Contributions to the cytogenetics of the Neotropical fish fauna

Abstract Brazilian fish cytogenetics started as early as the seventies in three pioneering research groups, located at the Universidade Estadual Paulista (UNESP, Botucatu, SP), Universidade Federal de São Carlos (UFSCar, São Carlos, SP) and Universidade de São Paulo (USP, São Paulo, SP). Investigations that have been conducted in these groups led to the discovery of a huge chromosomal and genomic biodiversity among Neotropical fishes. Besides, they also provided the expansion of this research area, with the genesis of several other South American research groups, in view of a number of dissertations and doctoral theses developed over years. The current authors were encouraged to make their thesis catalog accessible from a public source, in order to share informations on the taxa and subject matter analyzed. Some of the key contributions to evolutionary fish cytogenetics are also being highligthed.


Introduction
About 13,000 freshwater fish species are now recognized, 50% of them living in the Neotropical region (Reis et al. 2003), which emphasizes the significant parcel of the ichthyological diversity enclosed in this particular world region. Of course, this was one of the main reasons that attracted the attention of some Brazilian researchers, fostering the investigation on cytogenetics of Neotropical fishes.
Brazilian fish cytogenetics started in the early 70s, with three pioneering research groups located at the Universidade Estadual Paulista (UNESP, Botucatu, SP), Universidade Federal de São Carlos (UFSCar, São Carlos, SP) and Universidade de São Paulo (USP, São Paulo, SP). During this time, a lot of significant evolutionary and cytotaxonomic contributions were achieved, improving the knowledge on the biodiversity inside the rich Neotropical ichthyofauna.
The development of methodological approaches was certainly a key step for obtaining good chromosomal preparations and for improving fish cytogenetics. In this sense, the direct chromosome preparation from kidney cells, adapted in our early studies since 70s and recently revised , was largely utilized over years. In addition, the progressive application of conventional banding techniques (C, Ag-NORs, DAPI, CMA 3 staining), as well as more advanced methodologies combining cytogenetic and molecular procedures (chromosome mapping of DNA sequences by FISH, whole chromosome painting -WCP and comparative genomic hybridization -CGH) were essential tools in understanding the fish genome organization, particularly regarding to sex chromosome evolution and biodiversity investigations.
Although primarily and mainly devoted to freshwater species, the chromosomal analyses were also expanded to marine fishes, which is now the particular focus of some laboratories. From 1986 to now, successful biennual symposiums on fish cytogenetics are ongoing at different Brazilian regions. From some years ago, the discipline of genetics was also added to such meetings, with an expressive participation of professionals, students, as well as foreign invited researchers.
The catalogue of student theses, supervised in the Laboratory of Fish Cytogenetics of the Universidade Federal de São Carlos, comprises 42 doctoral theses and 52 master dissertations from 1981 to 2016. Informations about their corresponding students, taxa and matter subjects are available in the present communication, considering that not all the resulting data have been published. Theses/dissertations produced were assembled by taxonomic groups, according to Reis et al. (2003), regardless of their chronology. This criterion provides an overview of the different studied groups, considering that several families, genera and species have wider distribution and were subjected of more extensive investigations, being analyzed by different authors. The "taxa analyzed" item makes explicit when different populations, as well as different karyomorphs (karyotypes with distinct characteristics from each other) of a given species were investigated. The term "species group" was used for cases of specimens showing morphological similarities to a given valid species, but missing a proper taxonomic revision by the time they were studied.
Significantly, more than 20 research groups, nowadays located in different Brazilian regions, and also in Argentina, have emerged from such studies. These new researchers, along with those that have been emerged from the other pioneer laboratories, are now also engaged on fish chromosomal investigations. This was a preponderant condition for the big expansion experienced by the Brazilian fish cytogenetics.
The "Final Remarks" highlights some key contributions to fish evolutionary cytogenetics from MSc and PhD theses produced, as well as from other results that were led by our research team, some of them with significant colaborations of other national and international research groups. Laboratory

Catalogue of MSc Dissertations and PhD Theses
Note: Titles of Theses and Dissertations maintain the taxonomic and/or systematic data as they were originally employed. The classification of some species and genera were later updated by review studies (Reis et al. 2003;Oliveira et al. 2011;FishBase) (

Final remarks
Two general trends were found among the Neotropical fishes regarding the karyotype evolution. In fact, a significant number of families were characterized by conservative karyotypes, in contrast to others with highly divergent ones. Parodontidae, Anostomidae and Prochilodontidae species, for example, exhibit relatively homogeneous karyotypes at the macrostructural level, contrasting with the high chromosomal diversity found among Erythrinidae and Characidae species (Bertollo et al. 1986). It is noteworthy that karyotype features appear to be correlated with their lifestyle and ecological habits, since more dispersive and migratory species usually disclose more stable karyotypes when compared to those with low vagility and organized in small local populations (Bertollo et al. 1986;Blanco et al.2011;Oliveira et al. 2015). Indeed, many local populations were evidenced as having particular karyotypes, pointing to a large number of species complexes and the cryptic biodiversity present in the Neotropical fish fauna, as especially highlighted in the Characidae and Erythrinidae families (Moreira-Filho and Bertollo1991; Bertollo 2007;Cioffi et al. 2012a). In fact, many sympatric, or even syntopic, karyomorphs do not indicate hybridization at the chromosomal level, indicating the absence of gene flow among them and, consequently, corroborating the status of species complexes for some current nominal species . Reports on chromosomal polymorphisms (Giuliano-Caetano and Bertollo 1988;Vicari et al. 2003;Pazza et al. 2006Pazza et al. , 2008Mariotto et al. 2009), natural triploidy (Morelli et al. 1983;Venere and Galetti Jr. 1985;Giuliano-Caetano and Bertollo 1990;Centofante et al. 2001;Garcia et al. 2003) and broad karyotype evolution by centric fissions (Feldberg et al. 1993), were also emphasized for distinct fish groups. Noteworthy is also the cytogenetic contribution for biogeographical analyzes, clarifying the current fish fauna distribution in some important Brazilian river basins. In this sense, native species, as well as invasive ones due to dispersal events or breakdown of geographic isolation, were clearly identified by chromosomal investigations (Peres et al. 2009;Blanco et al. 2010;Silva et al. 2010;Perez et al. 2012). As a significant example, Astyanax bimaculatus from two important Brazilian watersheds, namely the São Francisco and Grande rivers share similar morphological characteristics. However, specimens from each one of such rivers were well characterized by their particular chromosomal features. In the early 1960s, a tributary of the Grande River was artificially transposed into the São Francisco river basin, with the consequent breakdown of the geographic isolation of their respective fish fauna. As a consequence, cytogenetic investigation was able to identify representatives of A. bimaculatus from both basins living in sympatry in the transposition region, as well as individuals with intermediate karyotypes in view of the resulting secondary hybrid zone in such region (Peres et al. 2012).
Over the years, a particular emphasis has been directed on the characterization and the evolutionary process of sex chromosomes. A larger number of Neotropical fish species with well differentiated sex chromosomes occur in comparison to other world regions (Moreira-Filho et al. 1993), carrying simple (ZZ/ZW, XX/XY) and multiple (X 1 X 1 X 2 X 2 /X 1 X 2 Y, XX/XY 1 Y 2 , ZZ/ZW 1 W 2 ) sex chromosome systems (Centofante et al. 2002;Cioffi et al. 2012b), in addition to some others disclosing a nascent or early stage of differentiation Freitas et al. in press). Usually, sex chromosomes occur as a particular feature for some species within a specific fish group, as exemplified in the Erythrinidae, Parodontidae, Anostomidae and Crenuchidae families (Galetti Jr. et al. 1981, 1995Moreira-Filho et al. 1985Molina et al. 1998;Centofante et al. 2001Centofante et al. , 2003Bertollo et al. 2000;Bertollo 2007;Vicari et al. 2008;Cioffi et al. 2013). As a singular exception, all species of the Triportheus genus (Triportheidae) share a same ZZ/ZW sex chromosome system (Artoni et al. 2001(Artoni et al. , 2002Diniz et al. 2008), constituting a special model to investigate the evolution of the sex chromosomes among lower vertebrates. The modern molecular cytogenetics was a key step for understanding the evolutionary process of the sex chromosomes among fishes. This way, the significative role of several classes of repetitive DNAs in the differentiation path of the sex pair, both at its inicial stage Freitas et al. 2017) or more advanced ones (Cioffi et al. , 2011a(Cioffi et al. , b, 2012bYano et al. 2014a, b), was clearly highlighted. Notably, whole chromosome painting (WCP) and comparative genomic hybridization (CGH) were able to demonstrate that fish sex chromosomes can have an independent origin even among closely related species (Cioffi et al. 2011c, d;2013) or, alternatively, a common origin within particular monophyletic groups (Yano et al. 2016).
Besides sex chromosomes, supernumerary or B chromosomes comprise another special feature that stands out in the Neotropical fishes. Such additional elements can be i) as large as the biggest chromosome pair of the karyotype, ii) medium-sized, iii) very small iv) or even characterized as microchromosomes. Two particular models, represented by Astyanax scabripinnis and Prochilodus lineatus, have been subjected to continuous analyses over years. A. scabripinnis has some morphologically differentiated B chromosomes, although a large and similar in size to the first chromosome pair of the karyotype is the most frequent one (Moreira-Filho et al. 2004). Its origin as an isochromosome was demonstrated by both standard and molecular cytogenetic, including meiotic data (Vicente et al. 1996;Mestriner et al. 2000). A continuous population analysis showed that Bs display a particular dynamism related to environmental and sex conditions in A. scabripinnis. Indeed, it is noteworthy their gradual decrease in frequency from higher to lower altitudes, until the complete absence in the latter ones (Néo et al. 2000). In addition, an evident sex ratio distortion is associated with these chromosomes. In fact, the mean number of Bs in males is only about 27% of the female one, which matches the male population frequency (Vicente et al. 1996), suggesting that B chromosomes may play a role on sex determination in this species.
Constrasting with A. scabripinnis, P. lineatus bears a number of very small B chromosomes (Pauls and Bertollo 1983), which also have an intraspecific origin as indicated by molecular cytogenetic and chromosomal banding (Jesus et al. 2003;Artoni et al. 2006). Remarkably, the frequency of these chromosomes was changed over years in close association with their transmission dynamics. In this sense, the average number of Bs increased twice along a time period indicating an accumulation mechanism, but without evidences of additional changes after that. Significantly, the mitotic instability of Bs declined almost 400 times during this same period, reaching a stable transmission. This way, it is likely that the mitotic stabilization was a key process for neutralizing the accumulation process (Cavallaro et al. 2000).
Nowadays, many of such issues so far investigated, in addition to additional approaches on fish biology, are going in advancing in the light of chromosomal, cytogenomic and molecular methodologies currently available. It is hoped that these procedures can provide additional and important advances for the Neotropical fish fauna evolutionary history.