Chromosomal analysis of Centromochlus heckelii (Siluriformes: Auchenipteridae), with a contribution to Centromochlus definition

Historically, there are divergences in the species allocation between Centromochlus and Tatia. This study aimed to generate the first cytogenetic data about Centromochlus and, by analyzing a population of Centromochlus heckelii from the Amazon River basin, to contribute as evidence to a historical taxonomic dilemma. Diploid number of 46 chromosomes and a heteromorphic pair was found in the female karyotypes, thus characterizing a ZZ/ZW sex chromosome system. Pale blocks of heterochromatin were located in centromeric regions of some chromosomes; however, the exclusive female chromosome (W) is almost entirely heterochromatic. AgNORs were detected in terminal position on the short arms of one acrocentric pair in males and two chromosome pairs in females, the acrocentric plus the sex chromosome pair. Notable differences between Centromochlus heckelii and previous data about species of Tatia are: lower diploid number, presence of a sex chromosome system and multiple AgNORs in Centromochlus, while species of Tatia have simple AgNORs and the absence of acrocentric chromosomes. Results in this study show that chromosomal markers could contribute as evidence to taxonomic delimitation studies.


INTRODUCTION
The Neotropical region is characterized by great biodiversity, and South America has the richest freshwater and marine ichthyofauna in the world (Reis et al., 2016). Siluriformes, popularly known as catfishes, comprises the second largest group of Neotropical freshwater fish, composed by 39 families and about 4,000 valid species (Fricke et al., 2020).
Auchenipteridae consists of 26 genera and 126 species (Fricke et al., 2020) divided into two subfamilies (Centromochlinae and Auchenipterinae), which present some historical internal phylogenetic incongruities (Ferraris, 1996;Soares-Porto, 1998). This family gathers small to medium size fishes, with suborbital groove to lodge maxillary bone and anal fin of male with intromittent organ (Calegari et al., 2019). Auchenipterids are inseminating fishes, a distinctive characteristic also present in Astroblepidae and Scoloplacidae (Spadella et al., 2006;2012). Moreover, they also present internal insemination and a remarkable sexual dimorphism related to anal fin modification and other parts of the body, such as dorsal and pelvic fins, and maxillary barbels (Birindelli, 2014).
Centromochlus had species morphologically very heterogeneous, some of which more similar to other Centromochlinae genera (Birindelli, 2014). Several species have been relocated throughout the entire taxonomy history of the family, mostly between Centromochlus and Tatia. Prior to the most recent phylogenetic study on this group (i.e., Calegari et al., 2019), seven of the 17 species in Centromochlus were not initially described in the genus (Fricke et al., 2020).
Considering the confuse taxonomy history of Centromochlinae genera (see taxonomic history section in Calegari et al., 2019) and the availability of published chromosomal 4/12 ni.bio.br | scielo.br/ni data for Tatia (Lui et al., 2013a), this study aimed to describe the first chromosomal data for Centromochlus. In addition, comparative differences in chromosomal data of Tatia and Centromochlus are herein discussed, and how it can contribute to the allocation of species.

MATERIAL AND METHODS
Twelve adult individuals (four males and eight females) of Centromochlus heckelii were collected from Solimões River, a tributary to Amazon basin, city of Manaus (AM) (03°09'12''S, 59°54'20''W) with fishing trawls. The specimens were kept alive in the Catalão Floating Support Base of the Instituto Nacional de Pesquisas da Amazônia -INPA. The specimens were euthanized by clove oil overdose (Griffiths, 2000) for anterior renal cells removal and preparation of mitotic chromosome cell suspensions (Bertollo et al., 1978). The slides were stained with Giemsa 5% for karyotype studies (Levan et al., 1964) and de-stained with fixative solution 3:1 of methanol and acetic acid, respectively. Afterwards, they underwent C-banding treatment and stained with a solution containing 20µl of anti-fading solution and 0.7 µl propidium iodide (Lui et al., 2012) to determine heterochromatin distribution pattern (Sumner, 1972). The slides were de-stained again and subsequently stained by silver nitrate impregnation (Howell, Black, 1980) to detect nucleolus organizing regions (AgNORs), in sequential analysis. The specimens were deposited in the Coleção Zoológica de Peixes do Instituto Nacional de Pesquisas da Amazônia (INPA) under the voucher number INPA 57942.

RESULTS
The diploid number found in Centromochlus heckelli was of 46 chromosomes for males and females. Thus 14 metacentric chromosomes, 6 submetacentric, 6 subtelocentric and 20 acrocentric chromosomes in males; and 15 metacentric, 6 submetacentric, 5 subtelocentric and 20 acrocentric chromosomes in females (Figs. 1A-C). The difference in karyotype formulas is due to the presence of a heteromorphic pair in the female karyotype, composed of a subtelocentric chromosome (Z) and a large metacentric chromosome (W), which stands out in the karyotype for being the largest in the complement (Fig. 1A). The ZZ chromosomal pair in males corresponds to two subtelocentric chromosomes, which are equivalente to pair 12 (Fig. 1C). Thus, this species presents female heterogamety with a ZZ/ZW sex chromosome system. Heterochromatin was observed in the centromeric regions of some metacentric and acrocentric chromosomes, with pale blocks (Figs. 1B-D; Fig. 2). The Z chromosome shows heterochromatin proximal in the long arm, while the female exclusive chromosome (W) is almost entirely heterochromatic (Figs. 1B-D). AgNORs were detected in two chromosomal pairs in females, both in the short arm terminal position, in the acrocentric pair 20 and in the sex determination corresponding pair (Z and W) (Fig. 1, in box).
Considering the eight species already studied in Auchenipteridae, this is the first report of a sex chromosome system. The superfamily Doradoidea is composed by 6/12 ni.bio.br | scielo.br/ni Auchenipteridae and Doradidae, and it is a monophyletic clade supported by many synapomorphies by both morphological and molecular characters (Birindelli, 2014;Callegari et al., 2019). A recent study was the first to describe the presence of a sex chromosome system in a Doradidae species, also a ZZ/ZW system based on the analysis of Tenellus trimaculatus (Boulenger, 1898) (Takagui et al., 2017). Even though they correspond to the same heteromorphic chromosome system, it is evident that the origin events are independent from one another due to the phylogenetic distance between the taxa. Recently, a multiple sex system X 1 X 1 X 2 X 2 /X 1 Y 1 X 2 Y 2 has also been described in Bunocephalus coracoideus (Cope, 1874) (e.g. , Ferreira et al., 2016), a species of Aspredinidae, which differs from those afore mentioned by a male heterogamety. Aspredinidae is considered sister group of the superfamily Doradoidea (Sullivan et al., 2006;Calegari et al., 2019).
Among Siluriformes, most reports of sex chromosomes were in Loricariidae species (e.g., Michele et al., 1977;Oliveira et al., 2007;Favarato et al., 2015), rarely described in other families. Moreover, it is observed that female heterogametic sex chromosome systems are less common than male heterogametic ones (Takagui et al., 2017). It is noticeable that when a clade has a single type of sex determining mechanism, further analysis may show independent origins of analogous mechanisms (Mank, Avise, 2009), as suggested for salmonid XY systems (e.g., Woram et al., 2003) and Oryzias' multiple systems (e.g., Tanaka et al., 2007), as it obviously corresponds to these Doradoidea species.
The heterochromatin distribution pattern of Centromochlus heckelii differs from the usual of Auchenipteridae. In this species, blocks were verified in the centromeric regions of some metacentric and acrocentric chromosomes (Fig. 1B-D). Most species of Auchenipteridae have terminal blocks but rarely in the centromeric region (e.g., Ravedutti, Júlio Jr, 2001;Lui et al., 2010;Lui et al., 2013a,b;Lui et al., 2015). Furthermore, there is the presence of a conspicuous proximal block on the long arm of the Z chromosome, and the W chromosome is almost completely heterochromatic (Fig.  2). Apparent blocks were also observed in the interstitial region of a submetacentric pair in Tatia neivai (Lui et al., 2013a), similar to the one observed on Z chromosome in Centromochlus heckelii but on chromosomes of different morphology (Fig. 2).
The majority of gonochoristic fish species have homomorphic sex chromosomes, and heteromorphic sex chromosomes have been found in roughly 10% of the karyotyped species (Devlin, Nagahama, 2002). According to Charlesworth et al. (2005), differentiation of sex chromosomes occurs initially by the accumulation of sex-specific alleles in only one of the proto-sex chromosomes. Afterwards, mechanisms that suppress recombination by chromosomal rearrangements and/or accumulation of repetitive DNA sequences among proto-sex chromosome homologues are triggered (Cioffi et al., 2017).
The fact that W is fully heterochromatic and larger than Z, the second having only one proximal heterochromatin block on the long arm, suggests that the emergence of this large chromosome may have occurred from two probable mechanisms: (1) heterochromatinization of the W chromosome, which would explain the almost inexistence of euchromatin regions when compared to Z, with evolutionary dynamics similar to the upper vertebrate W chromosomes (Cioffi et al., 2017). In such cases, W is almost entirely heterochromatic and Z has only one block restricted to the pericentric or telomeric regions, as observed in lower vertebrates, like Characidium Reinhardt, 1867 species (e.g., Vicari et al., 2008;Scacchetti et al., 2015). (2) Heterochromatin amplification, FIGURE 2 | Idiograms representing the karyotype and locations of heterochromatin and AgNORs in Centromochlus heckelii (present study) in comparison to Tatia neivai and Tatia jaracatia (Lui et al., 2013a). m: metacentric chromosomes; sm: submetacentric chromosomes; st: subtelocentric chromosomes; a: acrocentric chromosomes.
Multiple AgNORs are described for the first time in Auchenipteridae. Regarding the species of Tatia, which have greater historical taxonomic incongruities with Centromochlus, AgNORs are in terminal position on the short arm of a subtelocentric pair (Lui et al., 2013a), similar to populations of Trachelyopterus galeatus (Linnaeus, 1766) (Lui et al., 2009;Lui et al., 2010) and Glanidium ribeiroi Haseman, 1911(Lui et al., 2015. In Doradidae, AgNORs vary in number and morphology of the carrier pair, yet simple AgNORs in terminal position is considered standard for the family (Milhomem et al., 2008;Takagui et al., 2017).
Centromochlus heckelii had AgNORs in two pairs, including the sex determining pair, which may come from translocation between the chromosomal pairs. During interphase, chromosomes retain their individuality and occupy distinct territories in the nucleus. This conformation, called the "Rabl Model", occurs because each intherphase chromosome follows the course of a primary nuclear filament, where secondary and tertiary filaments extend as lateral projections and form a three-dimensional structure. The centromeres are preferably at a specific nucleus site and the telomeres tend to be at an opposite site, especially on the periphery of the nucleus. As a result, a close arrangement between non-homologous chromosomes may facilitate translocation of rDNA genes between equilocal telomeric regions (Cremer et al., 1982;Cremer, Cremer, 2010).
According to Calegari et al. (2019), Centromochlus and Tatia have represented the 8/12 ni.bio.br | scielo.br/ni most complex genera of the subfamily, with a high amount of homoplasies, caused primarily by many species descriptions and reallocations between these genera, undermining taxonomic limits, which highlights the need for alternative tools for species delimitation. Therefore, cytogenetics among Centromochlinae genera showed to be a supporting tool for taxonomy and perhaps phylogenetic informative, assisting the understanding of species allocation. While comparing Centromochlus heckelii and Tatia data, four notable differences are detected: (1) difference in diploid number, (2) absence of acrocentric chromosomes in Tatia, (3) presence of a sex chromosome system in C. heckelii and (4) multiple AgNORs in C. heckelii (Tab. 1). The data presented here, when compared to the literature, show simple chromosomal markers capable of pointing out distinct characteristics among genera. It is important to say that markers were found from classical cytogenetic techniques with potential to assist in future investigations in taxonomy and phylogenetic studies amid Tatia and Centromochlus.