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. 2013 Dec 22;67(2):215–222. doi: 10.1007/s10616-013-9676-x

Comparative cytogenetics between two species of the family Pseudopimelodidae (Siluriformes): occurrence of natural triploidy and supernumerary chromosomes

Juceli Gonzalez Gouveia 1, Vivian Patrícia Oliveira de Moraes 1, Larissa Bettin Pires 1, Renata da Rosa 1, Ana Lúcia Dias 1,✉
PMCID: PMC4329307  PMID: 24363190

Abstract

The Pseudopimelodidae family comprises 35 species however, cytogenetic studies have been performed in only six species. This study uncovered karyotypic data on Pseudopimelodus pulcher and Microglanis cottoides. Both species possessed 2n = 54, with 20m + 16sm + 10st + 8a and FN = 100 for P. pulcher and 30m + 14sm + 6st + 4a and FN = 104 for M. cottoides. A female of M. cottoides with 45m + 21sm + 9st + 6a (2n = 81) plus two extra small chromosomes was found, indicating a natural triploidy with supernumerary chromosomes. The formation of the polyploid individual seems to have come from a diploid female gamete, due to the presence of a marker chromosome pair partially heterochromatic presents only in females and common to that exemplar. This triploid female showed three chromosomes with nitrate staining (AgNOR), 18S rDNA probe and chromomycin A3 (CMA3) staining. AgNORs were observed on pairs 12 and 23 in P. pulcher and pair 24 in M. cottoides, results that were confirmed with an 18S rDNA probe and CMA3 fluorochrome. These are the first chromosomal data for P. pulcher and provide the first description of natural triploidy with the presence of supernumerary chromosomes in this family and emphasizing well the chromosomal rearrangements diversification between this species.

Keywords: Extra chromosomes, Heterochromatin, Pisces, Polyploidy

Introduction

The family Pseudopimelodidae is widely distributed in South America and is considered the least known family among the naked Neotropical freshwater catfishes (Shibatta 2003). According to Ferraris (2007) the family consists of 30 species and recently new species have been described: Microglanis carlae (Alcaraz et al. 2008); Microglanis minutus (Ottoni et al. 2010); Microglanis robustus (Ruiz and Shibatta 2010); Microglanis oliveirai and Microglanis xylographicus (Ruiz and Shibatta 2011).

Karyotypic descriptions in the Pseudopimelodidae family have been performed on six species of four distinct genera: Cephalosilurus apurensis (Mees 1978); Microglanis aff. cottoides, M. cottoides (Boulenger 1891); cc (cited as M. cottoides) Shibatta and Benine (2005); Pseudopimelodus bufonius (Valenciennes 1840); Pseudopimelodus mangurus (Valenciennes 1835) and Lophiosilurus alexandri Steindachner 1876 (Table 1). These analyses revealed 2n = 54 in all of the specimens analyzed, indicating a conserved karyotypic evolution in relation to the diploid number.

Table 1.

Cytogenetics data on species of the family Pseudopimelodidae

Species Localty 2n Karyotypic formula FN NOR 18S rDNA Reference
Cephalosilurus apurensis Orinoco river/Venezuela 54 6m + 28sm + 14st + 6a _ Pair 19 st, p, terminal _ Martinez et al. (2008)
Lophiosilurus alexandri São Francisco river 54 16m + 18sm + 10st + 10a _ 1 pair sm, p, terminal 1 pair sm, p, terminal Marques et al. (2008)
Microglanis aff. cottoides Cavalo stream, Jaraguá do Sul river/SC 54 10m + 32sm + 10st + 2a _ Pair 23 st, p and pair 22 st, q, terminal _ Martinez et al. (2008)
Microglanis garavelloi (cited M. cottoides) Araquá and Capivara river 54 22m + 20sm + 12st 96 Pair 1 m, q, terminal _ Vissotto et al. (1999)
Microglanis cottoides Forquetinha river/RS 54 30m + 14sm + 6st + 4a 104 Pair 24 st, p, terminal Pair 24 st, p, terminal Present study
Pseudopimelodus bufonius Trade aquarium/Amazonia 54 12m + 30sm + 12st _ Pairs 9, 10 and 11 sm, p, terminal _ Martinez et al. (2008)
Pseudopimelodus mangurus Mogi-Guaçu river/SP 54 6m + 26sm + 12st + 10a _ Pair 19 st, p, terminal _ Martinez et al. (2004)
Pseudopimelodus pulcher Laranjinha river/PR 54 20m + 16sm + 10st + 8a 100 Pair 12 sm and pair 23 st, p, terminal Pair 12 sm and pair 23 st, p, terminal Present study
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2n diploid number, FN fundamental number, NOR nucleolus organizer regions type: m, metacentric, sm submetacentric, st subtelocentric, a, acrocentric, p short arm, q long arm

Heterochromatin in Pseudopimelodidae appears as heterochromatic blocks usually distributed in pericentromeric and terminal regions of some chromosomes (Vissotto et al. 1999; Martinez et al. 2008; Marques et al. 2008). The AgNORs in this group of fish can be single, as in L. alexandri (Marques et al. 2008), with only one pair bearing this site, or multiple, as in P. bufonius (Martinez et al. 2008) with two pairs AgNORs.

Fluorescence in situ hybridization (FISH) has been performed only on L. alexandri by Marques et al. (2008). The authors confirmed the occurrence of single NORs in this species using the 18S rDNA probe; the 5S rDNA site, which was found in another pair of chromosomes, is not syntenic to the NOR. Marques et al. (2008) also used chromomycin A3 (GC specific) and 4′-6-diamino-2-phenylindole (DAPI) (AT specific) fluorochromes in L. alexandri and Martinez et al. (2004) in Pseudopimelodus mangurus; and the two species showed CMA3+ signals corresponding to the AgNORs.

The current paper revealed karyotypic data for Pseudopimelodus pulcher (Boulenger 1887) and M. cottoides in comparison with other family species, and will aid in understanding the karyotypic diversity of the group. The data for P. pulcher are novel, and we present the first description of natural triploidy and supernumerary chromosomes in the family in M. cottoides.

Materials and methods

We analysed five specimens of P. pulcher (Boulenger 1887) (two males, two females and one sex unidentified) from the Laranjinha River of the Paranapanema Basin, located in the city of Ribeirão do Pinhal/PR/Brazil (23°24′9.11″S and 50°27′16.4″W) and twelve specimens of M. cottoides (Boulenger 1891) (five males and seven females) from the Forquetinha River, located in the city of Forquetinha/RS/Brazil (29°24′21.8″S and 52°03′18.3″W) in the Patos Lagoon Hydrographic System/RS (Fig. 1). Specimens were deposited in the Museum of Zoology of the Universidade Estadual de Londrina under vouchers 6033 (M. cottoides) and 5767 (P. pulcher). The samples were collected with the permission of the Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis, protocol number 12,280/2.

Fig. 1.

Fig. 1

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Map of Brazil indicating the collection sites: a Laranjinha river—Paranapanema basin/Paraná, b Forquetinha river—Patos lagoon hydrographic system/Rio Grande do Sul

Mitotic chromosomes were obtained by direct preparation after removal of the kidney, as described by Bertollo et al. (1978). The chromosomes were organised according to Levan et al. (1964), with modifications, to determine the fundamental number (FN). Metacentric (m), submetacentric (sm) and subtelocentric (st) chromosomes were considered biarmed, and acrocentric (a) chromosomes were considered uniarmed. The distribution of heterochromatin was analysed by Giemsa C-banding (Sumner 1972). Silver nitrate staining of the active nucleolar organizer regions (AgNOR) was performed according to the method of Howel and Black (1980). The GC- and AT-rich bands were detected with CMA3 and DAPI, respectively, according to the technique described by Schweizer (1980). In addition, the FISH technique was carried out following the protocol reported by Pinkel et al. (1986), with modifications, along with an 18S rDNA probe designed for Prochilodus argenteus (Hatanaka and Galetti 2004).

Results

The diploid number of P. pulcher was 54 with a karyotypic formula of 20m + 16sm + 10st + 8a and FN = 100 (Fig. 2a).

Fig. 2.

Fig. 2

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Karyotypes of a Pseudopimelodus pulcher, b, c Microglanis cottoides diploid and triploid females, respectively. The boxes contain the chromosome NOR-bearing pairs with silver nitrate staining, CMA3 and FISH with 18S rDNA probe. The scale bar represents 5 μm

AgNORs were observed on the short arm of a submetacentric chromosome pair (pair 12) and on one pair of subtelocentric chromosomes (pair 23). These regions were coincident with the 18S rDNA probe after the FISH (Fig. 2a-box). The fluorochrome staining also presented four chromosomes coincident the NORs, with fluorescent signals for CMA3 (Fig. 2a-box).

The heterochromatin was distributed in the pericentromeric and terminal regions of some chromosomes and was positive for both CMA3 and DAPI after fluorochrome staining (Fig. 3h and i); one large-sized submetacentric pair, probably pair 12 of the NOR, showed heterochromatic markings in the pericentromeric and terminal regions of the short arm (Fig. 3h); the pericentromeric region was DAPI+ and CMA3+ and the terminal region was CMA3+ (Fig. 3i).

Fig. 3.

Fig. 3

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Somatic metaphases with C-banding of Microglanis cottoides: (a–b) diploid male, (c–d) diploid female and (e–g) triploid female and Pseudopimelodus pulcher (h–i). C-banding stained with Giemsa (a, c, e, f, h) and overlapping DAPI/CMA3 (b, d, g). The arrowheads in cells of M. cottoides (a–g) indicate the NOR-bearing pairs, and the arrows indicate the two chromosomes with the heterochromatic block in the diploid (c–d) and triploid (f–g) females; in the diploid male (a–b), the arrows indicate chromosome pair one without the heterochromatin block. In (e), the asterisk indicates the two supernumerary chromosomes that are totally heterochromatic; in (h), the asterisk indicates the heterochromatic chromosome pair with AT and GC sites in the pericentromeric region and GC in the terminal region. The scale bar represents 5 μm

The diploid number of M. cottoides also was of 2n = 54 with 30m + 14sm + 6st + 4a and a FN equal to 104 (Fig. 2b). A female of M. cottoides showed a karyotypic formula of 45m + 21sm + 9st + 6a totalizing 81 chromosomes corresponding to a triploid genome, plus two extra chromosomes of the small metacentric type (Fig. 2c).

The AgNOR of the M. cottoides diploid individuals was localized on the short arm of subtelocentric pair 24, and of the triploid individual was located in three subtelocentric chromosomes (24). In both karyotypes (diploid and triploid), the NOR was coincident with 18S rDNA probe, and was also positive for CMA3 fluorochrome staining (Fig. 2 b and c-box).

The heterochromatin in diploid individuals of M. cottoides was distributed in the pericentromeric and terminal regions of some chromosomes in both sexes (Fig. 3 a and c), however, all females presented a large block of interstitial heterochromatin in the 1st chromosome pair (Fig. 3c), not observed in any of the male individuals of M. cottoides (Fig. 3a). All heterochromatic regions of diploid individuals were DAPI+ and CMA3−, including interstitial heterochromatin in the 1st chromosome pair of the female diploid (Fig. 3 b and d).

The heterochromatin in triploid individual also was distributed in the pericentromeric and terminal regions of some chromosomes and in two out of three chromosomes number one of the complement was observed a large block of heterochromatin (Fig. 3 e, f, g), as the 1st chromosome pair of the diploid female; these chromosomes after staining with DAPI proved to be much more evident and positive for this fluorochrome and negative for CMA3 (Fig. 3g). The supernumerary chromosomes of the triploid individual appeared totally heterochromatic (Fig. 3e).

The NOR of pair 24 was C-band positive in all individuals (diploid and triploid), revealing to be heterochromatic in the terminal region of the short arm (Fig. 3 a–g).

Discussion

Both P. pulcher and M. cottoides showed diploid numbers of 54 chromosomes, corroborating the 2n described for the Pseudopimelodidae species that have been cytogenetically studied to date (Table 1). Although only a few species have already been karyotypically analyzed (7 of the 35 described species in the family), the diploid number observed in the analyzed species indicates conservative chromosome evolution in this group of fish.

Previously, Pseudopimelodidae belonged to the family Pimelodidae, divided into three monophyletic subfamilies: Pimelodinae, Heptapterinae and Pseudopimelodinae according to Shibatta (2003). As they have different morphological characteristics, according to the classification of Reis et al. (2003), most Pimelodidae were divided into two other families: Heptapteridae described by Bockmann and Guazzelli (2003) and Pseudopimelodidae described by Shibatta (2003). Swarça et al. (2007) in a cytogenetic revision showed that cytogenetic data corroborate this separation into three distinct families, separating them according to the diploid number, being most Pimelodidae species with 2n = 56, most Heptapteridae species with 2n = 58 and Pseudopimelodidae with predominance of 2n = 54, as confirmed in the present study (Table 1).

The exemplar of M. cottoides triploidy (3n = 81 + 2), is the first report of such an event in the Pseudopimelodidae family. Natural triploidy has been previously described in several species of neotropical fish (Malacrida et al. 2003; Garcia et al. 2003; Kantek et al. 2007; Tsuda et al. 2010). This event might be related to changes in environmental temperatures, facilitating the retention of the second polar body during the meiotic division and leading to the formation of a diploid gamete, which, after being fertilised, would give rise to a triploid individual (Cuellar and Uyeno 1972). Triploidy is induced in fish farms, usually by heat shock, with the aim of increasing growth in juveniles, extending the survival of individuals and improving growth in adult fish, and it is also useful for controlling overpopulation (Tiwary et al. 2004).

Despite its conserved diploid number, the family Pseudopimelodidae shows great variability, with the occurrence of multiple and single NORs (both in species of different genera and different species within the same genus) as well as variation in their location (Table 1). The differences observed among species of the same genus, such as the single NORs of P. mangurus (Martinez et al. 2004) and the multiple NORs of P. pulcher, confirm that these regions are suitable chromosome markers that may be species-specific, and the significant variability indicates that these regions do not follow a pattern within the family. According to Galetti (1998), a single chromosome pair bearing 45S rDNA is considered a primitive character shared among fish. Despite the small number of species studied, most Pseudopimelodidae present single NORs, which may be considered a plesiomorphic character shared among the groups from which the multiple NORs are derived. This possibility could be confirmed by the analysis of additional species from this family.

The NORs of M. cottoides and P. pulcher were confirmed by FISH using an 18S rDNA probe. These regions were also positive for CMA3 staining and are therefore GC-rich. Fluorochrome staining had previously been performed only on P. mangurus (Martinez et al. 2004) and L. alexandri (Marques et al. 2008), in which the NORs were also coincident, a finding that was confirmed in the latter by FISH with an 18S rDNA probe.

The triploid M. cottoides specimen presented three NOR-bearing chromosomes, similar to previously reported cases of triploidy in fish (Maistro et al. 1994; Tsuda et al. 2010; Silva et al. 2011) confirmed either by impregnation with silver nitrate and CMA3 or by hybridization with an 18S rDNA probe without the inactivation of ribosomal genes.

The triploid M. cottoides female exhibited an interstitial heterochromatic block on two of the three copies of chromosome one, similar to that observed on the first chromosome pair of the diploid female, and both individuals presented DAPI+ heterochromatin. This feature may be a chromosome marker, confirming the female as a bearer of a diploid gamete because there was no evidence of interstitial heterochromatin on the first chromosome pair in any of the males. This finding suggests that the two heterochromatic chromosomes of the triploid female originated from a diploid female gamete and the euchromatic chromosome one from the haploid male gamete.

Supernumerary chromosomes were observed only in triploid individuals of M. cottoides, which were totally heterochromatic. Cases of triploidy associated with B chromosomes have already been reported in Astyanax scabripinnis Jenyns 1842 (Maistro et al. 1994) and Curimata modesta Walbaum 1792 (Venere and Galetti 1985). The fact that these B chromosomes are shown only in the triploid M. cottoides individual, indicate that the occurrence of extra chromosomes constitute a unique condition present in the triploid specimen and that they are a consequence of the same event that led to the formation of the triploid fish, as suggested by Pansonato-Alves et al. (2011) in a triploid Characidium cf. zebra (Crenuchidae) specimen.

The presence of a small amount of heterochromatin in M. cottoides and P. pulcher, where only a few chromosomes showed positive bands, has also been described for Microglanis garavelloi (cited as M. cottoides in Vissotto et al. 1999), P. mangurus (Martinez et al. 2004), C. apurensis, Microglanis aff. cottoides and P. bufonius (Martinez et al. 2008), suggesting that this may be a characteristic of the family. All of the M. cottoides individuals (diploid and triploid) exhibited C-band-positive NORs, which were previously observed by Martinez et al. (2008) in C. apurensis and P. mangurus as well as in Microglanis aff. cottoides (Martinez et al. 2004).

After C-band treatment with CMA3 and DAPI fluorochromes, both GC and AT-rich heterochromatin were observed in P. pulcher; however, a large submetacentric pair, most likely pair 12 of the NOR, revealed pericentromeric GC and AT-rich and terminal, only GC-rich, showing that these regions differ from one another in relation to the heterochromatin base composition in this chromosome, which might suggest a chromosome marker in this species.

This study provides novel information on this fish family, including the first cytogenetic data on P. pulcher and supernumerary chromosomes and natural triploidy in M. cottoides population from the Patos Lagoon Hydrographic System/RS. The study of the heterochromatin allowed to infer about possible formation of triploidy, with fertilization of the diploid female gamete, due to the identification of a cytogenetic marker in this specie. Furthermore, this demonstrates the importance of cytogenetic analysis of other species of this group, suggesting different evolutionary paths in which chromosomal rearrangements may have played an important role in the karyotypic evolution of the family.

Acknowledgments

The authors are grateful to Dra. Lucia Giuliano Caetano and Dr. Alberto Sergio Fenocchio for the review of this manuscript and to MSc Bruno Ambrozio Galindo for the collection of Pseudopimelodus pulcher specimens. This research was supported by a grant from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). The researcher received permission from Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis (IBAMA) to collect fish specimens.

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