Origin of B chromosomes in Characidium alipioi (Characiformes, Crenuchidae) and its relationship with supernumerary chromosomes in other Characidium species

Abstract B chromosomes are apparently dispensable components found in the genomes of many species that are mainly composed of repetitive DNA sequences. Among the numerous questions concerning B chromosomes, the origin of these elements has been widely studied. To date, supernumerary chromosomes have been identified in approximately 60 species of fish, including species of the genus Characidium Reinhardt, 1867 in which these elements appear to have independently originated. In this study, we used molecular cytogenetic techniques to investigate the origin of B chromosomes in a population of Characidium alipioi Travassos, 1955 and determine their relationship with the extra chromosomes of other species of the genus. The results showed that the B chromosomes of Characidium alipioi had an intraspecific origin, apparently originated independently in relation to the B chromosomes of Characidium gomesi Travassos, 1956 Characidium pterostictum Gomes, 1947 and Characidium oiticicai Travassos, 1967, since they do not share specific DNA sequences, as well as their possible ancestral chromosomes and belong to different phylogenetic clades. The shared sequences between the supernumerary chromosomes and the autosommal sm pair indicate the origin of these chromosomes.


Introduction
B or supernumerary chromosomes are extra genomic elements in addition to the standard chromosomal set (A) and are found in approximately 15% of eukaryotic organisms (Camacho 2005). In general, B chromosomes are derived from the A chromosomes of their own species (intraspecific origin) or closely related species (interspecific origin) (Banaei-Moghaddam et al. 2015). The intraspecific origin of B chromosomes was demonstrated in maize (Lamb et al. 2005;Peng et al. 2011), locusts (Teruel et al. 2010), rye (Martis et al. 2012) and fishes (Mestriner et al. 2000;Silva et al. 2013;Valente et al. 2014;Utsunomia et al. 2016). On the other hand, there are also cases in which B chromosomes may have arisen spontaneously in response to new genomic conditions such as interspecific hybridization, which has been observed in grasses of the genus Coix Linné, 1753 (Sapre and Deshepande 1987), the fish Poecilia formosa Girard, 1859 (Schartl et al. 1995) and the wasp Nasonia vitripennis Walker, 1836 (McAllister andWerren 1997).
A significant evolutionary feature of B chromosomes is the accumulation of repetitive DNA sequences (Camacho 2005;Houben et al. 2014;Banaei-Moghaddam et al. 2015). The cytogenetic and/or cytogenomic data about this type of DNA have been very informative for understanding the origin and evolution of B chromosomes. The findings using fluorescence in situ hybridization (FISH) techniques and genomic sequences analysis showed the intraspecific origin of B chromosomes and the presence and expression of intact genes in these elements in fishes (Silva et al. 2013Utsunomia et al. 2016;Valente et al. 2014), as also observed in rye (Banei-Moghaddam et al. 2015).
Among genomes of fish species bearing B chromosomes, the genus Characidium Reinhardt, 1867 exhibits interesting cytogenetic features; the B chromosomes probably originated independently in the different species of this group, whereas the heteromorphic ZZ/ZW sex chromosomes seem to have originated once in the genus. Therefore, the B chromosomes of C. oiticicai Travassos, 1967originated interspecifically, whereas in C. gomesi Travassos, 1956and C. pterostictum Gomes, 1947, these elements have an intraspecific origin from the sex chromosomes (Pansonato-Alves et al. 2014, Serrano et al. 2016). In the present study, a novel occurrence of B chromosomes is described for C. alipioi, a species in which these elements were not found until now. In addition, the origin and evolution of these elements were studied using conventional cytogenetic techniques, including C-banding, microdissection, chromosome painting and fluorescence in situ hybridization with repetitive DNA probes.

Origin of the fishes/individuals, karyotype analysis and DNA extraction
A total of 19 C. alipioi samples were analyzed (9 females and 10 males) from the Ribeirao Grande river, Paraíba do Sul River Basin, Pindamonhangaba, São Paulo (22°49'00.3"S 45°25'23.7") ( Table 1). The animals were collected in accordance with Brazilian envi- To perform the cytogenetic preparations, the animals were anesthetized and dissected, and mitotic chromosome preparations were obtained following the protocol of Foresti et al. (1981). C-banding was performed according to the protocol described by Sumner (1972). Chromosome morphology was determined according to Levan et al. (1964), and the chromosomes were classified as metacentric (m), submetacentrics (sm) and subtelocentric (st) and organized in the karyotype by descending size.
DNA extraction was performed using the Wizard Genomic DNA Purification Kit (Promega) according to the manufacturer's instructions.

Microdissection and preparation of chromosomal probe
Chromosome microdissection was performed in an Eppendorf TransferMan NK2 micromanipulator coupled with a Zeiss Axiovert 100 microscope. For chromosome painting, ten B chromosomes were microdissected from the cytogenetic preparations of the samples from each species (C. alipioi, C. gomesi and C. oiticicai) carrying one extra chromosome. The probes for C. alipioi, C. gomesi and C. oiticicai denoted CaB, CgB and CoB, respectively.
Microdissected DNA from each species was placed into a tube containing 9 µL of DNase-free ultrapure water and amplified using the GenomePlex Single Cell Whole Genome Amplification Kit (wga4 Sigma) (Gribble et al. 2004). After the initial amplification, the DNA probes CaB, CgB and CoB were generated and were then labeled with Digoxigenin-11-dUTP (Roche Applied Science) using the GenomePlex Whole Genome Amplification Reamplification Kit (wga3 Sigma) according to the manufacturer's protocol.

Fluorescent in situ hybridization (FISH)
For the FISH experiments, the prehybridization conditions were performed according to procedures described by Pinkel et al. (1986). Posthybridization washes were performed according to the applied probe: (i) the slides probed with rDNAs, snDNA, histone and B chromosome probes were washed in 0.2× SSC/15% formamide for 20 min at 42°C, followed by a second wash in 0.1× SSC for 15 min at 60°C and a final wash at room temperature in 4× SSC/0.5% Tween for 10 min; probe detection was performed using avidin-FITC and anti-digoxigenin-rhodamine; and (ii) the slides probed with oligonucleotides were washed in 2× SSC for 5 min, followed by a second wash in 1× PBS for 1 min. Chromosomes were counterstained with DAPI (Vector Laboratories, Burlingame, Calif., USA). The images were digitally captured using Image Pro Plus 6.0 software (Media Cybernetics) with the appropriate filters on an epifluorescence microscope (Olympus BX61) equipped with an Olympus DP70 camera. The final composition of the images was performed using Adobe Photoshop CS6 image editor software with the image and uniform size scales.

Analysis of mitochondrial DNA
Analysis of mitochondrial DNA was performed in order to make a correct identification of the specimens, besides that, the position of the clades could be informative to discuss aspects of the origin of the B chromosomes in the genus. The average distance analysis of the COI and CytB sequences obtained in this study and other species taken from GenBank showed high similarity between the C. alipioi sequences of Santa Bárbara do Tugúrio-MG and the specimens analyzed here (Suppl. materials 1, 2), which confirmed the taxonomic status of the analyzed samples (Figure 1).

Chromosomal analysis
The analyzed C. alipioi individuals showed diploid chromosome number 2n = 50 and karyotype composed of 32 m + 18 sm with heteromorphic ZZ/ZW sex chromosomes, which was similar to the findings in other species of the genus (Figure 2). In addition, cells bearing 0-4 B chromosomes were observed in 45% of the individuals (Table 1). The B chromosomes were mitotically unstable, once the number of these chromosomes can variete between cells of the same individual. C-banding showed constitutive heterochromatin blocks in the pericentromeric region of all chromosomes and a conspicuous distal block in the longer arm of the Z chromosome. The W and B chromosomes were entirely heterochromatic (Figure 2b).

Chromosome painting
Chromosome painting performed in the C. alipioi chromosomes with the CaB probe showed signals on the entire length of the B chromosomes and the pericentromeric region of the sm chromosome pair nº 19 of the A complement (Figure 3a). On the other hand, this same probe did not reveal any signals on the C. gomesi and C. oiticicai chromosomes (Figure 3b and c). Similar findings were observed by hybridization with the CoB probe in C. alipioi chromosomes (Figure 3e). Conversely, the CgB probe showed hybridization signals on the W chromosome of C. alipioi (Figure 3d).    (Figure 5a and b), which had lower abundance and weak hybridization signals. Furthermore, the intense signals of the microsatellite (GAG) 10 showed preferential accumulation on the W and B chromosomes of this species (Figure 5c).

Discussion
The occurrence of B chromosomes in Characidium alipioi genome was revealed for the first time in this study, particularly since the population of C. alipioi of Santa Bárbara do Tugúrio-MG analyzed by Pansonato-Alves et al. (2014) showed no extra chromosomes. The presence of these elements in only certain populations was observed in other species of the genus Characidium such as C. zebra, C. pterostictum and C. gomesi (Pansonato-Alves et al. 2014). Our data does not allow to conclude about the dynamics or relationship of these chromosomes between populations; however, at least two mechanisms, which are based on the findings of two model organisms have been proposed to be involved in the absence of B chromosomes in certain populations: geographical barriers that limit the spread of individuals with these elements in Eyprepocnemis plorans Charpentier, 1825 (Cabrero et al. 1997, Manrique-Poyato et al. 2015 and the close relationship between temperature and rainfall factors that could influence the variations in the presence of B chromosomes, as observed in Myrmeleotettix maculatus Thunberg, 1815 (Hewitt and John 1967;Hewitt and Ruscoe 1971).
The individuals of the same species and location included in this study were analyzed in a previous study by Centofante et al. (2003); however, these authors did not detect B chromosomes in their analysis. Although cytogenetic studies in Characidium have revealed the occurrence of supernumerary chromosomes restricted to certain populations of some species (Pansonato-Alves et al. 2014), it should be noted that the geographical proximity of the sampling sites and the time between the present analysis and the analysis by Centofante et al. (2003) suggest that the two samples do not belong to different populations. Similarly, it can't be stated that the B chromosomes have arisen in this population after the first study. However, because some individuals with no B chromosomes were also identified in the present study, it is likely that the samples analyzed in the 2003 study were formed by individuals who do not carry these extra elements.
Chromosome painting using the B chromosomes of C. alipioi (CAB) as the probe indicated shared sequences between the A and B complements and, more specifically, the pericentromeric region of a sm chromosome pair (pair No. 19). This result probably reflects a relationship between these chromosomes and intraspecific origin of the supernumerary chromosomes in genome of this species and that pair No. 19 is likely the chromosome of its origin. Previous studies have identified the origin of B chromosomes from the A complement of the host species (Bugrov et al. 2003;De Jesus et al. 2003;Lamb et al. 2005;Teruel et al. 2010;Peng et al. 2011;Silva et al. 2014;Valente et al. 2014;Utsunomia et al. 2016); however, in a few cases it was possible to identify the ancestral chromosome, such as in the grasshopper (Teruel et al. 2010;Bueno et al. 2013) and in two fish species Astyanax paranae Eigenmann, 1914(Silva et al. 2014 and Moenkhausia sanctaefilomenae Steindachner, 1907. The intraspecific origin of B chromosomes has been reported in other species of the genus Characidium, namely C. pterostictum, C. gomesi and Characidium sp. aff. C. vidalli (Pansonato-Alves et al. 2014;Schacchetti et al. 2015). However, because in these species this chromosome originated from the Z and W chromosomes and the CaB probe in the present study showed no signals on these chromosomes, these supernumerary elements do not seem to share the same ancestral supernumerary chromosomes that are present in C. alipioi. In addition, the C. alipioi B chromosomes showed no homology with the supernumeraries of C. gomesi and C. oiticicai, which are two species whose B chromosomes are apparently composed of different types of repetitive DNA and have different origins (Pansonato-Alves et al. 2014). Therefore, it possible that the supernumerary elements in C. alipioi originated independently from the other types of B chromosomes reported in the representatives of this genus, and the phylogenetic position of these species at different clades (Figure 1) supports this assumption. However, these conclusions should be made with caution because the probes obtained by microdissection are composed of anonymous sequences, and the amplification method (GenomePlex) may favor specific sequences of repetitive DNA that are present in the B chromosomes and absent on other A chromosomes. In this context, it cannot be ruled out that other sequences shared between A and B chromosomes are not represented in the probes used in this study, which was previously noted by Pansonato-Alves et al. (2014).
Hybridizations with microsatellite DNA sequences demonstrated the presence of these repetitive elements in B chromosomes. (GA) 15 and (CA) 15 are both dispersed with conspicuous blocks in the terminal regions of the A chromosomes and are less abundant in the supernumerary chromosomes. Moreover, a clear accumulation was observed with respect to the (GAG) 10 sequence. Notably, the hybridization with the (GAG) 10 probe revealed a preferential accumulation in B and W chromosomes in C. alipioi. Similarly, microsatellites were detected in the B chromosomes of maize (Ananiev et al. 2005), rye (Langdon et al. 2000) and locusts (Milani and Cabral-de-Mello 2014;Ruiz-Ruano et al. 2015). Given that the CaB probe did not paint the C. alipioi sex chromosomes, a possible explanation for the accumulation of (GAG) 10 in the B and W chromosomes would be that the CaB probe does not contain this microsatellite, which probably occurred due to the amplification method used in this study, as mentioned above. However, if it is considered that the accumulation mechanisms of this type of repetitive DNA permit its fixation on certain chromosomes due to its non-recombinant nature and preferential accumulation in heterochromatic regions, as observed in other studies (Cuadrado and Jouve 2011, Lohe et al. 1993, Scacchetti et al. 2015, then the distribution of microsatellites in these chromosomes may not reflect the aspects of its origin, but its accumulation after the appearance of the B chromosomes.
Our present results extend the knowledge of the structure and composition of B chromosomes between representatives of the Characidium genus, particularly in C. alipioi. In addition, the shared sequences between the A and B chromosomes of this species suggests an intraspecific origin of these chromosomes that is independent from the B chromosomes of other congeneric species. These observations reinforce the idea that this fish group is an interesting model to study the origin and structure of B chromosomes.