Several repetitive elements have previously been isolated from several Leporinus species. These elements exhibited wide diversity with respect to chromosomal location and distribution [5, 12–15]. The organisational patterns of these repetitive sequences demonstrate the dynamics of the fish genome, despite the apparent chromosomal stability that has been observed in the Anostomidae family since the first cytogenetic studies were performed in the 1980s (E.L. Silva, personal communication).
The predominantly dispersed distribution pattern of the LeSpe II element revealed by Southern blot analysis was consistent with the genomic organisation of most, if not all, previously analysed LINEs. Moreover, this characteristic distribution pattern differed from that of other previously described repetitive elements of the anostomids. LINE elements comprise a class of Non-LTR retrotransposons, which are an important group of repetitive DNA elements widely studied in many organisms (see ). The accumulation of LeSpe II near the telomeres of almost all chromosomes of the analysed anostomids was similar to the distribution pattern observed for the LINE CiLINE2 . Higashiyama et al.  suggested that because these elements display terminal distribution, they may play a role in the stabilisation of the chromosomes on which they reside, in some circumstances, or they might form a structural cap to protect the terminal portions of the chromosomes. In fact, the sex chromosome system of L. elongatus is considered to still be undergoing evolution ; thus the absence of LeSpe II favours an environment of instability, which is required for ongoing evolution.
In some groups of vertebrates, dispersed elements are arranged in clusters and blocks that can easily be visualised on chromosomes [19, 20], but in some fish species, these elements have a widely scattered distribution pattern on all of the chromosomes [19, 21]. These sequences have been associated with the evolution of the genome size of the host organism . Indeed, these sequences elicit alterations in gene function through the process of insertion, inducing chromosomal rearrangements and the production of coding and non-coding material, thereby allowing new genes or new regulatory sequences to emerge . These elements are therefore potentially associated with speciation events .
A series of repetitive DNA elements isolated from the Leporinus genus have been used as probes in chromosome mapping. All of these DNA families presented different distribution patterns, adding unique features to some species, including sex-specific sequences and species-specific repeated elements. Nakayama et al.  described two different sequences, including one found in both Z and W chromosomes and the other representing a second family specific to the W chromosome. LeSpe I repetitive DNA was described as a sex-specific dispersed repetitive element with distinct distribution patterns on two exclusive female chromosomes, named W1 and W2 by Parise-Maltempi et al. . This sequence was used to probe chromosomal preparations from L. macrocephalus, L. obtusidens and L. friderici, which displayed positive signals only on the W chromosomes of L. macrocephalus and L. obtusidens. Here, we examined the distribution patterns of LeSpe I in these species, as well in L. striatus, L. lacustris, Schizodon borellii and S. isognathus, which are all from different hydrographic basins, and we corroborated the sex chromosome specificities of this sequence. Besides the repetitive sequences associated with differentiated sex chromosomes, also a satellite DNA, LeSma I, has also been isolated from L. elongatus. This satellite DNA, which is linked to nucleolar organiser regions (classified as chromosomes Z2 and W2 by Parise-Maltempi et al. ), is exclusively found in L. elongatus, in both male and female individuals.
The simple sex chromosome system originated from a pair of autosomes that, for some reason, stopped recombining and gradually diverged from each other . This process of sex-chromosome evolution has attracted considerable interest over the years, and an important question has been what the evolutionary forces are that act to make a pair of autosomes cease recombining in one sex, eventually leading to the formation of two discrete chromosome types .
A series of heterochromatin acquisitions may have driven the morphological differentiation of simple sex chromosomes found in some Leporinus species to the multiple sex chromosome system observed in L. elongatus. Considering the direct relationship between the LeSpe I element and sex chromosome differentiation, at least in L. elongatus, L. macrocephalus and L. obtusidens, the presence of LeSpe II in all of the anostomids studied here, including those without differentiated sex chromosomes (such as Leporinus friderici, L. striatus, L. lacustris, Schizodon borellii and S. isognathus), may represent an acquisition that is more ancient than that of the remaining repetitive elements already described in this family.
In the L. elongatus genome, the LeSpe II element is absent from the sex chromosomes (W1, Z2 and W2), but it is widely distributed in the remaining autosomes. The absence of this element on W1, Z2 and W2 may have contributed to the fixing of the other repetitive elements. During another stage of differentiation, the accumulation of LeSpe I and LeSma I sequences may have modified the accumulation of repetitive sequences in the L. elongatus genome [5, 14], and a remarkable event such as a rearrangement may have promoted the formation of a multiple sex system in this species, since another heteromorphic pair (Z2 and W2) was found only in this species .
In general, the acquisition of heterochromatin, which resulted in the development of a series of important characteristics unique to anostomids, is the most prominent route for chromosomal differentiation in this family [27–29]. Our results revealed amazing new characteristics of the Anostomidae genome and the results of the present study provide new insights into the pathways of chromosomal diversification of the species in this family and will be useful for future comparative genomic studies. The fishes, in general, comprise a basal group of vertebrates with a wide distribution of sex chromosome types that were brought about by events that resulted among others alterations, in changes in or multiplication of repetitive sequences. Therefore, this study also provides important insights into the evolution of the vertebrate genome.