Samples of the two morphologically different but still undescribed species of Peckoltia named Peckoltia sp. 3 Jarumã and Peckoltia sp. 4 Caripetuba, after the rivers they were collected in different hydrographic points in the Tocantins River basins of northern Brazil were analyzed (Figure 1). The taxonomic identification of the sample was checked using the identification key proposed by Armbruster and Lujan (2016), Armbruster (2008). The results show that the specimens do not fit into any of the species already Figure 1described. The collection points, number of individuals, sex, and voucher of deposits in the zoological collection are shown in Table 2. The samples were obtained under a permanent field permit obtained by JCP (number 13248 from “Instituto Chico Mendes de Conservação da Biodiversidade”). The Cytogenetics Laboratory from UFPA has permit number 19/2003 from the Ministry of Environment for sample transport and permit 52/2003 to use the samples for research. The Ethics Committee (Comitê de Ética Animal da Universidade Federal do Pará) approved this research (Permit 68/2015). The specimens have been deposited in the ichthyological collection of the Museu Paraense Emílio Goeldii (MPEG) (Belém, Brazil).

Mitotic chromosomes were obtained from kidney cells after in vivo colchicine treatment as described (Bertollo et al., 1978). The animals were anesthetized with eugenol and subsequently sacrificed for the removal of kidney cells. Metaphases were analyzed by conventional Giemsa, C-banding (Sumner, 1972) and AgNO₃ staining (Howell and Black, 1980). Fluorescence in situ hybridization (FISH) was undertaken as described (Martins and Galetti, 1999) using a general telomere probe for vertebrates, 18S rDNA, 5S rDNA, and U1 snDNA probes.

DNA extraction was performed using PureLink Genomic DNA Mini Kit (Invitrogen) following the manufacturer’s instructions. The probes were obtained from a PCR using genomic DNA of Peckoltia sp. 3 Jarumã and Peckoltia sp. 4 Caripetuba with primers previously described for 18S rDNA (Hatanaka and Galetti Jr, 2004), for 5S rDNA (Suarez et al., 2017) and U1 snDNA (Cabral-de Melo et al., 2012). These probes were labeled by nick-translation with biotin or digoxigenin. Telomeric probes were obtained from PCR using the set of primers F-5′(TTAGGG)₅-3′ and R-5′(CCCTAA)₅-3′ followed by labeling with Digoxigenin-11-dUTP (Roche Applied Science^®) (Ijdo et al., 1991). Fluorescence in situ hybridization (FISH) was performed as described by Martins and Galetti (1999) using the following stringency conditions: 2.5 ng/μL of each probe, 50% formamide, 2 x SSC, 10% dextran sulfate, and hybridization at 42°C for 16 h. Fluorescent signals were detected using Streptavidin Alexa Fluor 488 (Molecular Probes, Carlsbad, CA, United States) and anti-digoxigenin rhodamine Fab fragments (Roche Applied Science, Penzberg, Germany). Chromosomes were counterstained with 0.2 μg/ml of 4′6-diamidino-2-phenylindole (DAPI) in Vectashield mounting medium (Vector, Burlingame, CA, United States).

At least 30 metaphases Giemsa-stained per individual were analyzed for confirming the diploid number, karyotypic structure, and chromosomal markers. Cytogenetic images of Giemsa-stained chromosomes were obtained using an Olympus BX41 microscope (bright field/phase) with a digital camera CCD 1300QDS and analyzed using GenASIs software version 7.2.7.34276 from ASI (Applied Spectral Imaging). FISH images were obtained using a Nikon H550S microscope and analyzed using the Nis-Elements software. Images were adjusted using Adobe Photoshop CS6 software. Chromosomal morphology was classified according to literature (Levan et al., 1964), with adaptations.

Cytogenetic information for the species of Peckoltia is described in Table 1 and, despite the occurrence of 2n = 52 chromosomes, all species of Peckoltia show different KF. These differences can be explained by inversions or translocations, which represent important mechanisms of karyotypic diversification in Loricariidae (Kavalco et al., 2005; Mariotto et al., 2011). Alternatively, centromeric repositioning has also been proposed to cause variations in chromosome morphology with no change in diploid number (Montefalcone et al., 1999; Rocchi et al., 2012). However, the occurrence of this mechanism in Loricariidae karyotypes still needs deep investigation.

The Loricariidae has extensive chromosomal diversity, with variation in diploid number from 34 to 96 chromosomes, with a putative ancestral karyotype showing 2n = 54 chromosomes (Artoni and Bertollo, 2001). The reduction to 2n = 52 was probably due to a Robertsonian fusion in an ancient common ancestor in Ancistrini representatives with no ITS manutention (Bueno et al., 2018).

In Loricariidae, it is expected the presence of heterochromatic regions distributed in blocks on few chromosomes (Ziemniczak et al., 2012). Interestingly, in Peckoltia, extensive heterochromatic blocks are observed, some occupying a large part of the long arms of submetacentric/subtelocentric chromosomes (de Souza et al., 2009, present study). The presence of large heterochromatic blocks on morphologically similar chromosomes may suggest a shared character in Peckoltia karyotypes, as proposed previously (de Souza et al., 2009). However, it is known that heterochromatic regions are characterized by great diversity in highly repetitive DNA content (Dimitri et al., 2009) and may not reflect chromosomal homologies in Peckoltia species, as visualized among the species analyzed here by FISH with repetitive sequences. Noteworthy, the distribution of heterochromatin, and different repetitive sequences, observed in pairs 24 in Peckoltia sp. 3 Jarumã and 21 in Peckoltia sp. 4 Caripetuba, makes these chromosomes good cytotaxonomic markers, and both represent unique characteristics in each of these species.

Other plesiomorphic conditions for Loricariidae include the 18S and 5S rDNA sequences in a single pair of meta/submetacentric chromosomes (Ziemniczak et al., 2012). However, in the Ancistrini tribe, both the synteny and non-synteny between the 18S and 5S sequences are commonly observed (Mariotto et al., 2011; Ribeiro et al., 2015; Favarato et al., 2016; Prizon et al., 2016; Pety et al., 2018), showing the huge chromosome sites variation in the karyotypes of this group (Pansonato-Alves et al., 2013; Bueno et al., 2014; Pety et al., 2018). The mapping of 18S and the 5S sequences in the karyotypes here described is in agreement with that observed in other species of Peckoltia; in which extensive dispersion of these genes is observed (Pety et al., 2018) (Table 1). This dispersion of rDNA in the genomes of Loricariidae can be explained either by the association of these genes with other repetitive sequences, including transposable elements or by the evolutionary breakpoint regions close to rDNA sites promoting chromosome rearrangements (Barros et al., 2017; Glugoski et al., 2018, 2020; Deon et al., 2020). Furthermore, the heterochromatic condition involving clusters of rDNA suggests that other repetitive DNA classes, around 45S and 5S rDNA sequences, may promote their chromosomal dispersion in the Peckoltia species analyzed here, as shown for other species of Loricariidae (Glugoski et al., 2018; Deon et al., 2020).

In fish, the snRNA genes have shown great diversity of the pattern of chromosomal localization (Úbeda-Manzanaro et al., 2010; Cabral-de-Melo et al., 2012; Scacchetti et al., 2015; Yano et al., 2017). In this work, the U1 snDNA sequence was mapped for the first time in Loricariidae species, showing location in a pair of submetacentric chromosomes in both species, in addition to an extra site in one of the homologues of pair 21 of Peckoltia sp. 4 Caripetuba (Figure 3D). The snDNA sequences are considerably more stable at the chromosomal level when compared to rDNA (Cabral-de-Melo et al., 2012). However, we observed that among the karyotypes of Peckoltia analyzed here, the U1 snDNA probes show variation in the number of chromosomal sites similar to that observed for the rDNA (Table 1). These data indicate that these gene families can be equally dynamic in the genomes of species of Peckoltia. Several chromosomal sites of rDNA and snDNA sequences are observed in different groups of fish, such as species of the Loricariidae, Cichlidae and Anostomidae families; the emergence of new chromosomal sites is related to the association of these sequences with active mobile elements in these organisms (Kapitonov and Jurka, 2006; Cabral-de-Melo et al., 2012; Dulz et al., 2020). Future analyzes of rDNA and snDNA nucleotide sequences will be essential to verify the possible involvement of transposable elements in the movement of these sequences in the genomes of Peckoltia species.

The putative ancestral karyotype for Loricariidae has 2n = 54, a single NOR and gene synteny for 5S and 18S rDNA sequences (Ziemniczak et al., 2012). The tribes belonging to the Hypostominae subfamily share a common ancestor (Armbruster, 2004; Lujan et al., 2015) that possibly had a 2n = 52 chromosomes (Bueno et al., 2018). Thus, variations in the 2n, multiple NOR and synteny break between 5S and 18S would represent derived characteristics that can be apomorphic or homoplasic. Analyses involving 18S and 5S rDNA and U1 snDNA show the importance of these sequences as markers of karyotype diversification in the Peckoltia genus.

Phylogenetic analyzes support the monophyly of the Peckoltia genus (Lujan et al., 2015, 2017; Roxo et al., 2019). A phylogeny for the Peckoltia clade proposed by Lujan et al. (2017), based on the concatenated sequences of two mitochondrial genes (16S, Cyt b) and three nuclear genes (RAG1, RAG2, MyH6), has two well-defined branches, which are sister groups and two branches with non-defined relationships. One of the defined branches presents P. vittata (single NOR, synteny of the 5S with 18S) (Pety et al., 2017), P. compta, P. braueri and P. lineola (karyotypes not described); and the other branch with P. sabaji (multiple NORs, non-synteny of the 5S with 18S) (Pety et al., 2018), P. furcata and P. relictum (karyotypes not described). Noteworthy, most of the specimens from the branch with P. vittata are on the right bank of the lower Amazon River, and those from the branch with P. sabaji are on the left bank. The karyotype of Peckoltia sp. 4 Caripetuba would be more similar to that of Peckoltia sp. 1 and 2 previously described (de Souza et al., 2009), collected on the left bank of the Amazon River and with karyotypic characteristics derived from the ancestral karyotype proposed for Loricariidae. Thus, it is possible that these derived features, such as multiple rDNA and NORs sites, have arisen in this region (Figure 4).

In the present work, the karyotype of Peckoltia sp. 4 Caripetuba has several derived characteristics, being found on the right bank of the Amazon River. This fact can be explained if we consider the paleogeography of the region. The continental portion of Abaetetuba (Rio Jarumã) comprises the Barreiras Formation (Miocene). In turn, the Post-Barreiras Formation (Plio-Pleistocene) filled the paleovale of the Tocantins River, diverting this river (which originally crossed Marajó Island to its northern portion) and thus splitting Marajó Island from the mainland, originating Rio Pará and the islands in front of the city of Abaetetuba-PA (Figure 4), where the Caripetuba River is found (Rossetti and Valeriano, 2007). Therefore, the rivers where the karyotype of Peckoltia sp. 4 Caripetuba is found are in islands considerably newer than the one where Peckoltia sp. 3 Jarumã is found. These islands are covered by the Post-Barreiras Formation, being connected to the western portion of Marajó Island (Tatumi et al., 2007), not related with the continental region where the Jarumã River is located. The Marajó Island, in turn, is a communication corridor, connecting biodiversity on the left bank of the Amazon River with biodiversity on the right bank (Fernandes et al., 1995). This fact may explain why the most recent karyotype (Peckoltia sp. 4 Caripetuba) is located in this region. It will be important to test by molecular markers whether the Peckoltia-associated morphotypes analyzed in this study and by de Souza et al. (2009), which have derived karyotypes, belong to the P. sabaji branch.

This study describes the karyotypes of two undescribed species of Peckoltia and compares them with available chromosomal data for the genus. The maintenance of the same 2n in the species of Peckoltia may suggest that this genus has conserved karyotypes; however, the variations observed in the KF, NOR, heterochromatin and 18S and 5S rDNA sequences between the karyotypes of the species in this study compared to those previously described suggest great interspecific diversity in the genus. Furthermore, the differential localization of the U1 snDNA sequence among the karyotypes described here corroborates the involvement of repetitive sequences in the diversification of the genomes of these species. Therefore, due to the considerable cytogenetic diversity, species-specific characters were observed, showing great potential for identifying distinct taxonomic lineages in Peckoltia, in addition to demonstrating that the karyotypic variation in this genus is much greater than conventional staining suggests. Future analyzes considering the geographic distribution of Peckoltia species with primitive versus derived karyotypic characteristics compared to molecular phylogenies may provide relevant information about its evolutionary history.