Neotropical Ichthyology
Original article
https://doi.org/10.1590/1982-0224-2020-0103
Chromosome analysis in Saccodon
wagneri (Characiformes) and insights
into the karyotype evolution of
Parodontidae
Correspondence:
Anna Rita Rossi
annarita.rossi@uniroma1.it
Submitted September 28, 2020
Accepted December 21, 2020
by Guillermo Ortí
Epub 22 Feb, 2021
Mauro Nirchio1,2, Maria Cecilia Masache1, Fabilene Gomes Paim3,
Marcelo de Bello Cioffi4, Orlando Moreira Filho4, Ramiro Barriga5,
Claudio Oliveira3 and Anna Rita Rossi6
Parodontidae is a relatively small group of Neotropical characiform fishes
consisting of three genera (Apareiodon, Parodon, and Saccodon) with 32 valid
species. A vast cytogenetic literature is available on Apareiodon and Parodon, but
to date, there is no cytogenetic data about Saccodon, a genus that contains only
three species with a trans-Andean distribution. In the present study the karyotype
of S. wagneri was described, based on both conventional (Giemsa staining, AgNOR, C-bands) and molecular (repetitive DNA mapping by fluorescent in situ
hybridization) methods. A diploid chromosome number of 2n = 54 was observed
in both sexes, and the presence of heteromorphic sex chromosomes of the ZZ/
ZW type was detected. The W chromosome has a terminal heterochromatin band
that occupies approximately half of the long arm, being this band approximately
half the size of the Z chromosome. The FISH assay showed a synteny of the
18S-rDNA and 5S-rDNA genes in the chromosome pair 14, and the absence
of interstitial telomeric sites. Our data reinforce the hypothesis of a conservative
karyotype structure in Parodontidae and suggest an ancient origin of the sex
chromosomes in the fishes of this family.
Keywords: Ag-NOR, Cytogenetics, FISH, Heterochromatin, ZW sex
chromosomes.
1 Universidad Técnica de Machala, Av. Panamericana
mauro.nirchio@gmail.com; (MCM) mcme1794@gmail.com.
km
5.5,
Vía
Pasaje,
Machala,
El
Oro,
Ecuador.
(MN)
2 Escuela de Ciencias Aplicadas del Mar, Núcleo de Nueva Esparta, Universidad de Oriente, Apartado 174, Porlamar, Isla de
Margarita, Venezuela.
Online version ISSN 1982-0224
Print version ISSN 1679-6225
Neotrop. Ichthyol.
vol. 19, no. 1, Maringá 2021
3 Departamento de Morfologia, Instituto de Biociências Universidade Estadual Paulista - UNESP, 18618-689 Botucatu, SP, Brazil.
(FGP) fabillene@yahoo.com.br; (CO) claudio.oliveira@unesp.br.
4 Departamento de Genética e Evolução, Universidade Federal de São Carlos, 13565-090 São Carlos, SP, Brazil.
(MBC) mbcioffi@ufscar.br; (OMF) omfilho@ufscar.br.
5 Instituto de Ciencias Biológicas, Escuela Politécnica Nacional, Quito Ecuador. ramiro.barriga@epn.edu.ec.
6 Dipartimento di Biologia e Biotecnologie “C. Darwin”, Sapienza - Università di Roma, Via Alfonso Borelli 50, 00161 Rome, Italy.
annarita.rossi@uniroma1.it (corresponding author).
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Cytogenetic analysis of Saccodon wagneri
Parodontidae é um grupo relativamente pequeno de peixes caraciformes
neotropicais que consiste em três gêneros (Apareiodon, Parodon e Saccodon)
com 32 espécies válidas. Uma vasta literatura citogenética está disponível
sobre Apareiodon e Parodon, mas até o momento não há dados citogenéticos
sobre Saccodon, um gênero que contém apenas três espécies com distribuição
transandina. No presente estudo foi descrito o cariótipo de S. wagneri, baseado
em métodos convencionais (coloração de Giemsa, Ag-NOR, bandas C) e
moleculares (mapeamento de DNA repetitivo por hibridização fluorescente
in situ). Um número cromossômico diplóide de 2n = 54 foi observado, e a
presença de cromossomos sexuais heteromórficos do tipo ZZ/ZW foi revelada.
O cromossomo W possui uma banda terminal heterocromática que ocupa
aproximadamente metade do braço longo, sendo esta banda aproximadamente
a metade do tamanho do cromossomo Z. O ensaio FISH mostrou uma sintenia
dos genes 18S-rDNA e 5S-rDNA no par de cromossomos 14, e a ausência de
sítios teloméricos intersticiais. Nossos dados reforçam a hipótese de uma estrutura
cariotípica conservadora em Parodontidae e sugerem uma origem ancestral dos
cromossomos sexuais nos peixes desta família.
Palavras-chave: Ag-RON, Citogenética, Cromossomos sexuais ZW, FISH,
Heterocromatina.
INTRODUCTION
The Neotropical region has the largest repository of freshwater fish species that
correspond to about 16% of the world’s fish biodiversity (Albert, Reis, 2011; Reis et al.,
2016). This biodiversity has enormous ecological relevance and economic importance, as
many of these species represent a fishery and aquaculture resource (Hilsdorf, Hallerman,
2017). One of the most represented fish groups present in the hydrographic basins of
this geographic region is Characiformes. This order includes exclusively freshwater
fishes distributed in both Africa and America and shows its greatest diversity in the
Neotropical Region (Malabarba, 1998; Nelson et al., 2016). Characiformes comprises
2,081 valid species grouped into 23 families, mostly in Characidae (1,214 species)
(Fricke et al., 2020a). Parodontidae is a relatively small family distributed throughout
South America and part of Panama (Nelson et al., 2016), and includes 32 species (Fricke
et al., 2020a) organized in three genera: Apareiodon Eigenmann, 1916 (N = 15), Parodon
Valenciennes, 1850 (N = 14) and Saccodon Kner, 1863 (N = 3) that differ due to some
subtle morphological characters (Pavanelli, 2003).
Cytogenetic studies in Parodontidae cover about 50% of recognized valid species (Tab.
1), representing only two genera: Apareiodon and Parodon. Although the available data show
that these fishes have a conserved diploid number (2n) of 54 chromosomes, differences in
the number of chromosome arms (FN) and extensive variation in the position of 18S and
5S rDNA sites exist. Besides this, species with proto sex chromosomes are found together
with others characterized by ZZ/ZW and ZZ/ZW1W2 multiple sex chromosome systems
(Tab. 1). Sex chromosomes show different sizes among the Parodontidae species (Moreira-
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Mauro Nirchio, Maria C. Masache, Fabilene G. Paim, Marcelo B. Cioffi, Orlando M. Filho, Ramiro Barriga, Claudio Oliveira and Anna R. Rossi
Filho et al., 1993; Rosa et al., 2006; Vicari et al., 2006; Bellafronte et al., 2009), but in
all the ZW species, the W chromosome is a subtelocentric chromosome almost entirely
heterochromatic, whereas the Z is smaller and usually shows heterochromatic regions only
in the distal segmental portion of its short arms. The use of satellite DNA and transposable
elements as probes showed that the differentiation of the sex chromosomes in the family
is associated with the accumulation of these repeated sequences (Bellafronte et al., 2011;
Schemberger et al., 2011, 2016; Nascimento et al., 2018).
The genus Saccodon is cytogenetically unexplored and contains only three valid species:
S. dariensis (Meek & Hildebrand, 1913) distributed in Colombia and Panama, S. terminalis
(Eigenmann & Henn, 1914) that lives in the Daule River Basin in Ecuador, and S. wagneri
Kner, 1863 that inhabits the coastal basins of Ecuador and northern Peru (Pavanelli,
TABLE 1 | Cytogenetic characteristics in Parodontidae. 1. Moreira Filho et al., 1980; 2. Moreira Filho et al., 1985; 3. Jesus et al., 1999; 4. Jorge,
Moreira-Filho, 2000; 5. Bellafronte et al., 2009; 6. Bellafronte et al., 2011; 7. Schemberger et al., 2011; 8. Leite, Maistro, 2004; 9. Calgaro et al.,
2004; 10. Traldi et al., 2016; 11. Traldi et al., 2019; 12. Bellafronte et al., 2012; 13. Moreira-Filho et al., 1984; 14. Vicari et al., 2006; 15. Santos et al.,
2019; 16. Rosa et al., 2006; 17. Moreira-Filho et al., 1993; 18. Centofante et al., 2002; 19. Bellafronte et al., 2005; 20. Paula et al., 2017.
Genus/Species
Locality/River, State
2n
Karyotype formula
Male
Sex
system
Ribosomal genebearing chromosomes
18S rDNA
(pair)
Female
Reference
5S rDNA
(pair)
Apareiodon
A. affinis (Steindachner,
1879)
Rio Passa Cinco (SP) Brazil
54/55 50 m/sm, 4 st
51 m/sm, 4 st
ZZ/
ZW1W2
26 (st)
A. affinis
Rio Sapucai (MG), Brazil
54/55 50 m/sm, 4 st
51 m/sm, 4 st
ZZ/
ZW1W2
26 (st)
A. affinis
Rio Paraná (MP) Argentina
54/55
40–42 m/sm,
12 st
43/47 m/sm,
8–12 st
Absence
A. argenteus Pavanelli &
Britski, 2003
Rio Araguaia (GO) Brazil
54
54 m/sm
54 m/sm
Absence
A. argenteus
Rio Araguaia (GO) Brazil
54
54 m/sm
54 m/sm
Absence
10, 11
A. cavalcante Pavanelli &
Britski, 2003
Rio Araguaia (GO) Brazil
54 52 m/sm, 2 st
52 m/sm, 2 st
Absence
10, 11
A. davisi Fowler, 1941
Rio Salgado (CE) Brazil
54
54 m/sm
54 m/sm
Absence
4, 9, 14, 17
(m)
4, 9, 14,
17(m)
10, 11
A. hasemani Eigenmann,
1916
Rio São Francisco (MG)
Brazil
54
54 m/sm
54 m/sm
ZZ/ZW
7, 22 ,11
(st)
14 (m)
12
A. ibitiensis Amaral
Campos, 1944
Ribeirão Araras (MG) Brazil
54 50 m/sm, 4 st
50 m/sm, 4 st
ZZ/ZW
14 (m), 26
(st)
9 (m)
5, 6
A. ibitiensis
Rio Passa Cinco (SP) Brazil
54 50 m/sm, 4 st
50 m/sm, 4 st
Absence
26 (st)
A. ibitiensis
Rio Passa Cinco (SP) Brazil
54 50 m/sm, 4 st
50 m/sm, 4 st
ZZ/ZW
6, 14, 15,
26 (st)
A. ibitiensis
Rio Piumi (MG) Brazil
54 50 m/sm, 4 st
50 m/sm, 4 st
ZZ/ZW
7
A. ibitiensis
Rio Verde (PR) Brazil
54 48 m/sm, 6 st
47 m/sm, 7 st
ZZ/ZW
7
A. machrisi Travassos, 1947
Rio Araguaia (GO) Brazil
54 52 m/sm, 2 st
52 m/sm, 2 st
Absence
10
A. piracicabae (Eigenmann,
1907)
Rio Passa-Cinco/Mogi-Guaçu
(SP) Brazil
54 52 m/sm, 2 st
52 m/sm, 2 st
Absence
27 (st)
A. piracicabae
Rio Piumhi (MG) Brazil
54 52 m/sm, 2 st
52 m/sm, 2 st
Absence
27 (st)
Apareiodon sp.
Rio Verde (PR) Brazil
54 48 m/sm, 6 st
47 m/sm, 7 st
ZZ/ZW
25 (st)
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8 (m)
1–7
8
9
2 (m)
18 (m)
10
4
9 (m)
9 (m)
5, 6
2, 3, 6, 13
7
9 (m)
5, 6, 14
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Cytogenetic analysis of Saccodon wagneri
TABLE 1 | (Continued)
Genus/Species
Locality/River, State
2n
Karyotype formula
Male
Sex
system
Ribosomal genebearing chromosomes
18S rDNA
(pair)
Female
Reference
5S rDNA
(pair)
Apareiodon sp.
Rio Aripuanã (MT) Brazil
54 50 m/sm, 4 st
50 m/sm, 4 st
ZZ/ZW
2, 5, 9, 26,
27 (m)
Apareiodon sp. A
Rio Frio (MG) Brazil
54 50 m/sm, 4 st
50 m/sm, 4 st
Absence
26 (st)
4
Apareiodon sp. B
Rio Barreiro (MG) Brazil
54 50 m/sm, 4 st
50 m/sm, 4 st
Absence
26 (st)
45
Apareiodon sp. C
Rio Araguaia (GO) Brazil
54 52 m/sm, 2 st
52 m/sm, 2 st
Absence
6 (m)
15
4
27 (st),
additional
14 (m)
A. vittatus Garavello, 1977
Rio Jordão (PR) Brazil
54 52 m/sm, 2 st
52 m/sm, 2 st
Absence
9 (m)
4, 6
A. vittatus
Rio Jordão (PR) Brazil
54 52 m/sm, 2 st
52 m/sm, 2 st
Absence
A. vladii Pavanelli, 2006
Rio Piquiri (PR) Brazil
54 50 m/sm, 4 st
50 m/sm, 4 st
ZZ/ZW
A. vladii
Rio Piquiri (PR) Brazil
54 50 m/sm, 4 st
50 m/sm, 4 st
ZZ/ZW
P. hilarii Rheinhardt, 1867
Córrego do Porta (MG)
Brazil
54
54 m/sm
53 m/sm, 1 st
ZZ/ZW
16 (m)
11 (m)
6, 7, 17
P. moreirai Ingenito &
Buckup, 2005
Córrego Paiol Grande (SP)
Brazil
54
54 m/sm
54 m/sm
ZZ/ZW
15 (m)
11 (m)
6, 7, 18
P. nasus Kner, 1859
Rio Passa Cinco (SP) Brazil
54 48 m/sm, 6 st
48 m/sm, 6 st
Absence
25 (st)
25 (st)
2, 6, 13, 19
P. nasus
Rio Três Bocas (PR) Brazil
54
54 m/sm
54 m/sm
Absence
2 (m)
P. nasus
Rio Paraguai (MT) Brazil
54 50 m/sm, 4 st
50 m/sm, 4 st
Absence
P. pongoensis (Allen, 1942)
Rio Taquaralzinho (MT)
Brazil
54 50 m/sm, 4 st
50 m/sm, 4 st
Absence
Parodon cf. pongoensis
Rio Araguaia (GO) Brazil
54 50 m/sm, 4 st
50 m/sm, 4 st
Absence
Río Bonito (El Guabo) / El
Oro Ecuador
54
32 m, 16 sm,
6 st
31 m, 16 sm,
7 st
ZZ/ZW
7
26 (st)
9 (m),
additional
3 (m)
6, 16
7
Parodon
20
7
2 (m)
9 (m)
6, 7
10, 11
Saccodon
S. wagneri Kner, 1863
14 (m)
14 (m)
Present
study
2003). This last one was previously known as S. cranocephalum Thominot, 1882 and
Parodon ecuadoriensis Eigenmann & Henn, 1914, now considered synonym (Fricke et al.,
2020b). Saccodon wagneri is adapted to live in rivers that flow rapidly with rocky bottoms
near the mountains and generally above 100 m altitude (Roberts, 1974), often forming
schools when swims in rapid waters (Glodek, 1978). Saccodon wagneri specimens easily
adapt to confinement in aquariums where they eat algae and even balanced food, so that
they could also be considered as aquarium fish, similarly to other Parodontiidae. Indeed,
some species of this family as Apareiodon affinis (Steindachner, 1879), Parodon pongoensis
(Allen, 1942), and P. suborbitalis Valenciennes, 1850, are included in the pet trade (Prang,
2008) and advertised on websites dedicated to the sale of aquarium fish (https://www.
aquariumglaser.de/en/fish-archives/apareiodon_affinis_en/).
In the present study, we performed a cytogenetic survey of S. wagneri based on
both conventional (Giemsa staining, silver staining, C-banding) and molecular
(repetitive DNA mapping methods). The study aims to verify whether morphologically
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Mauro Nirchio, Maria C. Masache, Fabilene G. Paim, Marcelo B. Cioffi, Orlando M. Filho, Ramiro Barriga, Claudio Oliveira and Anna R. Rossi
differentiated sex chromosomes, that are present in some Apareiodon and Parodon
species, can be identified also in the genus Saccodon and whether chromosome number
and main karyotype structure are conserved in this genus. A comparative analysis of
cytogenetic data on this species and the remaining Parodontidae is presented here.
MATERIAL AND METHODS
Eleven individuals (2 males and 9 females) of S. wagneri, from the Río Bonito, El Guabo,
El Oro Province, 03°07’55”S 79°45’00”W, were sampled (Fig. 1). The fishes were
collected with cast nets and placed in plastic bags filled up to a third of their capacity
with water and oxygen the remaining two thirds, transported in cardboard boxes to
the laboratory where they were confined in aquariums provided with constant aeration
until they were processed.
Mitotic chromosomes were obtained from kidney cells suspension following the
conventional air-drying method (Nirchio, Oliveira, 2006). The animals were stimulated
to increase the number of metaphases with an injection of yeast-glucose suspension
(Lozano et al., 1988) in the caudal peduncle 48 h before being processed. Each fish was
injected with 0.0125% colchicine (1.0 ml/100 g of body weight) 50 min before being
sacrificed with an overdose of benzocaine (Leary et al., 2013).
Voucher specimens are preserved and deposited in the Ichthyology Collection
of the Laboratório de Biologia e Genética de Peixes (LBP) of Universidade Estadual
Paulista, Botucatu, São Paulo, Brazil (UNESP) (collection numbers LBP 26871–
26874) and Universidad Técnica de Machala, El Oro, Ecuador (collection numbers
UTMACH–0398–0399).
FIGURE 1 | Map of Ecuador, highlighting the sampling site of Saccodon wagneri.
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Cytogenetic analysis of Saccodon wagneri
The metaphases were stained with 5% Giemsa solution to define the 2n and the
karyotype formula. C-positive heterochromatic regions were identified by the
C-banding procedure, following Sumner (1972), while the nucleolus organizer regions
(NORs) were identified using silver nitrate impregnation (Howell, Black, 1980) after
Giemsa staining.
The 5S rDNA and 18S rDNA (ribosomal genes), and telomeric repeats were mapped
onto chromosomes by fluorescence in situ hybridization (FISH) (Pinkel et al., 1986).
Probes were obtained and labeled by PCR from the genome of Hypsolebias flagellatus
(Costa, 2003) using the primers described by Pendas et al. (1995) for 5S rDNA,
Utsunomia et al. (2016) for 18S rDNA and Ijdo et al. (1991) for telomeric repeats. The
5S rDNA and telomeric probes were labeled with biotin-16-dUTP (2’-deoxyuridine
5’-triphosphate), and the 18S rDNA probes were labeled with digoxigenin-11-dUTP.
Signals were detected with fluorescein-conjugated avidin (Sigma-Aldrich, www.sigmaaldrich.com) and antidigoxigenin–rhodamine conjugate (Roche Diagnostics, www.
roche.com), respectively. Chromosomes were counterstained with 4,6-diamidino-2phenylindole included in the Vectashield mounting medium (Vector Laboratories, Ltd.,
Peterborough, UK).
Images capture of chromosome spread after Giemsa, silver staining (Ag-NORs),
and C-bands (constitutive heterochromatin), was performed under a CX31 Olympus
microscope equipped with a Moticam 10+ digital camera coupled to a Motic Images Plus
2.0 software. FISH metaphases were analyzed under an Olympus BX53 epifluorescence
microscope (Olympus Corporation, Ishikawa, Japan) with the appropriate filters; images
were captured with an Olympus DP73 digital camera coupled to cellSens Dimension
Software (Olympus) for image acquisition. Images were merged and edited to optimize
the brightness and contrast using the Photoshop CS5 program (Adobe Systems, www.
adobe.com). At least 30 metaphase spreads per individual were analyzed to confirm the
diploid number, karyotype structure and FISH results. Chromosomes were classified
as metacentric (m), submetacentric (sm), or subtelocentric (st) according to their arm
ratios (Levan et al., 1964).
RESULTS
The diploid number of S. wagneri is 2n = 54 chromosomes for males and females,
although differences in the FN are present between sexes. Indeed, the karyotype is
composed of 31m + 16sm + 7st chromosomes in females, with FN = 101 (Fig. 2A),
and of 32m + 16sm + 6st, FN = 102 (Fig. 2B) in males. This is due to the presence of
morphologically differentiated sex chromosomes, i.e., to a heteromorphic ZZ/ZW sex
chromosome system. The Z chromosome is submetacentric while the W is metacentric
and almost twice as large as the Z (Fig. 2).
Sequential Giemsa and silver nitrate staining revealed a single pair of Ag-NOR
positive marks located at the tip of the short arms of a small metacentric chromosome
pair, probably pair 14 (Fig. 2, boxes).
C-banding revealed regions of centromeric heterochromatin in most chromosomes,
as well as the presence of interstitial and terminal C-positive bands (Fig. 3). A large
heterochromatic block is present on the half-distal part of the long arms of W chromosome
in the female metaphases (Fig. 3B); a similar band is absent in the Z chromosome.
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Mauro Nirchio, Maria C. Masache, Fabilene G. Paim, Marcelo B. Cioffi, Orlando M. Filho, Ramiro Barriga, Claudio Oliveira and Anna R. Rossi
FIGURE 2 | Saccodon wagneri Giemsa karyotypes. A. Female; B. Male. Sex chromosomes are indicated. The NOR-carrying chromosomes,
after silver staining, are boxed.
FIGURE 3 | Saccodon wagneri C-banded metaphases. A. Female; B. Male. The arrows indicate the sex chromosomes.
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Cytogenetic analysis of Saccodon wagneri
In situ hybridization using the 18S rDNA probe confirmed the presence of a single
cluster of major ribosomal genes, localized on a small metacentric chromosome pair,
likely coinciding with the Ag-NOR signals. Minor ribosomal genes were located on
this same chromosome pair, just below the major rDNA cluster, in a syntenic condition
(Fig. 4A).
FISH with the telomeric repeat probe (TTAGGG)n (Fig. 4B) revealed hybridization
signals only in the telomeric regions of all chromosomes, without the presence of
interstitial telomeric sites (ITSs).
DISCUSSION
Recent characiform phylogenomic studies showed that Parodontidae originated about
70 million years ago (mya) and the first recognized cladogenesis occurred about 40 mya,
separating Saccodon (an exclusive trans-Andean group) from Parodon and Apareiodon
(wide-spread groups in the Neotropical region) (Bruno F. Melo, 2020, pers. comm.).
The cytogenetics data on Parodontidae reveal a conservative 2n = 54 karyotype, that
is composed predominantly of metacentric and submetacentric chromosomes (except
for Apareiodon affinis, where females present 2n = 55 due to the unique ZW1W2
sex system) (Moreira Filho et al., 1980) (Tab. 1). Results here obtained on Saccodon
wagneri reinforce this picture, despite the ancient divergence of this genus within the
family. Moreover, other Neotropical fishes closely related to Parodontidae, e.g., families
Anostomidae, Prochilodontidae, Chilodontidae, and Curimatidae (Betancur et al., 2019;
Bruno F. Melo, 2020, pers. comm.), also share this feature, i.e., almost all species with 54
FIGURE 4 | Saccodon wagneri metaphase plates after A. Double FISH with 5S rDNA (green-thin arrows) and 18S rDNA (red-thick arrows)
probes; B. FISH using telomeric probes showing positive signals in the terminal positions of all chromosomes.
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chromosomes, mainly metacentrics and submetacentrics, with a few exceptions (Arai,
2011). These data indicate an ancient origin of such a karyotype, whose conservatism
has been related to the population structures of these fishes, as they include many long
migratory species able to form large schools (Oliveira et al., 1988).
A morphologically well-differentiated ZZ/ZW sex chromosome system is present in
approximately half of all Parodon and Apareiodon species analyzed so far (Moreira-Filho
et al., 1993; Rosa et al., 2006; Vicari et al., 2006; Bellafronte et al., 2009; Kitano, Peichel,
2012). The occurrence of such sex system, characterized by an enlarged metacentric W
chromosome, in S. wagneri points to its old origin inside Parodontidae. Besides this, and
as frequently observed in higher vertebrates (Schartl et al., 2016), rather than showing
a size reduction, the sex-specific W chromosome in Parodontidae is larger than the
Z, because of a huge heterochromatin amplification. Despite these common features,
the W chromosomes have evolved to different shapes and sequence contents among
Parodontidae species. Two main questions remain unanswered, i.e., whether (i) the Z
and W chromosomes have a common origin, representing the same linkage group in all
species and (ii) the absence of sex chromosomes in some species may represent a derived
character, probably related to sex chromosomes turnovers, as already documented in
other fishes (Kitano, Peichel, 2012). Our data reinforce the hypothesis that this common
ZW system has an ancient origin and it seems possible that the putative absence in
some Parodontidae species would be related to subsequent specific chromosome
differentiation. Further studies will make it possible to confirm the validity of this
hypothesis.
In all the Parodontidae species studied so far, the presence of a single pair of NOR
bearing chromosomes is the common condition, with few exceptions (Bellafronte et
al., 2011). However, different locations of these genes have been observed among the
species, probably as the result of chromosomal rearrangements (pericentric inversion),
occurred along with the diversification of their karyotypes. The presence of multiple
sites reported in Apareiodon davisi (Traldi et al., 2016), and A. ibitiensis (Bellafronte et al.,
2009; Bellafronte et al., 2011) represents an exception, that has been attributed to the
presence of transposable elements (Bellafronte et al., 2011). The syntenic arrangement
of the 18S and the 5S rDNA genes detected in S. wagneri, has only been reported in two
other Parodontidae species, named A. davisi (Traldi et al., 2016) and P. nasus (Bellafronte
et al., 2005), and does not represent a common condition in fishes (Sochorová et al.,
2018). Indeed, the presence of these genes on different chromosomes/sites in fishes
and in the majority of vertebrates has been interpreted in the light of their functional
dynamics (Martins, Galetti, 1999) and efficiency in evolution processes associated with
multiple tandem arrays (Martins, Wasko, 2004).
FISH with the telomeric probe (TTAGGG)n in S. wagneri revealed hybridization
signals only in the telomeric regions of all chromosomes in females and males, without
Interstitials Telomeric Sequences (ITSs) that might result from the occurrence of recent
Robertsonian fusions or other chromosomal rearrangements (Ocalewicz, 2013). This
evidence, the common localization of constitutive heterochromatin (Moreira-Filho et
al., 1984; Jesus, 2000; Jesus, Moreira-Filho, 2000; Vicente et al., 2001, 2003; Centofante
et al., 2002; Bellafronte et al., 2005; Rosa et al., 2006; Vicari et al., 2006), and the constancy
of 2n suggest that diversification in Parodontidae karyotypes has not involved macrostructural reorganizations but rather microstructural ones.
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Cytogenetic analysis of Saccodon wagneri
In conclusion, our study, the first one to report cytogenetic data on a Saccodon species
both by conventional and molecular protocols, reinforces the hypothesis of karyotype
homeostasis in fishes of the family Parodontidae, by conserving the basic diploid number
and chromosome formulae. The synteny of both 18S and 5S rDNA found in S. wagneri
represents an uncommon trait, and its presence in species of the other two genera
(A. davisi and P. nasus), suggests its ancient origin, i.e., that this is a symplesiomorphic
character within the family. As an alternative hypothesis, this similarity could be due
to a homoplasic condition, obtained by parallelism. Further studies with chromosomal
painting, sequence analysis of microdissected sex chromosomes, and comparative
mapping of transposable elements will be useful to obtain a more complete picture of
the evolution of karyotype and sex chromosomes within Parodontidae.
ACKNOWLEDGMENTS
Mauro Nirchio received financial support from Centro de Investigación of Universidad
Técnica de Machala, Ecuador (GPR–GEN–155); CO received financial support from
Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP grants 2018/20610–1,
2016/09204–6, 2014/26508–3 and Conselho Nacional de Desenvolvimento Científico
e Tecnológico – CNPq proc. 306054/2006-0; ARR received financial support from
Università Sapienza (RP11816430E2E16A).
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AUTHOR’S CONTRIBUTION
Mauro Nirchio: Conceptualization, Formal analysis, Funding acquisition, Project administration,
Supervision, Validation, Writing-original draft, Writing-review and editing.
Maria Cecilia Masache: Visualization.
Fabilene Gomes Paim: Data curation, Investigation.
Marcelo de Bello Cioffi: Data curation, Visualization, Writing-original draft.
Orlando Moreira Filho: Methodology, Validation, Writing-original draft.
Ramiro Barriga: Methodology, Resources, Visualization.
Claudio Oliveira: Data curation, Funding acquisition, Validation, Writing-original draft.
Anna Rita Rossi: Conceptualization, Formal analysis, Funding acquisition, Validation, Writing-original
draft, Writing-review and editing.
ETHICAL STATEMENT
Procedures were performed in compliance with the Ethics Committee on Animal Experimentation (process
No 01/2020) of the Universidad Técnica de Machala.
COMPETING INTERESTS
This is an open access article under the terms of the
Creative Commons Attribution License, which permits
use, distribution and reproduction in any medium,
provided the original work is properly cited.
Distributed under
Creative Commons CC-BY 4.0
© 2021 The Authors.
Diversity and Distributions Published by SBI
The authors declare no competing interests.
HOW TO CITE THIS ARTICLE
•
Nirchio M, Masache MC, Paim FG, Cioffi MB, Moreira Filho O, Barriga R, Oliveira C,
Rossi AR. Chromosome analysis in Saccodon wagneri (Characiformes) and insights into the
karyotype evolution of Parodontidae. Neotrop Ichthyol. 2021; 19(1):e200103. https://doi.
org/10.1590/1982-0224-2020-0103
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