Allozyme and cytogenetic analysis in two species of Hypostomus (Siluriformes: Loricariidae) from the Paraguai River basin, Brazil: occurrence of B microchromosome and intrapopulation heterochromatic polymorphism in H. boulengeri

Abstract

Hypostomus is distributed by Central and South America basins, with diverse species with taxonomic conflicts. This way, the integration of auxiliary techniques contributes to understanding the systematics and phylogeny of the group. Thus, this study aimed to investigate the Hypostomus cochliodon and H. boulengeri from the Onça stream (Paraguai River basin) by allozyme and cytogenetic techniques. Hypostomus boulengeri showed a diploid number of 68 chromosomes (14m+14sm+18st+22a), multiple NOR revealed by Ag-NOR and 18S rDNA FISH, a polymorphism of heterochromatin in acrocentrics and the presence of B microchromosome. Hypostomus cochliodon showed a diploid number of 64 chromosomes (16m+26sm+14st+8a); despite the single NOR, some individuals showed NOR in both telomeres detected by Ag-NOR and 18S rDNA FISH. Isozyme identified two diagnostic loci (Idh-A and Gdh-A) between the two species and multiple loci with unique alleles in H. boulengeri. The genetic variability indicated by the mean heterozygosity (He) was 0.2461 and 0.0309 in H. boulengeri and H. cochliodon,respectively.Thus, this study reports the first cytogenetic data for H. boulengeri and the first isozymatic data for H. boulengeri and H. cochliodon. The two species presented evident cytogenetic and isoenzymatic differences with the obtaining of exclusive genetic markers providing support for future evolutionary studies in the group.

Keywords:
Ribosomal DNA; C-Banding; Diagnostic Loci; Isoenzymes

Resumo

Hypostomus está distribuído por bacias da América Central e do Sul, com grande diversidade de espécies com conflitos taxonômicos. Desta forma, a integração de técnicas auxiliares contribui para a compreensão da sistemática e filogenia do grupo. Assim, este estudo teve como objetivo investigar Hypostomus cochliodon e H. boulengeri do riacho Onça (bacia do rio Paraguai) por meio de técnicas aloenzimáticas e citogenéticas. As análises citogenéticas em H. boulengeri mostraram número cromossômico igual a 2n = 68 (14m+14sm+18st+22a), sistema de NOR múltiplo revelado por Ag-NOR e 18S-FISH, um polimorfismo de heterocromatina em acrocêntricos e a presença de microcromossomos Bs. Hypostomus cochliodon mostrou um número diploide de 64 cromossomos (16m+26sm+14st+8a); apesar do sistema de NOR simples, alguns indivíduos apresentaram NOR em ambos os telômeros detectados por Ag-NOR e 18S-FISH. Uma isozima identificou dois loci diagnósticos (Idh-A and Gdh-A) entre as duas espécies e múltiplos loci com alelos únicos em H. boulengeri. A variabilidade genética indicada pela heterozigosidade média (He) foi de 0,2461 e 0,0309 em H. boulengeri e H. cochliodon, respectivamente. Assim, este estudo relata os primeiros dados citogenéticos para H. boulengeri e os primeiros dados isoenzimáticos para H. boulengeri e H. cochliodon. As duas espécies apresentaram evidentes diferenças citogenéticas e isoenzimáticas com a obtenção de marcadores genéticos exclusivos fornecendo suporte para futuros estudos evolutivos no grupo.

Palavras chave:
DNA Ribossomal; Banda C; Loco diagnóstico; Isoenzimas

INTRODUCTION

Loricariidae is the most prominent family among the Siluriformes, distributed throughout Central and South America, and it is composed of fish popularly known as catfish. Among the subfamilies, Hypostominae has 500 species and 45 valid genera (Fricke et al., 2023), that despite the monophyly, present phylogenetic conflicts, mainly in genus Hypostomus Lacepède, 1803 which presents a large number of species with significant morphological variation (Zawadzki et al., 2001, 2008a; Armbruster, 2003, 2004; Reis et al., 2006; Ferraris, 2007; Cramer et al., 2011; Lujan et al., 2015; Roxo et al., 2019). Thus, the integration of phylogenetic techniques can elucidate taxonomic uncertainties; for example, integrative taxonomy associates molecular, cytogenetic, and morphological methods, which together contribute to aspects related to genetic variability and cryptic diversity of the genus (Pugedo et al., 2016; Dias, Zawadzki, 2018; Azevedo et al., 2021).

Studies using the allozyme technique to identify species of Hypostomus were carried out mainly in specimens present in the Paraná River basin, contributed to the evaluation of the genetic variability of populations, identification, and distinction of species, in addition to the inference of phylogenetic relationships and systematic approach of this genus that represents a complex subject (Zawadzki et al., 1999, 2002, 2004, 2008a; Paiva et al., 2005; Ito et al., 2009).

On the other hand, cytogenetic studies in Hypostomus show wide variation in chromosome number, ranging from 2n = 64 in H. faveolus Zawadzki, Birindelli & Lima, 2008, H. cochliodon Kner, 1854, H. soniae Hollanda Carvalho & Weber, 2005 (Bueno et al., 2013; Oliveira et al., 2019) to 2n = 84 in H. perdido Zawadzki, Tencatt & Froehlich, 2014 (Cereali et al., 2008; Zawadzki et al., 2014). In addition, interspecific and intraspecific karyotypic variations have been reported in different populations. This diversity has been attributed to chromosomal rearrangements throughout the karyotypic evolution of Hypostomus (Artoni, Bertollo, 2001; Bueno et al., 2012; Ferreira et al., 2019). In addition, inferences about the taxonomy and phylogeny have been made from the physical mapping of some regions of the chromosomes, such as the nucleolus organizer region (NOR), where the single NOR is considered a pleisiomorphic character, while multiple NOR located in the terminal region is the most commonly found character and deemed apomorphic character in the genus (Artoni, Bertollo, 1996, 2001; Alves et al., 2006; Rubert et al., 2016; Lorscheider et al., 2018). Moreover, constitutive heterochromatin co-localized with NOR sites may be involved in the dispersion of extra copies of rDNA genes along the genome in Hypostomus, where unequal mating events and amplification of heterochromatin would explain the occurrence of multiple NORs. Additionally, transposable elements have also been suggested as agents of dispersion of copies of these genes throughout the genome of the group (Bueno et al., 2014; Rubert et al., 2016; Lorscheider et al., 2018).

In addition, variations in the distribution of constitutive heterochromatin across the karyotype have made it possible to discuss the dispersion mechanisms of heterochromatin in different populations of Hypostomus (Artoni, Bertollo, 1999; Bitencourt et al., 2011; Traldi et al., 2012; Baumgärtner et al., 2014). Conspicuous blocks of heterochromatin located on the long arm of acrocentric chromosomes have been commonly found in the group, and even reports of polymorphism involving these chromosomes have been detected intra- and interpopulationally (Artoni, Bertollo, 1999, 2001; Rubert et al., 2011; Bitencourt et al., 2012). The hypothesis that the amplification of heterochromatic segments could have caused the dispersion of heterochromatin along the karyotype, in addition to other elements such as transposable elements and chromosomal rearrangements, was also suggested (Bitencourt et al., 2012; Traldi et al., 2012, 2019; Baumgärtner et al., 2014; Oliveira et al., 2015; Ferreira et al., 2019).

In this article, we aimed to expand on the cytogenetic and isozymatic data of H. cochliodon and H. boulengeri (Eigenmann & Kennedy, 1903) collected in a tributary of the Paraguai River. Here are the first cytogenetic data for H. boulengeri and the first isoenzymatic data for both species.

MATERIAL AND METHODS

Study area and sampling. The individuals of Hypostomus boulengeri and H. cochliodon(Figs. 1A, B) were collected in the Onça stream, a tributary of the Taquari River, upper Paraguai River basin, Mato Grosso do Sul (Coxim-MS; 18º30’S 54º40’W and 18º32’S 51º25’W). Specimens were anesthetized and sacrificed by immersion in eugenol, fixed in 10% formalin solution, and later preserved in 70% ethanol (Griffiths, 2000). Specimens of both species were deposited in the ichthyological collection of the Núcleo de Pesquisas em Limnologia, Ictiologia e Aquicultura (Nupélia) of the Universidade Estadual de Maringá (NUP 9821, H. boulengeri; NUP 9822, H. cochliodon).

Cytogenetic analysis. Ten individuals of H. boulengeri (three males; five females; two unidentified) and sixteen of H. cochliodon (seven males; eight females; one unidentified) were analyzed. Mitotic chromosomes were obtained from kidney cells, according to the technique described by Bertollo et al. (1978). The Nucleolar Organizer Regions (NOR) were detected by the silver nitrate staining (Howell, Black, 1980) and Fluorescent in situ Hybridization (FISH) technique, using 18S rDNA probes obtained from 18S rDNA fragments of Prochilodus argenteus Spix & Agassiz, 1829 (Hatanaka, Galetti, 2004), following the methodology described by Pinkel et al. (1986). The C-banding technique determined the heterochromatin distribution (Sumner, 1972) and stained with propidium iodide (Lui et al., 2012). The arms ratio, as proposed by Levan et al. (1964), established chromosome morphology and classified it as metacentric (m), submetacentric (sm), subtelocentric (st), and acrocentric (a).

FIGURE 1 |
Specimens of Hypostomus boulengeri (A) and H. cochliodon (B) from the Onça stream, upper Paraguai River basin. Scale bar = 100 mm.

Allozyme analysis. Muscle, liver, and heart samples were collected from both species and preserved at low temperatures (-20ºC). Starch gels (15%) were prepared using three different buffer systems in pH 7.4 (Murphy et al., 1996), each one specific for the other enzymatic systems and tissues (Tab. 1). Tissue samples were homogenized with 0.02 M Tris-HCl buffer, pH 7.5, and centrifuged at 25.000 rpm for 30 min at a low temperature. The protein extract was applied to the gel, subjected to continuous horizontal electrophoresis, and subsequently incubated in specific histochemical solutions (Murphy et al., 1996). The enzymatic systems were analyzed (Tab. 1), and the genetic interpretation of the zymograms was based on the quaternary structure of the enzymes, according to Ward et al. (1992). Data were analyzed using Popgene 1.31 (Yeh et al., 1999). Loci and alleles were named according to Simonsen (2012), and data was analyzed using Popgen 1:32 software (Yeh et al., 1997). Genetic variability was determined by calculating heterozygosity (expected and observed) according to Nei (1978). The identity (I) and the genetic distance (D) were calculated with the values of the allele frequencies. We employed the dendrogram (grouping method by the algorithm UPGMA- Unweighted Pair Group Method with Arithmetic Means) of the populations, assuming Hardy-Weinberg equilibrium.

TABLE 1 |
Allozymes analyzed in species Hypostomus boulengeri and H. cochliodon: enzyme name, enzyme commission (EC) number, tissues, and buffers. EC n - Enzyme Commission Number; L - liver; M - muscle; H - heart; TBE - Tris-borate-EDTA; TC - Tris-citrate; TEM - Tris -EDTA-maleate.

Enzyme (abbreviation)	no EC	Tissues	Buffers
Alcohol dehydrogenase (ADH)	1.1.1.1	L	TBE
Aspartate aminotransferase (AAT)	2.6.1.1	L	TEM
Acid Phosphatase (ACP)	3.1.3.2	L	TC
Glucose-3-phosphate dehydrogenase (G3PDH)	1.1.1.8	L	TC
Glucose-6-phosphate isomerase (GPI)	5.3.1.9	M, H	TC
Glucose dehydrogenase (GCDH)	1.1.1.118	L	TEM
Isocitrate dehydrogenase (IDH)	1.1.1.42	L, M, H	TC
Malate dehydrogenase (MDH)	1.1.1.37	L, M, H	TC
Superoxide dismutase (SOD)	1.15.1.1	L	TBE

RESULTS

Cytogenetic data. Individuals of Hypostomus boulengeri presented a diploid number of 2n = 68 distributed in 14m+22sm+10st+22a and a fundamental number (FN) equal to 114 (Fig. 2A). In addition to the basic karyotype, all male and female individuals presented a variation from zero to one B microchromosomes in the somatic cells without homology with the other chromosomes (Fig. 2A, in the box). These elements are smaller than any chromosome of the standard A complement and presented 6% of frequency in the cells analyzed (Tab. 2). Hypostomus cochliodon showed 2n = 64 with the karyotypic formula of 16m+22sm+18st+8a and FN = 120 (Fig. 3A). There were no karyotypic differences between males and females in both species.

FIGURE 2 |
Karyotypes of Hypostomus boulengeri subjected to A. Giemsa, B. C-banding and G. FISH with 18S rDNA probe (yellow). Polymorphism of the heterochromatin in pairs 24, 25, and 26 are shown in C, D, E, and F. The Ag-NOR-bearing chromosomes are boxed beside the karyotype stained with Giemsa. The B microchromosomes are boxed beside the karyotypes stained with Giemsa and C-banding. Scale bar = 10 µm.

FIGURE 3 |
Karyotypes of Hypostomus cochliodon subjected to A. Giemsa, B. C-banding, and C. FISH with 18S rDNA probe (yellow). The Ag-NOR-bearing chromosomes are boxed beside the karyotype stained with Giemsa. Note one of the homologs of pair 29 with marking in both telomeres after stained Ag-NOR (Box in A), C-banding (Box in B), and FISH with 18S rDNA probe (Box in C). Scale bar = 10 µm.

Analysis of the nucleolus organizer region performed with Ag-NOR and 18S rDNA FISH techniques in H. boulengeri showed a multiple NOR located on the short arm of three pairs of submetacentric chromosomes (pairs: 9, 10, and 14; Fig. 2A, in the box) and in the telomere region on the long arms of pair 24 (Fig. 2A, in the box). In H. cochliodon, the single NOR was detected on the long arm of the first pair of acrocentric chromosomes at the telomeric position (pair 29; Figs. 3A, C, in the boxes). However, in some individuals of this species, additional staining was detected in the short arm in one of the homologs of the NOR organizing pair (NORs in both telomeres, Figs. 3A, C, in the boxes).

TABLE 2 |
Frequency of B microchromosomes in Hypostomus boulengeri.

Individual	Cells analyzed	Cells with B microchromosomes
1	17	2 (11.76%)
2	4	0 (0%)
3	30	2 (6.7%)
4	35	1 (2.86%)
5	19	0 (0%)
6	62	4 (6.45%)
7	23	3 (13.04%)
8	3	0 (0%)
9	4	0 (0%)
10	3	0 (0%)
Total (Frequency)	200	12 (6.0%)

In H. boulengeri, the C-banding revealed pericentromeric heterochromatin blocks in most metacentric, submetacentric, and subtelocentric chromosomes and the short arm of pairs 9, 10, and 14 coinciding with the NOR regions (Fig. 2B). Furthermore, a conspicuous heterochromatic segment can also be observed in the long arm of some acrocentric chromosomes in both sexes (pairs: 24, 25, and 26), and the number of chromosomes containing this type of heterochromatin varied among individuals in the population (two to six chromosomes; Figs. 2C–F), characterizing a numerical polymorphism. Additionally, some individuals have heterochromatic B microchromosome (Fig. 2B, in the box). Heterochromatinin H. cochliodon was evidenced mainly in the pericentromeric regions of metacentric and submetacentric chromosomes and blocks on the short arm of the submetacentric and subtelocentric chromosomes (Fig. 3B). Further, the NOR region was C-banding positive (Fig. 3B, in the box).

Allozyme data. Nine enzymatic systems allowed the analysis of 15 loci of the species H. boulengeri and H. cochliodon, presenting 30 alleles; among these were diagnoses (Idh-A and Gcdh-A; Tab. 3). In H. boulengeri, several exclusive alleles were detected with variable frequencies at the loci: Aat-A-a and c, Adh-A-b and c, Gpi-B-a and d, G3pdh-A-b and c, G3pdh-B-b and Idh-B-a. Regarding the genetic variability of the two populations, values of 0.2461 and 0.0309 were found for the average expected heterozygosity (He) (Tab. 3) for H. boulengeri and H. cochliodon, respectively.

TABLE 3 |
Allelic frequencies were obtained from the polymorphic loci of the species analyzed in this study. Number of Hypostomus boulengeri analyzed - n (H. b.), number of H. cochliodon analyzed - n (H. c.), Loci - polymorphic loci, percentage of polymorphic loci (P%), number of alleles per locus (K), average heterozygosity obtained (Ho) and expected (He). In parentheses are the respective standard deviations.

Loci	Allelic	Hypostomus boulengeri	n (H. b.)	Hypostomus cochliodon	n (H. c.)
Aat-A	a	0.0526	19	—	24
	b	0.5789		1.0000
	c	0.3684		—
Aat-B	a	1.0000	20	1.0000	22
Acp-A	a	1.0000	20	1.0000	24
Adh-A	a	0.3000	20	1.0000	24
	b	0.6500		—
	c	0.0500		—
Gcdh-A	a	1.0000	20	—	24
	b	—		1.0000
Gpi-A	a	1.0000	20	1.0000	24
Gpi-B	a	0.2250	20	—	24
	b	0.4000		0.6522
	c	0.2000		0.3478
	d	0.1750		—
G3pdh-A	a	0.5750	20	1.0000	24
	b	0.3250		—
	c	0.1000		—
G3pdh-B	a	0.2000	20	1.0000	24
	b	0.8000		—
Idh-A	a	—	20	1.0000	24
	b	0.2000		—
	c	0.6500		—
	d	0.1500		—
Idh-B	a	0.6000	20	—	22
	b	0.4000		1.0000
Mdh-A	a	1.0000	20	1.0000	24
Mdh-B	a	1.0000	20	1.0000	24
Mdh-C	a	1.0000	20	1.0000	24
Sod-A	a	1.0000	20	1.0000	24
P		7		1
P%		46.67		6.67
K		1.8667		1.0667
Ho		0.0981 (0.1702)		0.0000 (0.0000)
He		0.2461 (0.2836)		0.0309 (0.1171)

DISCUSSION

Cytogenetics analysis.Hypostomus cochliodon from the Onça stream showed a diploid number (2n = 64) similar to other cytogenetically characterized populations. However, the karyotype formula, FN, nucleolar organizer pair’s location, and constitutive heterochromatin distribution detected in the present study differed (Tab. 4). Although this species belongs to the H. cochliodon group (Ambruster, 2003), considered a monophyletic clade with 20 valid species distributed throughout South America (Tencatt et al., 2014), cytogenetic studies are scarce. Bueno et al. (2013) related cytogenetic data of Hypostomus species with their respective geographic distributions along the watershed, considering that H. cochliodon is one of the species with the highest diploid number widely distributed in the North basin (Paraguai and Amazonia), despite the great diversity of species spread across these basins, cytogenetic data on karyotypic variety are also scarce for the genus. The present study extends the cytogenetic data of Hypostomus belonging to the Paraguai basin; in addition, H. boulengeri presented 68 chromosomes. Therefore, it corroborates recent studies that demonstrate that in the southern basins, the species of Hypostomus contain a high number of chromosomes (Bueno et al., 2013; Becker et al., 2014; Rubert et al., 2016; Ferreira et al., 2019). Thus, it is necessary to increase such data to understand the karyotypic evolution of the group; in addition, together with morphological and molecular studies, they can help to understand the systematics and phylogeny of this species (Becker et al., 2014; Rubert et al., 2016; Ferreira et al., 2019).

Furthermore, the present study shows the first cytogenetic description of H. boulengeri by detecting a constitutive heterochromatin polymorphism involving acrocentric chromosomes that presented conspicuous heterochromatin blocks (Figs. 2C–F). This type of heterochromatin pattern in acrocentrics was also found in other species of Hypostomus (Artoni, Bertollo, 1999; Kavalco et al., 2004; Baumgärtner et al., 2014; Oliveira et al., 2015; Ferreira et al., 2019). In addition, in some populations, polymorphisms related to this type of heterochromatin distribution pattern in acrocentric chromosomes were also observed, suggesting that the amplification of heterochromatic regions originated the intra and interpopulational variations in the genus (Traldi et al., 2012; Baumgärtner et al., 2014; Oliveira et al., 2015; Ferreira et al., 2019). In H. regani (Ihering, 1905), also collected from the Onça stream, a chromosomal heteromorphism was detected by the C-banding technique, which allowed the distinction of two karyotypes, suggesting that the origin of this heteromorphism occurred from the amplification of heterochromatin that allowed the difference of two karyotypes (Ferreira et al., 2019). In H. strigaticeps (Regan, 1908), from the upper Paraná River basin, heterochromatin amplification supposedly caused the interpopulation polymorphism, considered that the unequal crossing over processes and the proximity of homologous segments in the interphase nucleus would probably facilitate unequal exchanges and dispersion of heterochromatin and that such events could be involved in the amplification process of this region by the genome (Baumgärtner et al., 2014).

TABLE 4 |
Comparison among cytogenetic studies in the species Hypostomus cochliodon. FN: Fundamental number; m: metacentric; sm: submetacentric; st: subtelocentric; a: acrocentric.

Species/ Sampling site	Diploid Number	Karyotype	FN	NOR System/ NOR pair	Heterochromatin distribution	References
H. cochliodon/ Iguaçu River	64	12m+16sm+16st+20a	108	Simple/ 28	-	Bueno et al. (2013)
H. aff. cochliodon/ Esparramo stream	64	18m+20sm+26st/a	102	Multiple/ 22, 26	large heterochromatic blocks in pairs: 20, 21 e 22	Becker et al. (2014)
H. aff. cochliodon/ Pitaluga stream	64	18m+20sm+26st/a	102	Multiple/ 22, 26	large heterochromatic blocks in pairs: 20, 21 e 22	Becker et al. (2014)
H. cochliodon/ Piraputanga River	64	16m 20sm 28st-a	100	Multiple	-	Rubert et al. (2016)
H. cochliodon/ Onça stream	64	16m+22sm+18st+ 8a	120	Simple/ 29	absence of large heterochromatic blocks in pairs	Present study

In addition, the association of transposable elements (TEs) to heterochromatin would promote its reorganization due to the ability of TEs to disperse throughout the genome, thus contributing to chromosomal evolution (Baumgärtner et al., 2014). Rex1 transposable elements (TEs) were associated with heterochromatin in H. ancistroides (Ihering, 1911), and H. nigromaculatus (Schubart, 1964). Transposable elements (TEs) Rex1 were found to be associated with heterochromatin in H. ancistroides and H. nigromaculatus. Accumulation of TEs in some chromosomes of Hypostomus species indicates the involvement of these elements with the organization of constitutive heterochromatin (Pansonato-Alves et al., 2013; Traldi et al., 2019). Thus, the heterochromatin polymorphism involving acrocentric chromosomes detected in H. boulengeri in the present study that could occurred by amplifying the constitutive heterochromatin, unequal crossing-over and/or transposable elements associated with heterochromatin, which plays an essential role in the karyotypic evolution of Hypostomus.

Regarding the B microchromosome detected in H. boulengeri in the present study, this type of chromosome is uncommon in Hypostomus, with B chromosomes being observed only in Hypostomus sp. from Xingu-3 (Milhomem et al., 2010) and Hypostomus sp. 3 (Cereali et al., 2008). In both studies described previously, the frequency of this chromosome in the populations analyzed was not mentioned. In five individuals of H. boulengeri, these chromosomes were present in 6% of the cells analyzed (Tab. 2), showing an inter and intra-individual variabilities of these elements, suggesting mitotic instability, probably due to their non-Mendelian behavior that may be related to chromosomal non-disjunction during meiosis, leading to uneven segregation of genetic material between germ cells (Rosa et al., 2014).

Furthermore, the B microchromosome observed in H. boulengeri were completely heterochromatic, while in Hypostomus sp. from Xingu-3 (Milhomem et al., 2010) and Hypostomus sp. 3 (Cereali et al., 2008), these B microchromosomes were neither heterochromatic. This indicates that these B microchromosomes can have a different DNA composition, mainly concerning repetitive sequences. In other species of the family Loricariidae, B chromosomes are rarely found, having been reported only in Hisonotus leucofrenatus (Miranda Ribeiro, 1908)(Andreata et al., 1993), Loricaria sp. and Proloricaria prolixa (Isbrücker & Nijssen, 1978) (Scavone, Júlio-Jr, 1994), Neoplecostomus paranensis Langeani, 1990 (Alves et al., 1999), Rineloricaria pentamaculata Langeani & de Araujo, 1994 (Porto et al., 2010) and Harttia longipinna Langeani, Oyakawa & Montoya-Burgos, 2001(Blanco et al., 2012).

Regarding the nucleolar organizer region, although most individuals of H. cochliodon presented a number and location of the NOR, considered conserved in Hypostomus (Artoni, Bertollo, 1996, 2001; Kavalco et al., 2005; Alves et al., 2006; Cereali et al., 2008; Milhomem et al., 2010; Bitencourt et al., 2011, Martinez et al., 2011; Rubert et al., 2011; Lorscheider et al.,2018), some individuals showed an additional NOR site (NORs in both telomeres) in one of the homologs of the NOR organizer pair. Hypostomus with NORs in both telomeres has been reported in H. cochliodon, H. hermanni (Ihering, 1905), H. albopunctatus (Regan, 1980), and H. aff. paulinus (Ihering, 1905) (Rubert et al., 2016). In some fish species such as in the genus Psalidodon Eigenmann, 1911 (Mantovani et al., 2005; Fernandes, Martins-Santos, 2006; Fernandes et al., 2009; Abelini et al., 2014), Hoplias malabaricus (Bloch, 1794) (Cioffi et al., 2009; Blanco et al., 2010), Pyrrhulina cf. australis (Oliveira et al., 1991) and Poecilia latipunctata Meek, 1904 (Galetti, Rash, 1993), NOR in both telomeres has been reported.

For the variation in the distribution of 18S rDNA sites in the Loricariidae, it has been suggested that the dispersion of such sites throughout the genome could jointly or/and separately have contributed to the karyotypic evolution of the group (Porto et al., 2011, 2014a,b; Rubert et al., 2016). Rubert et al. (2016) observed interspecific variation in four species of Hypostomus (H. cochliodon, H. hermanni, H. albopunctatus, and H. aff. paulinus); it was proposed that the association between heterochromatin and rDNA sites contributes to the occurrence of unequal crossing-over generating new rDNA loci. In addition, the proximity between telomeres in the interphase nucleus would facilitate the translocation of some copies of the rDNA genes located in telomeric regions, resulting in the translocation/transfer of genetic material among the chromosomes (Schweizer, Loidl, 1987; Fernandes, Martins-Santos, 2006; Cioffi et al., 2010; Porto et al., 2014a; Rubert et al., 2016). The transposable elements associated with rDNA copies may also contribute to the dissemination of these genes due to their ability to disperse throughout the genome in fish (Silva et al., 2011; Piscor et al., 2013; Bueno et al., 2014; Rubert et al., 2016).

We suggest that in H. cochliodon, the NOR in both telomeres is a derived character. The NOR sites on the long arm probably occurred duplication or amplification and were later inserted in a new region of the same chromosome (a short arm of pair 29). Thus, the NOR in both telomeres in H. cochliodon and the multiple NOR in H. boulengeri corroborate the data for other species, characterized as apomorphies in Hypostomus(Lorscheider et al., 2018). Rubert et al. (2016) suggest that intrinsic genus factors led to different karyoevolutionary mechanisms and would explain the NOR variability, the chromosomal behavior, and the dispersion of specific rearrangements that occurred differently in each population.

Allozyme analysis. Concerning the isozyme analysis, the two diagnostic loci and the exclusive alleles for H. boulengeri showed a distinction between the two species. Allozyme studies among populations of this group have made it possible to identify genetic differences that contribute to the differentiation between them. Renesto et al. (2007) identified diagnostic loci for species H. boulengeri and H. cochliodon from the upper Paraguai River basin (sAat-2, Idh-2 and Mdhp-B) that differed from those found in the present work (Idh-A and Gdh-A). Furthermore, the sAat-2 locus separated H. boulengeri and H. cochliodon from seven other species (H. latifrons Weber, 1986, H. regani, Hypostomus sp. 1, Hypostomus sp. 2, Hypostomus sp. 3, H. cf. latirostris, and Pterygoplichthys ambrosettii (Holmberg, 1893) from the Manso River (Manso Reservoir) and the Cuiabá River. The Idh-2 locus reported by Renesto et al. (2007) is equivalent to the Idh-B locus of the present study, while the Mdhp (malic enzyme) was not analyzed.

The average expected heterozygosity (He) of H. boulengeri (He = 0.2461) was the highest ever verified among the species of this genus studied by isozyme analysis. The highest He values previously found was 0.199 for H. hermanni from the Ivaí River (upper Paraná River basin; Paiva, 2006). For H. boulengeri in the present work, the value of He represents more than four times the expected average heterozygosity value for fish (He = 0.051), obtained by Ward et al. (1992). However, Renesto et al. (2007) verified in H. boulengeri from the upper Paraguai River basin that He equals 0.078. Distinct values were found between populations of H. margaritifer (Regan, 1908) from the Itaipu reservoir in Paraná (He = 0.104) and from the Corumbá Reservoir in Goiás (He = 0.061) (Zawadzki et al., 2002). Hypostomus cochliodon revealed a He value of 0.0309, similar to that found for the population of this same species collected in the Itaipu reservoir (0.039) (Zawadzki et al., 2005), however with a lower He value (0.070) described for H. cochliodon of the upper Paraguai River (Renesto et al., 2007).

A study of three populations of H. regani from the Corumbá, Itaipu, and Manso reservoirs showed that they differed in terms of heterozygosity values, 0.0527, 0.0712, and 0.0317, respectively (Zawadzki et al., 2008b). Thus, heterozygosity is a measure of genetic variability, which can be similar or variable between populations of the same species of Hypostomus. Several biotic and abiotic factors are proposed in the literature. They may be involved in the process of genetic differentiation of this group, such as natural inbreeding barriers that impede gene flow, differences in temperature, water velocity, food resources, and reproductive strategies (Zawadzki et al., 1999, 2002, 2005, 2008b; Paiva, 2006; Ito et al., 2009).

There are few studies on the biology of Neotropical fish, especially those from the Paraguai River basin. Thus, there is difficulty in correlating multivariate biological factors with greater or lesser heterozygosity in these fish. Although it is impossible to confirm the causes of this high genetic variability, it is known that it is crucial because, as expressed by Vida (1994), “the future of maintaining species diversity lies in the genetic diversity of species. Generally, the greater genetic diversity maintained, the greater adaptability and the probability of species survival in a changing environment”.

The present study presents cytogenetic and isozymatic data of H. cochliodon and H. boulengeri collected in a tributary of the Paraguai River offered the first cytogenetic data for H. boulengeri and the first isozymatic data for both species, with the detection of two diagnostic loci, exclusive alleles and high genetic variability for H. boulengeri, in addition, the two species presented evident cytogenetic and isoenzymatic differences with the obtaining of exclusive genetic markers providing support for future evolutionary studies in the group.

ACKNOWLEDGEMENTS

We authors thankNúcleo de Pesquisas em Limnologia, Ictiologia e Aquicultura (Nupélia) for logistic support. This study was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

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ADDITIONAL NOTES

• HOW TO CITE THIS ARTICLE

  Paiva S, Porto FE, Codognotto FJ, Fernandes CA, Rossi MMV, Borin-Carvalho LA, Portela-Castro ALB, Zawadzki CH, Renesto E, Martins-Santos IC.Allozyme and cytogenetic analysis in two species of Hypostomus (Siluriformes: Loricariidae) from the Paraguay River basin, Brazil: occurrence of B microchromosomes and intrapopulation heterochromatic polymorphism in H. boulengeri. Neotrop Ichthyol. 2024; 22(2):e230117. https://doi.org/10.1590/1982-0224-2023-0117

Edited-by

Claudio Oliveira

Publication Dates

• Publication in this collection
  22 July 2024
• Date of issue
  2024

History

• Received
  24 Oct 2023
• Accepted
  21 May 2024