Archolaemus janeae (Gymnotiformes, Teleostei): First insights into karyotype and repetitive DNA distribution in two populations of the Amazon

Abstract Archolaemus, one of the five genera of Neotropical freshwater fish of the family Sternopygidae (Gymnotiformes), was long considered a monotypic genus represented by Archolaemus blax. Currently, it consists of six species, most of them occurring in the Amazon region. There are no cytogenetic data for species of this genus. In the present study, we used classical cytogenetics (conventional staining and C‐banding) and molecular cytogenetics (probes of telomeric sequences and multigenic families 18S rDNA, 5S rDNA, and U2 snDNA) to study the karyotype of Archolaemus janeae from Xingu and Tapajós rivers in the state of Pará (Brazil). The results showed that the two populations have identical karyotypes with 46 chromosomes: four submetacentric and 42 acrocentric (2n = 46; 4m/sm + 42a). Constitutive heterochromatin occurs in the centromeric region of all chromosomes, in addition to small bands in the interstitial and distal regions of some pairs. The 18S rDNA occurs in the distal region of the short arm of pair 2; the 5S rDNA occurs in five chromosome pairs; and the U2 snDNA sequence occurs in chromosome pairs 3, 6, and 13. No interstitial telomeric sequence was observed. These results show karyotypic similarity between the studied populations suggesting the existence of a single species and are of great importance as a reference for future cytotaxonomic studies of the genus.


| INTRODUC TI ON
Gymnotiformes, a diverse order of Neotropical electric fish, has more than 250 species widely distributed in Central and South America, with great diversity and abundance in the Amazon Basin (Albert, 2001;Albert & Crampton, 2005;Ferraris et al., 2017;Fricke et al., 2021).
Based on morphological analysis, Vari et al. (2012) consider that Archolaemus comprises six species, of which five (A. blax, Archolaemus ferreirai, Archolaemus janeae, Archolaemus luciae, and Archolaemus santosi; Vari et al., 2012) are distributed in the Amazon Basin and its tributaries and one (Archolaemus orientalis Stewart, Vari et al., 2012) occurs in the São Francisco River basin of eastern Brazil.
To date, cytogenetic study for this genus is still lacking.

Sternopygus macrurus
In this article, we report for the first time the karyotype of Archolaemus janeae from Xingu and Tapajos rivers (both in Pará state), Brazilian Amazon. As A. janeae is originally known from populations in these rivers (e.g., Vari et al., 2012), we compared the karyotypes found in both rivers to test the previous hypothesis that those populations belong to a single species. We also relate the cytogenetic data to those previously reported for Sternopygidae.

| Samples
About the sample of Archolaemus janeae (Figure 1) used in the present study (Table 1), six specimens were collected in the Xingu River, municipality of Altamira, Pará, Brazil. The specimens were collected with the aid of an electric discharge detector and nylon handles.
Probes were labeled by PCR-based incorporation of biotinylated dUTP (Invitrogen) or nick translation with a BioNick kit (Invitrogen) for biotin staining, and with a Dig-Nick kit (Roche) for digoxigenin staining. Images were captured using a Nikon H550S Fluorescence Photomicroscope equipped with the Nis-Elements Software, and the karyotypes were organized using Adobe Photoshop CC2018.
Chromosomes were classified into two groups, metacentric/submetacentric (m/sm) and subtelocentric/acrocentric (st/a), and arranged in decreasing order of size for each group (adapted from measurements in Levan et al., 1964).

| RE SULTS
The specimens of Archolaemus janeae from the Xingu and Tapajos rivers have the same karyotype, with no difference in any of the markers used. The species has 46 chromosomes (Figure 2a, Tapajos sample; 2b, Xingu sample) and a karyotype formula composed by four bi-armed and 42 acrocentric chromosomes (2n = 46, 4m/ sm + 42a), without a cytogenetically visible sex system. F I G U R E 1 Specimens of Archolaemus janeae (a) from locality 1 (above) and locality 2 below; map showing the collection locations of the samples (b). The map was made using QGIS v. 3.10.7. The shape files containing country limits and hydrography were obtained from DIVA-GIS [Hijmans et al., 2004] Figure S1; 3e, Xingu).

| D ISCUSS I ON
Archolaemus janeae samples from both localities studied present the same karyotype (2n = 46, 4m/sm + 42st/a) being similar for all the markers used, despite being in different rivers (Table 1 and Figure 1).  (Fernandes et al., 2010;Fernandes et al., 2020). Heteromorphism in the size of the NOR adjacent to a heterochromatic block has also been described for two other genera of family Sternopygidae: Eigenmannia (Silva et al., 2009 and Sternopygus Silva et al., 2008).
The multiple 5S rDNA sites found in Archolaemus janeae fusion (Glugoski et al., 2018). Alternatively, the variation in the number of sites for 5S can be explained by its presence close to fragile sites (Barros et al., 2017) and evolutionary breakpoint regions (Deon et al., 2020), leading to homologous and nonhomologous repair mechanisms such as Robertsonian fusions (Barros et al., 2017). In a study of Eigenmannia aff. trilineata, the 5S rDNA was found colocated with the snDNA U2 cluster (Araya-Jaime et al., 2017). In Archolaemus janeae, however, as well as in Gymnotus sylvius, Gymnotus inaequi-

labiatus, Gymnotus pantanal, Gymnotus javari, Gymnotus carapo, and
Gymnotus pantherinus , colocation of these sequences is not seen. The cluster number appears to be conserved in six species of Gymnotus, and only one has multiple sites . Studies using U2 snRNA are still scarce for fish, especially for order Gymnotiformes.
To date, cytogenetic information has been available for species from three of the six genera of Sternopygidae. Among them, Eigenmannia has been the most studied: A high karyotype diversity is seen between species of this genus, with 2n ranging from 28 to  Fernandes et al., 2020;Sene et al., 2014;Silva et al., 2009Silva et al., , 2015. It has been proposed that populations of Eigenmannia experience isolation processes due to genetic drift caused by their low mobility and population density rates and that this facilitates the rapid fixation of structural or numerical chromosomal rearrangements (Araya-Jaime et al., 2017;Silva et al., 2009). Sternopygus macrurus has a relatively more conserved karyotype (2n = 46, 30m/16sm; Silva et al., 2008); no difference in karyotype was found between specimens from several locations in the Amazon basin, but some differences in the karyotype formula were found between samples from São Francisco River (32m/14sm) and the Parana River (28m/18sm) described by Almeida-Toledo et al. (1993). In contrast, Rhabdolichops was reported to have high karyotype variability, with 2n ranging from 54 to 74 (Suárez et al., 2017). The data presented here for Archolaemus demonstrate a 2n = 46 conserved karyotype. As both sampled rivers make up most of the geographic distribution of the genus, more studies on Archolaemus cytogenetics probably will show the same karyotype.
Although the 2n = 46 for Archolaemus is present in Sternopygus and Eigenmannia, the karyotype composition differs, showing the occurrence of intrachromosomal rearrangements.
As we had the diploid numbers of each genus in the molecular phylogeny published by Tagliacollo et al. (2016) for Sternopygidae ( Figure 4), it can be seen that 2n = 46 is present in three of the four genera analyzed and distributed throughout the phylogeny, suggesting that the 2n = 46 may be ancestral for this family and that chromosomal rearrangements have occurred in each genus.

ACK N OWLED G M ENTS
We would like to thank Dr. Willam Oliveira da Silva for making the map in Figure 1.

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest. writing-review and editing (equal). Cleusa Yoshiko Nagamachi:

DATA AVA I L A B I L I T Y S TAT E M E N T
All data used in this research are available in the article. The authors are available for any further explanation.