA analysis can generate distortions in the results when reticulate events are present since it represents a maternally inherited marker (Toews, Brelsford, 2012). Thesampling of nuclear markers is required to overcome such inheritance biases and provide a more reliable perspective on the evolutionary history of the species under study (Tollesfrud et al., 2009). Furthermore, incomplete lineage sorting, introgression, and the effect of homoplasy can be better resolved with the use of several genetic markers (Mirarab et al., 2016; Simmons, Gatesy, 2021).

Using complexity reduction techniques that take advantage of the high throughput of NGS technologies is one of the primary methods used to access the diversity and genetic structure of these species at the nuclear level. This approach allows for the cost-effective acquisition of a large number of independent genetic markers across the genome (Davey et al., 2011). Among the complexity reduction methods, double digest restriction-site associated DNA (ddRAD) sequencing is capable of generating a high quantity of genome-wide molecular markers suitable for phylogenetics and population structure analysis (Peterson et al., 2012; Dierickx et al., 2015; Kozlov et al., 2017; Edet et al., 2018; Ivanov et al., 2021; Fahey et al., 2022; Kriuchkova et al., 2023). In ddRAD sequencing, the DNA is digested by two distinct restriction enzymes before sequencing, generating small fragments which can be used as molecular markers without the need for a reference genome (Peterson et al., 2012). DArTseq sequencing (Diversity Arrays Technology) is a ddRAD variation that allows for the enrichment of hypomethylated regions (Jaccoud et al., 2001; Kilian et al., 2012). Since molecular cytogenetics approaches primarily focus on repetitive and hypermethylated regions, DArTseq is a useful complementary strategy for cytogenetics investigations in population genetics, phylogenetic, and phylogeographic inferences (e.g., Cioffi et al., 2019; de Oliveira et al., 2019; Panthum et al., 2021; Schimek et al., 2022).

The Lebiasinidae includes seven genera (Lebiasina Cuvier & Valenciennes, 1847, Piabucina Cuvier & Valenciennes, 1850, Derhamia Géry & Zarske, 2002, Nannostomus Günther, 1872, Pyrrhulina Valenciennes, 1846, Copella Myers, 1956, and Copeina Fowler, 1906) and 74 valid species that are native to the Neotropical region and widely spread in South and Central America (Costa Rica and Panama), except for Chile (Fricke et al., 2024). Two subfamilies are recognized based on morphological characters: Lebiasininae, which includes Derhamia, Lebiasina, and Piabucina, and Pyrrhulininae, the most varied clade, encompassing the genera Nannostomus, Copeina, Copella, and Pyrrhulina (Weitzman, Cobb, 1975; Géry, Zarske, 2002). Unpublished phylogenetic analysis, however, recovers Derhamia as a basal taxon within the Pyrrhulininae (e.g., Netto-Ferreira, 2010; Marinho, 2014). In general, Lebiasinidae is composed of small-sized fishes that range in length from 1.5 to 7.0 cm and exhibit a wide diversity of body forms and colors, making them very appealing to aquarium hobbyists (Weitzman, Vari, 1988; Weitzman, Weitzman, 2003). Even with this diversity, some of the proposed species are very similar, therefore, taxonomic identification of species is sometimes difficult, demanding the use of additional techniques such as cytogenetics or barcoding (Xu et al., 2024). In the same way, issues of phylogenetic placement also occurred. For a long time, the phylogenetic relationships of Lebiasinidae were uncertain (Ortí, Meyer, 1997; Buckup, 1998; Calcagnotto et al., 2005; Oliveira et al., 2011; Arcila et al., 2017; de Pinna et al., 2018; Betancur-R et al., 2019; Mirande, 2019; Cassemiro et al., 2023; Near, Thacker, 2024). Phylogenetic reconstruction based on morphological characters recovered Lebiasinidae closely related to Erythrinidae, Ctenoluciidae, and Hepsetidae (Buckup, 1998) and Tarumanidae (de Pinna et al., 2018). However, phylogenies based on molecular data recovered Lebiasinidae as relatedto Serrasalmidae, Erythrinidae, and Hepsetidae (Ortí, Meyer, 1997) or Ctenoluciidae. Combined data have recurrently indicated Lebiasinidae as a sister taxon to Ctenoluciidae, with divergence around 60 million years ago (mya) (Calcagnotto et al., 2005; Nakatani et al., 2011; Oliveira et al., 2011; Rabosky et al., 2013; Arcila et al., 2017; Betancur-R et al., 2019; Mirande, 2019; Cassemiro et al., 2023; Near, Thacker, 2024).

Because of their small size, karyotypes and other cytogenetic data are still scarce for Lebiasinidae (Arai, 2011). The main challenge with the cytogenetics of such small fish isobtainingaccurate information on the number and quantity of chromosome spreads (De Moraes et al., 2019). As a result, the studies were limited to describing haploid and/or diploid chromosome numbers in some species (Scheel, 1973; Oliveira et al., 1991; Arai, 2011). Thanks to improvements in methods, studies using both classical and molecular cytogenetics have made a considerable difference in our understanding of the karyotype of this group of fishes (De Moraes et al., 2017, 2019, 2021, 2023; Sassi et al., 2019, 2020; Toma et al., 2019; Sember et al., 2020; Ferreira et al., 2022; Leite et al., 2022). Such investigations included the first cytogenetic data for Lebiasina, Copeina, Nannostomus, and Pyrrhulina and proposed trends in karyotype evolution.Here, to gain insights into the evolution of Lebiasinidae fishes, we describe for the first time the karyotype of Copella callolepis (Regan, 1912), filling an important gap in the cytogeneticknowledge of the family. We also used complexity reduction methods to produce a set of single nucleotide polymorphic sites (SNPs) to reconstruct the evolutionary relationships of some taxa within the family. With the assistance of these new data, we assessed the chromosomal evolution patterns of Lebiasinidae and proposed their most likely evolutionary pathways.

Material and methods

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Sampling. Thirteen individuals (11♂ and 2♀) of Copella callolepis were collected in Tefé – AM (03°25’50.7”S 54°44’54.8”W) and taxonomically analyzed to confirm the species based on the analysis of meristic characters following Marinho (2014). A list of all individuals used in cytogenetics and sequencing analysis is presented in Tab. 1. Sampling sites for all species are presented in Fig. 1, andthe map was produced using the software QGis v. 3.4.4 (https://qgis.org), Inkscape 0.92 (https://inkscape.org), and Adobe Photoshop CC 2020 (San Jose, CA, USA).

TABLE 1 | Lebiasinidae species analyzed in this study. Diploid number (2n), detailed sampling site location, and geographical coordinates with the respective references. INPA, Instituto Nacional de Pesquisas da Amazônia, Manaus; LEC, Laboratório de Citogenética Evolutiva, Universidade Federal de São Carlos, São Carlos; MZUSP, Museu de Zoologia da Universidade de São Paulo, São Paulo.

Species	2n	Sampling site	Geographical coordinates	Voucher	References
Boulengerella lateristriga	36	Novo Airão, AM, Brazil	02°37′28.5′′S 60°58′16.8′′W	INPA-ICT 053246	Souza e Sousa et al. (2017)
Copeina guttata	42	Tefé River, AM, Brazil	03°39’49.5”S 64°59’40”W	MZUSP 124915	Toma et al. (2019)
Copella callolepis	36	Tefé River, AM, Brazil	03°25’50.7"S 54°44’54.8"W	LEC 23341	Present study
Lebiasina bimaculata	36	Arenillas River, El Oro, Ecuador	03°30’57.2”S 80°03’44.2”W	MZUSP 124457	Sassi et al. (2019)
Lebiasina melanoguttata	36	Cachoeira da Serra, PA, Brazil	08°46′ 59.4”S 54°58’26.9”W	MZUSP 124625	Sassi et al. (2019)
Lebiasina minuta	36	Cachoeira da Serra, PA, Brazil	08°44’39.0"S 55°02’03.0"W	MZUSP 126519	Leite et al. (2022)
Nannostomus anduzei	22	Zamula Stream, Barcelos, AM, Brazil	00°04’57.5"S 67°06’23.8"W	LEC 23359	De Moraes et al. (2023)
Nannostomus beckfordi	44	Agenor Stream, AM, Brazil	02°55’53.9"S 59°58’30.7"W	MZUSP 123071	Sember et al. (2020)
Nannostomus eques	36	Cuieiras River, AM, Brazil	02°47’58.1"S 60°29’19.8"W	MZUSP 123079	Sember et al. (2020)
Nannostomus marginatus	42	Adolpho Ducke Reserve, AM, Brazil	02°55’53.9"S 59°58’30.7"W	MZUSP 123083	Sember et al. (2020)
Nannostomus unifasciatus	22	Cuieiras River, AM, Brazil	02°47’58.1"S 60°29’19.8"W	MZUSP 123084	Sember et al. (2020)
Pyrrhulina aff. australis	40	Branco River, MT, Brazil	15°11’28.0"S 57°41’30.7"W	MZUSP 119079	De Moraes et al. (2017)
Pyrrhulina aff. marilynae	40	12 de Outubro Stream, MT, Brazil	12°58’41.0"S 60°00’34.0"W	MZUSP 119077	De Moraes et al. (2021)
Pyrrhulina sp.	40	Alto Alegre do Parecis, RO,  Brazil	12°11’58.0"S 61°46’47.4"W	MZUSP 123080	De Moraes et al. (2021)

FIGURE 1| Map of South America with sampling sites indicated marked as dots. Each color represents a distinct species, as indicated on the legend in the left corner. An Copella callolepis individual is presented above the legend (photo by José L. O. Birindelli).

Cytogenetics procedures. Tissues (liver, kidney, spleen, and gills) were collected using a stereomicroscopegiven the small size of the species. Chromosome preparation from kidney fragments followed the air-drying technique (Bertollo, 1978). Following the dissection, the complete fish bodies were preserved in 100% ethanol and deposited in the tissue bank of the Laboratório de Citogenética Evolutiva (LEC), Universidade Federal de São Carlos (São Carlos, São Paulo, Brazil). Constitutive heterochromatin was revealed by the standard C-banding procedure (Sumner, 1972). The 5S and 18S rDNA probes were isolated from the Hoplias malabaricus (Bloch, 1794) genome, following Pendás et al.(1994) and Cioffi et al.(2009), respectively. The 5S rDNA probe included 120 base pairs (bp) of the 5S rDNA gene and 200 bp of the non-transcribed spacer (NTS) (Pendás et al., 1994). The 18S rDNA probe was composed of a 1400-bp-long segment of the 18S rDNA (Cioffi et al., 2009). Both probes were directly labeled with the Nick Translation Mix Kit (Jena Bioscience, Jena, Germany) according to the manufacturer’s instructions: the 18S rDNA probe with Atto488-dUTP (green fluorescence) and the 5S rDNA with Atto550-dUTP (red fluorescence). Both rDNA sequences were mapped by fluorescence in situ hybridization (FISH) with high stringency conditions following Pinkel et al.(1986) and modifications by Sassi et al.(2022). Briefly, metaphase chromosomes were treated with RNAse A (40 µg/mL) for 1.5 h at 37 °C and the metaphase chromosomes were denatured in 70% formamide/2xSSC at 72 °C for 3 min. A total of 20 µL of the hybridization mixture (2.5 ng/L probes, 50% deionized formamide, and 10% dextran sulfate) was then applied to the slides, and the hybridization process occurred overnight at 37 °C in a humidified chamber. The first post-hybridization wash was performed with 1xSSC for 5 min at 65 °C in a shaker, followed by 4xSSC/Tween for 5 min at room temperature. Chromosomes were counterstained with DAPI in Vectashield (Vector Laboratories, Burlingame, CA, USA).

Sequencing and SNP calling. We selected a total of 41 individuals from 13 Lebiasinidae species and Boulengerella lateristriga (Boulenger, 1895) (Ctenoluciidae) for sequencing (Tab. 1). We used the Ctenoluciidae species as our outgroup since this family is consistently recovered as the sister of Lebiasinidae in both morphological and molecular-based phylogenies (Calcagnotto et al., 2005; Nakatani et al., 2011; Oliveira et al., 2011; Rabosky et al., 2013; Arcila et al., 2017; Betancur-R et al., 2019; Mirande, 2019; Cassemiro et al., 2023; Near, Thacker, 2024). From the 13 Lebiasinidae species, three of them were classified as aff. or sp., given the uncertainty about their taxonomic classification, which impairs the complete classification as a current valid species from the low number of individuals sampled. These classifications of aff., cf. and sp. are also supported by differences in cytogenetic characteristics previously described by De Moraes et al.(2017, 2019). We selected a pool of three individuals from each species for sequencing. From each individual, we extracted a small fragment of liver or muscle and stored them in 100% ethanol. We performed the DNA extraction according to the protocol described by Sambrook, Russell (2001). DNA quality was assessed using a NanoDrop spectrophotometer (ThermoFisher Scientific, Branchburg, NJ,USA) and then sent the samples to Diversity Arrays Technology Pty Ltd. (Canberra, Australia), for the DArTseq procedure.

DArTseq is a sequencing technique related to ddRAD that relies on two distinct restriction enzymes employed to select fragments for sequencing across the whole genome: a frequent cutter and a nuclease with affinity for hypomethylated regions, generally PstI and SphI (Kilian et al., 2012). The sequencing was performed on the Illumina HiSeq 2500 platform, using three individuals per species (except for Pyrrhulina aff. australis). A third of the sequences are analyzed twice as technical replicates, and from these replicates a measure of repeatability is calculated. After sequencing and demultiplexing the reads, all generated adapters were trimmed, and sequences went through a quality filtering process. The quality of the reads was checked, and the resulting reads were processed with proprietary DArT software to guarantee the reliability of the data. The reads were cluster reads among individuals, resulting in high-quality de novo sequences. The repeatability measure is used to filter the sequences, keeping only those with low error rate and high quality. From these sequences, a final dataset comprising Single Nucleotide Polymorphisms (SNPs) was generated, with SNPs coded as 1 for heterozygotes, 0 for reference homozygotes, and 2 for alternate homozygotes. We have filtered out all monomorphic loci present on the dataset and converted the data to PHYLIP with dartR v. 2.9.7 package (Gruber et al., 2018).

Principal Component Analysis. To provide an overview of the distribution of genetic diversity among samples, we carried out a Principal Component Analysis (PCA). We performed the analysis with two distinct datasets, the first comprising data from all sampled species, and the second only Nannostomus data. We imported the SNP matrix as a genlight object, a data structure format from the adegenet package, included in the dartR package, to efficiently store and manipulate large-scale genetic marker data such as the SNPs herein used, including the combined information on the genotypes and individual metadata. The PCA was then performed using dartR v. 2.9.7 (Gruber et al., 2018).

Phylogenetic analysis and Neighbor-net. We extracted and concatenated 692 sequences (each with a maximum size of up to 69 bp) with the “gl2fasta” function of the package dartR v. 2.9.7 (Gruber et al., 2018), resulting in a dataset 38.25 kb in length for phylogenetic analysis. To better understand the genetic relationships between sampled Lebiasinidae species, we estimated a maximum likelihood species tree using RAxML-NG (Stamatakis, 2014). Before the analysis, we concatenated all sequences from each sample in a PHYLIP format using the dartR package. Initially, we conducted a model test to define which substitution model best adjusted to our data. We estimated the substitution model with the maximum likelihood approach using ModelTest-NG (Darriba et al., 2020) with a template parameter adjusted to RAxML-NG, datatype set as DNA, and seed number defined as 12345. We set all remaining parameters as default. We considered the model with the smallest Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC) the best model. The phylogenetic analysis was conducted using the GTR+Gamma+I model with four rate categories, setting parameters to generate 20 distinct starting trees (10 random starting trees and 10 parsimony trees). A total of 100 bootstrap replicates were performed. We used a random seed number of 12345 for both the parsimony starting trees and the bootstraps. The support values of the bootstrap trees were mapped to the best tree in RAxML-NG with the support command. The consensus tree was exported with FigTree v. 1.4.4. To test for any signals of reticulation, which includes hybridization, introgression, or other events that generate deviation from the bifurcating tree-like evolution, we also implemented a NeighborNet network analysis (Bryant, Moulton, 2004) in SplitsTree software.

Results​

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Cytogenetic analyses. The diploid chromosome number in all analyzed males of Copella callolepis was 2n = 36 (Fig. 2). The karyotype consisted predominantly of acrocentric chromosomes except for two subtelocentric chromosomes (Fig. 2A), being the karyotype formula defined as 2st+34a. The C-positive heterochromatin blocks are found in the pericentromeric region of almost all chromosomes (Fig. 2B).

FIGURE 2| Karyotypes of Copella callolepis according to different cytogenetic protocols. Giemsa staining (A), C-banding (B), and dual-color FISH with 18S (green) and 5S (red) rDNA probes (C). Chromosomes are counterstained with DAPI (blue). Scale bar = 10 µm.

Multiple 5S rDNA and 18S rDNA sites were found in C. callolepis karyotype. For the 5S rDNA, positive signals were found in the centromeric regions of pairs 5, 9, and 14, while for 18S rDNA, only at the pericentromeric region of pairs 6 and 12 (Figs. 2C, 3E). The comparative analysis on the chromosomal distribution of rDNA in both Lebiasinidae and Ctenoluciidae (Figs. 3A–E) revealed species-specific patterns of accumulation, with multiple sites of 18S rDNA observed in almost all genera of both families (except for Copeina and Lebiasina bimaculata Valenciennes, 1847). On the other hand, the 5S rDNA has a simple distribution (a single pair) in all Boulengerella species, but it is spread into more pairs on most Lebiasinidae karyotypes. Only L. bimaculata presents a simple distribution for both rDNA sequences (Fig. 3B).

FIGURE 3| Schematic representation of chromosomes of Lebiasinidae and Ctenoluciidae species, highlighting the positions of the 18S rDNA (green) and 5S rDNA (red). The small box highlights a sex chromosome system in Pyrrhulina semifasciata, while the bigger box highlights the Ctenoluciidae members (the outgroup). FISH data here compiled in the idiograms refers to the mappings conducted by Souza e Sousa et al.,2017; De Moraes et al., 2017, 2019, 2021, 2023; Sassi et al.,2019; Toma et al.,2019; Sember et al.,2020; Leite et al.,2022. Letters correspond to the genera: (A) Boulengerella, (B) Lebiasina, (C) Nannostomus, (D) Copeina, (E) Copella, and (F) Pyrrhulina.

Principal Component Analysis. The principal component analysis shows mostly a clear clustering of samples at the genera level, evidencing the genetic differentiation between the species examined (Fig. 4A). Some individuals of different species, however, also show close proximity, such as L. melanoguttata Netto-Ferreira, 2012 and L. minuta Netto-Ferreira, 2012; some individuals from all Pyrrhulina species; and Nannostomus eques Steindachner, 1876 and N. beckfordi Günther, 1872. The PCA comprising only Nannostomus samples (Fig. 4B) helps to unravel the differentiation between N. beckfordi, N. eques, and N. unifasciatus Steindachner, 1876, which formed a cluster in the full dataset PCA. As expected, the clustering patterns obtained in PCAs highly agree with the phylogenetic structure.

FIGURE 4| Principal component analysis results. Each circle represents an individual, and species are colored differently according to the names. Percentage values indicate the explanatory capability of each axis. A. Displays the results for the PCA analysis with the full dataset; B. Displays the results for the dataset comprising only Nannostomus samples.

Phylogenetic analysis and Neighbor-net. The estimated phylogeny is presented in Fig. 5A. Individuals of the same species are collapsed together to simplify visualization, and the full tree is presented in the supplementary material (Fig. S1).The tree depicts the relationships among sampled Lebiasinidae species and that withinthe outgroup (B. lateristriga). Two well-supported primary lineages are recovered. The first lineage comprises the genera Pyrrhulina, Copella, and Copeina. Within this clade, Pyrrhulina species form a monophyletic group sister to Copella callolepis, while Copeina guttata (Steindachner, 1876) occupies a basal position relative to the Pyrrhulina–Copella clade. The second major lineage includes Lebiasina and Nannostomus. Within Lebiasina, the Ecuadorian species L. bimaculata is recovered as the sister lineage to a clade composed of the Brazilian species L. melanoguttata and L. minuta. Among Nannostomus, N. marginatus represents the earliest diverging lineage within the genus. Nannostomus eques is resolved as the sister taxon to a clade containing N. unifasciatus, that follows the less supported clade formed by the sister species N. anduzei Fernandez & Weitzman, 1987 and N. beckfordi. All major clades are supported by high bootstrap values, with nodes marked by diamonds indicating a bootstrap support value of 100. The overall topology reveals clear phylogenetic structure within the family, with strong support for both intergeneric and most of the intrageneric relationships.On the other hand,the NeighborNet network (Fig. 5B) presents a structure that is greatly congruentwith thephylogeny, but the position of some Nannostomus species is not identical, highlighting the difficulty of establishing the true relationships inside the genera. The network deviates from a tree-like structure, with several boxes mainly in between distinct genera, indicating conflicting signals.

FIGURE 5| Maximum likelihood phylogenetic tree (A), diamond symbols indicate nodes with bootstrap values of 100, while other bootstrap values are represented near nodes. Gray boxes indicate relevant cytogenetic features of each clade. NeighborNet network circles are colored in the same scheme as the phylogenetic tree, and each circle represents a sample that was collapsed into one in the phylogeny (B).

Discussion​

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This study presents a comprehensive analysis of the cytogenetics of Lebiasinidae, including the first karyotype description of Copella callolepis. In addition, by using molecular phylogenetic analysis and a NeighborNet network, we successfully reconstructed a phylogenetic tree, placing C. callolepis in a wider phylogenetic context for the first time and also providing a thorough understandingof the genetic relationships among Lebiasinidae species. Previous phylogenetic studies using molecular data were restricted to DNA barcoding in some species of Nannostomus (Benzaquem et al., 2015) or included few (usually two to four species) representatives in a broader phylogenetic context (e.g., Rabosky et al., 2013; Cassemiro et al., 2023), not making assumptions about the phylogenetic relationships inside Lebiasinidae. Based on this new information, we analyzed the chromosomal evolution patterns of Lebiasinidae and proposed their most likely evolutionary pathways.

Chromosomal features of Copella callolepis in the context of Lebiasinidae karyotype evolution. Based on our findings, C. callolepis preserved the proposed ancestral 2n for the family (2n = 36), but not its karyotype structure. In contrast to the ancestral karyotype, which was mostly made up of metacentric chromosomes, the karyotype of C. callolepis exhibited a large number of acrocentrics, which reinforces the important role of pericentric inversions in the karyotype evolution of the group. Similar scenarios are observed in other fishes, such as the giant trahiras Hoplias (Sassi et al., 2021), Nothobranchius killifishes (Krysanov et al., 2023),and Ictalurus catfishes (Waldbieser et al., 2023). Besides pericentric inversions, centromere repositioning (Schubert, 2018) might also explain the high divergence of acrocentrics between karyotypes of related species. Indeed, a molecular study in the medaka fish Oryzias javanicus suggests the repositioning of centromere-associated repeats (repetitive DNA) as the main mechanism for the increase of acrocentrics in karyotype, rather than pericentric inversions (Ansai et al., 2023). In addition to acrocentrics, the karyotype of C. callolepis has a single pair of large subtelocentric chromosomes, which is very similar in shape and size to pair 5 of Pyrrhulina marylinae Netto-Ferreira & Marinho, 2013, pair 1 of Pyrrhulina sp., and pair 2 of P. obermulleri Myers, 1926 and Pyrrhulina cf. laeta (Cope, 1872) (De Moraes et al., 2021), suggesting that it may have originated before the split of Pyrrhulina and Copella (Fig. 5). The presence of metacentric and submetacentric chromosomes suggests that the karyotype of Copeina guttata retained other features of the basal karyotype of the family (Toma et al., 2019). However, given their similar sizes, it’s also possible that the subtelocentric pair observed in Pyrrhulina and Copella is also present in the karyotype of Copeina guttata, either as pair 2, 3, or 4. To shed light on this scenario, additional investigations employing whole-chromosome painting are needed.

While the 2n of other Copella species ranges from 24 to 44 chromosomes (Scheel, 1973; Arai, 2011), these studies were merely descriptive and omitted important details like the karyotype formula or the number of individuals karyotyped, which impairs their reproducibility. However, such diploid number variation indicates that a series of rearrangements have taken place during the chromosomal evolution of the genus, which might have included chromosome fissions, fusions, translocations, and inversions, as also observed in other lebiasinids (Sember et al., 2020; De Moraes et al., 2021, 2023). This chromosomal dynamism is further evidenced by the presence of multiple 5S and 18S rDNA sites, an unusual condition among teleosts (Gornung, 2013; Sochorová et al., 2018) which may be facilitated by transposable elements or pseudo-homologous regions promoting recombination among acrocentric chromosomes (Guarracino et al., 2023; Garcia et al., 2024). The association of rDNA with centromeric heterochromatin, as observed in C. callolepis, may point to total repression or a decrease of recombination in these regions (Roberts, 1965; Ellermeier et al., 2010), suggesting that TE-mediated dispersal could be a more likely mechanism behind rDNA amplification. While our study found no cytogenetic evidence of sex chromosomes in C. callolepis, the species pronounced sexual dimorphism and parental care behaviors (Marinho, Menezes, 2017) highlight the importance of further investigation. Although rDNA accumulation on sex chromosomes has been reported in several teleosts (Cioffi, Bertollo, 2012) including the putative ZZ/ZW of the lebiasinid L. bimaculata (Sassi et al., 2019), this pattern is not universal as shown by the absence of such sequences in the multiple sex chromosomes X₁X₂Y of P. semifasciata (De Moraes et al., 2019).

Trends in karyotype evolution of Lebiasinidae in light of new genomic data. According to TimeTree 5 (Kumar et al., 2022), a database that compiles phylogenetic information and divergence times of previous time-calibrated phylogenetic studies,the split between Lebiasina (Lebiasinidae) and Boulengerella (Ctenoluciidae) occurred around 70mya, while Lebiasina and Nannostomus divergedat 65 mya. By that time, with the uplifting of the Andes Mountain range, several river capture events occurred in South America, raising diversification rates and creating an extraordinary richness in freshwater fishes, mostly through allopatric speciation events (Boschman et al., 2023; Cassemiro et al., 2023). In contrast, the split between Pyrrhulina and Copella took place more recently, around 55 mya, during the Paleogene. This is associated with the Paleocene–Eocene thermal maximum, which raised temperatures and significantly increased carbon input into the ocean and atmosphere. Given the unique community structures identified in fossilized ray-finned fish, this split indicates the age of modern fishes (Haynes, Hönisch, 2020).

As most Lebiasinidae populations are small and allopatric, both hypotheses support the large cytogenetic diversity observed within the family. The conservation of the ancestral karyotype in Lebiasininae (which, according to morphological data, includes Derhamia, Lebiasina, and Piabucina) and extensive rearrangements leading to the formation of acrocentrics in the karyotypes of Pyrrhulininae (including Copeina, Copella, Nannostomus, and Pyrrhulina) were the two main evolutionary pathways that occurred in Lebiasinidae. As proposed by Sassi et al.,(2020), these two pathways comprise: i) the conservation of a plesiomorphic karyotype in the subfamily Lebiasininae with 2n = 36 metacentric/submetacentric chromosomes, and ii) the Pyrrhulininae subfamily exhibiting differences in diploid numbers and karyotypes mostly composed of acrocentric chromosomes in the majority of species (Sassi et al., 2020). In this scenario, most of Pyrrhulininae’s acrocentric chromosomes are the result of rearrangements such as centric fissions (Sassi et al., 2020). However, some exceptions within the subfamily have revealed further fusion events, resulting in metacentric chromosomes in some species and a decrease in 2n, as seen in P. marilynae (2n = 34), N. anduzei (2n = 22), and N. unifasciatus (2n = 22), the last two representing some of the lowest diploid chromosome numbers found among teleost fishes (Sember et al., 2020; De Moraes et al., 2021, 2023).

Recent molecular studies (e.g., Cassemiro et al., 2023), have challenged the traditional placement of Nannostomus within Pyrrhulininae, a view that is further supported by our phylogenetic analysis based on DArTseq sequences. Our results indicate that Nannostomus is more closely related to Lebiasina, and together they form a sister group to the clade composed of Copeina, Copella, and Pyrrhulina (Fig. 5). This topology suggests that Lebiasininae may comprise Lebiasina and Nannostomus (and possibly Derhamia and Piabucina, which were not included in our sampling), whereas Pyrrhulininae includes Copeina, Copella, and Pyrrhulina. These findings underscore the importance of integrating genomic, morphological, and cytogenetic datasets to resolve taxonomic inconsistencies. For example, apparent morphological similarities between Nannostomus and Pyrrhulina might reflect convergence or retention of ancestral traits, rather than close evolutionary affinity.

However, the Neighbor-net network highlights several conflicting signals between Nannostomus and Lebiasina species. A non-bifurcating structure may reflect reticulation events, such as introgression or hybridization, but clarifying this pattern would require targeted hybridization analyses and a more comprehensive sampling of Lebiasinidae species and populations. Although no existing phylogenetic study has included all recognized species of the family, many have provided robust hypotheses for intergeneric relationships. In this context, our results are consistent with those of Cassemiro et al.(2023), who recovered Pyrrhulina as sister to Copella, and Lebiasina as sister to Nannostomus. Additionally, recent mitogenomic data from Xu et al.(2024) on four Nannostomus species, three of which (N. marginatus, N. beckfordi, and N. unifasciatus) were also sampled in our study, support our findings, with N. beckfordi and N. unifasciatus recovered as closely related and N. marginatus positioned more distantly, in agreement with both our PCA and phylogenetic analyses (Figs. 4–5).

Using the previous and the herein phylogenetic tree, the inferred ancestral 2n of the family (36) (Souza, Sousa et al., 2017; Sassi et al., 2019) is observed in both Lebiasinidae subfamilies, shared by C. callolepis, N. eques, and all Lebiasina species karyotyped to date. This indicates that Lebiasinidae represents another Neotropical Characiformes family with extensive interspecific karyotype diversity, along with Erythrinidae (Bertollo, 2007; Cioffi et al., 2012) and Characidae (Arefjev, 1990; Pazza, Kavalco, 2007; Soto et al., 2018). Although the majority of Nannostomus and Pyrrhulina have higher diploid numbers and a karyotype dominated by acrocentric chromosomes, some species exhibit metacentric chromosomes that result from secondary fusions (Sember et al., 2020; De Moraes et al., 2023). This feature and the molecular phylogeny indicate that the two evolutionary pathways proposed by Sassi et al.(2020) may only partially reflect the evolutionary history of Lebiasinidae. With that in mind, we can now proposea primary pattern of high karyotypic reorganization for the whole Lebiasinidae, that results in acrocentrics dominating Pyrrhulininae karyotypes.

As for Lebiasininae, while the ancestral karyotype (i.e., 2n = 36 with meta- and submetacentric chromosomes) is fully conserved in Lebiasina (Sassi et al., 2019; Leite et al., 2022), it has experienced substantial modification in Nannostomus, leading to karyotypes made up of acrocentrics, which in certain species may undergo full secondary fusion into metacentrics. As was already indicated, a small number of studies on Copella species have previously reported the 2n with reliable results (Scheel, 1973; Arai, 2011) nonetheless, these studies, when combined with our findings, suggest that this genus also experiences processes similar to the evolution of Nannostomus karyotypes. Chromosomal rearrangements, such as inversions, fusions, fissions, and translocations, can play a fundamental role in reproductive isolation and speciation by altering the way that beneficial alleles are transposed through populations. Inversions, for instance, suppress recombination in heterozygotes, which can facilitate local adaptation and promote speciation by reducing gene flow between diverging populations (Berdan et al., 2023). Moreover, the accumulation of repetitive elements and heterochromatin, often associated with these rearrangements, can further reduce recombination and gene flow, reinforcing genomic divergence and enabling speciation processes to unfold (Cioffi, Bertollo, 2012). However, due to the resolution limitations inherent to cytogenetic analyses, it remains unclear whether the 2n = 36 observed in the C. callolepis described herein, as well as in N. eques, represents retention of the ancestral diploid number or a case of convergent evolution, an issue that warrants further investigation.

This study contributes to a growing body of work positioning Lebiasinidae among other Neotropical Characiformes (e.g., Erythrinidae, Characidae) as families with extraordinary karyotypic diversity (Bertollo, 2007; Pazza, Kavalco, 2007; Cioffi et al., 2012). However, full understanding of this diversity remains hampered by limited sampling. Future research should prioritize the cytogenetic and genomic characterization of underrepresented taxa such as Derhamia and Piabucina, as well as the application of advanced tools such as whole-chromosome painting and long-read sequencing. These efforts will be critical for clarifying chromosomal evolution, identifying sex-specific differences, and refining the phylogeny of Lebiasinidae. Populational studies will be also essential to complement these efforts by revealing intraspecific chromosomal variation, detecting potential cryptic species, and identifying populations with unique cytogenetic features, since the family have a complex taxonomy impairing the classification at species level in some cases as the Pyrrhulina species here included. Such studies can help clarify the extent and mechanisms of chromosomal rearrangements, assess their role in reproductive isolation, and test hypotheses about local adaptation and speciation.

Acknowledgments​

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The authors appreciate the contribution of José L. O. Birindelli (UEL) for providing the picture of a specimen of Copella callolepis used in Fig. 1.

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Authors

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Renata Luiza Rosa de Moraes^1,2, Fernando Henrique Santos de Souza^1,2, Geize Aparecida Deon^1,2, Manoela Maria Ferreira Marinho³, Marcelo de Bello Cioffi¹ and Francisco de Menezes Cavalcante Sassi^1,4

^[1]    Departamento de Genética e Evolução, Universidade Federal de São Carlos (UFSCar), Rodovia Washington Luís, km 235, C. P. 676, São Carlos 13565-905, SP, Brazil. (RLRM) rlrdm@hotmail.com, (FHSS) fernando_hsouza@outlook.com.br, (GAD) geizedeon@hotmail.com, (MBC) mbcioffi@ufscar.br, (FMCS) francisco.sassi@hotmail.com (corresponding author).

^[2]    Institute for Applied Ecology, University of Canberra, Bruce, ACT 2617, Australia.

^[3]    Instituto de Biociências, Universidade Federal do Mato Grosso do Sul, Cidade Universitária, Av. Costa e Silva, Campo Grande 79070-900, MS, Brazil. (MMFM) manoela.marinho@ufms.br.

^[4]    School of Life Sciences, Southwest University (SWU), 2 Tiansheng Road, Beibei District, Chongqing 400715, China.

Authors’ Contribution

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Renata Luiza Rosa de Moraes: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Supervision, Validation, Visualization, Writing-original draft, Writing-review and editing.

Fernando Henrique Santos de Souza: Data curation, Formal analysis, Investigation, Methodology, Software, Supervision, Validation, Visualization, Writing-original draft, Writing-review and editing.

Geize Aparecida Deon: Data curation, Formal analysis, Methodology, Validation, Writing-original draft, Writing-review and editing.

Manoela Maria Ferreira Marinho: Data curation, Formal analysis, Investigation, Methodology, Validation, Writing-original draft, Writing-review and editing.

Marcelo de Bello Cioffi: Conceptualization, Funding acquisition, Investigation, Project administration, Resources, Validation, Writing-original draft, Writing-review and editing.

Francisco de Menezes Cavalcante Sassi: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Supervision, Validation, Visualization, Writing-original draft, Writing-review and editing.

Ethical Statement​

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All individuals were collected under the authorization of the Brazilian environmental agency ICMBIO/SISBIO (License number 48628–14) and SISGEN (A96FF09). The experiments followed ethical standards, and anesthesia followed the Ethics Committee on Animal Experimentation of the Universidade Federal de São Carlos (Process number CEUA 7994170423).

Statement of Equal Contribution by the Authors

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Authors Renata Luiza Rosa de Moraes and Fernando Henrique Santos de Sousa contributed equally to the development of this article

Competing Interests

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The author declares no competing interests.

Data availability statement

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The author declares no competing interests.

Funding

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This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) grants 2019/25045–3 (RM); 2023/00955–2 (MBC) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) grant number 302928/2021–9 (MBC), 200247/2025–5 (FMCS). This study was also supported by INCT – Peixes, funded by MCTIC/CNPq (proc. 405706/2022–7), and by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, finance code 001).

How to cite this article

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Moraes RLR, Souza FHS, Deon GA, Marinho MMF, Cioffi MB, Sassi FMC. A new karyotype and a molecular phylogeny of Lebiasinidae (Teleostei: Characiformes) shed light on the chromosomal evolution of the family. Neotrop Ichthyol. 2025; 23(3):e250029. https://doi.org/10.1590/1982-0224-2025-0029

Copyright​

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This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Distributed under

Creative Commons CC-BY 4.0

© 2025 The Authors.

Diversity and Distributions Published by SBI

Accepted June 11, 2025

Submitted February 14, 2025

Epub October 20, 2025