CompCytogen 8(3): 21 1-222 (2014) COMPARATIVE = Areerrerewet open-access over

doi: 10.3897/CompCytogen.v8i3.7084 Kan Cyto genetics

www.pensoft.net/journals/compcytogen International Journal of Plant & Animal Cytogenetics,
I

Karyosystematics, and Molecular Systematics

Basic cytogenetics and physical mapping of 5S and |8S
ribosomal genes in Hoplias malabaricus (Osteichthyes,
Characiformes, Erythrinidae) from isolated
natural lagoons: a conserved karyomorph
along the Iguacu river basin

Gisele Gemi', Roberto Laridondo Lui', Fernando Rodrigo Treco?,
Leonardo Marcel Paiz?, Rafaela Maria Moresco', Vladimir Pavan Margarido'

I Centro de Ciéncias Biolégicas e da Saude, Universidade Estadual do Oeste do Parand, Rua Universitaria
2069, CEP: 85819-110, Cascavel, PR, Brazil 2 Universidade Paranaense, Avenida Julio Assis Cavalheiro
2000, CEP: 85601-000, Francisco Beltréo, PR, Brazil 3 Universidade Estadual de Maringd, Departamento
de Biologia, CEP: 87020-900 Maringd, PR, Brazil

Corresponding author: Vladimir Pavan Margarido (Vladimir. Margarido@unioeste.br)

Academic editor: G. Furgala-Selezniow | Received 20 January 2014 | Accepted 21 July 2014 | Published 12 August 2014
http://zoobank.org/57E7CDFB-09 10-4059-BC7F-B3DCB51170C0

Citation: Gemi G, Lui RL, Treco FR, Paiz LM, Moresco RM, Margarido VP (2014) Basic cytogenetics and physical
mapping of 5S and 18S ribosomal genes in Hoplias malabaricus (Osteichthyes, Characiformes, Erythrinidae) from isolated
natural lagoons: a conserved karyomorph along the Iguacu river basin. Comparative Cytogenetics 8(3): 211-222. doi:
10.3897/CompCytogen.v8i3.7084

Abstract

Erythrinidae include Neotropical teleost fish that are widely distributed in South America. Hoplias Gill,
1903 include two large groups: H. malabaricus Bloch, 1794 and H. lacerdae Miranda Ribeiro, 1908.
Hoplias malabaricus is characterized by remarkable karyotype diversity, with some karyomorphs widely
distributed geographically while others are more restricted to certain river basins. Cytogenetic analyzes
were performed in a population of Hoplias malabaricus from the Wildlife Refuge of Campos de Palmas,
the Iguacu River basin. The specimens showed diploid number of 42 chromosomes (24m+18sm) without
differentiated sex chromosomes system. The impregnation by silver nitrate showed multiple AgNORs.
Seven pairs (4, 7, 10, 13, 16, 20 and 21) carrying 18S rDNA were detected by FISH. Heterochromatin
was verified in the centromeric and pericentromeric region of most chromosomes and the terminal region
of some pairs. FISH with 5S rDNA probes showed two chromosome pairs carrying these sites in the
interstitial region (8 and 14). The data obtained in this study are similar to those found for two other

Copyright Gisele Gemi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

2:12; Gisele Gemi et al. / Comparative Cytogenetics 8(3): 211-222 (2014)

populations of H. malabaricus already studied in the basin of the Iguacu River, confirming the hypothesis
that this species is natural, not having been introduced, as well as having an intrinsic characteristic, such
as the largest number of sites of 18S rDNA.

Keywords
Chromosomal conservadorism, double-FISH, evolution, karyotype, rDNA

Introduction

The basin of the Iguacu River, located in the southern region of the State of Parana, is
comprises a drainage area of 69,373 square km and a length of 1,275 km in its main
riverbed. Its springs emerge from Serra do Mar and flow towards the First Plateau, or
Plateau of Curitiba, and to the Second and Third Plateau. In the latter, the Iguacu river
basin is bordered by the Plateau of Palmas at the border of the State of Santa Catarina
(Silva et al. 2001), where 79% belongs to the State of Parana, 19% to the State of Santa
Catarina and 2% to Argentina (Agostinho et al. 1997). The Iguacu river basin has a
low diversity of species and a high degree of endemism, with a total of 106 species, be-
ing 35 of Characiform, 46 of Siluriform and 11 Perciform (Baumgartner et al. 2012).
This endemism has as its main cause the appearance of the Iguacu Falls at the last part
of the flow (Agostinho et al. 2004). In addition to this large geographical barrier of 72
meters, other barriers that segment the Iguacu river were observed along its flow: Salto
Caiacanga (9 meters), Salto Grande (13 meters), Salto Santiago (40 meters) and Salto
Osorio (30 meters) (Maack 1981).

Erythrinidae are characterized by a sedentary lifestyle which consequently reduces
gene flow between the populations that inhabit the same basin since they do not over-
come obstacles, such as waterfalls (Blanco et al. 2010). This family is composed of
three genera: Erythrinus Scopoli, 1777, Hoplerythrinus Gill, 1896 and Hoplias Gill,
1903 (Gayet et al. 2003). Erythrinus is comprised of two species, E. erythrinus Bloch &
Schneider, 1801 and E. kesslerie Steindachner, 1877 (Oyakawa 2003). Hoplerythrinus
includes three species: H. cinereus Gill, 1858, H. gronovii Valenciennes, 1847 and H.
unitaeniatus Spix & Agassiz (Froese and Pauly 2014). Hoplias is the most widespread
in South America, composed of two large groups: Hoplias lacerdae Miranda Ribei-
ro, 1908 and Hoplias malabaricus Bloch, 1794, the first group containing six species
(Oyakawa and Mattox 2009), and the second is a classic case of cryptic species related
to chromosomal aspects (Bertollo et al. 2000).

According to Bertollo et al. (2000), Hoplias malabaricus is a Neotropical freshwater
species widely distributed and with great karyotype diversity (different karyomorph).
The chromosomal studies show diversity in diploid number from 39 to 42 chromo-
somes, differences in chromosomal formulas and presence (karyomorph B, D and G)
or absence (karyomorph A, C, E and F) of a sex chromosome system. According to this
author, H. malabaricus includes seven karyomorph, being some of them more widely
distributed, such as karyomorph A, C and F, and other restricted to only one or a few
sites, such as karyomorph B, D, E and F. Populations of this species from the basin

Basic cytogenetics and physical mapping of 5S and 18S ribosomal genes... 213

of the Iguacu river were previously analyzed by cytogenetic methods and two of these
karyomorph were detected (A and B) (Lemos et al. 2002, Vicari et al. 2003, Vicari et
al. 2006). The karyomorph A appears to be more widely distributed throughout this
basin, while the karyomorph B is restricted to only one population in a region next to
its riverbed side (Lemos et al. 2002).

With regards to the occurrence of H. malabaricus in the basin of the Iguacu river,
there is a controversy as to its origin in this location. According to Garavello et al.
(1997), H. malabaricus would not be a native species of the Iguacu river, which may
have been introduced from nearby basins. Subsequently, a study with chromosomal
populations of this species suggested that the karyomorph A is a native form of the
Iguacu River. In addition, the little karyotype diversity detected among the popula-
tions that were analyzed must be due to vicariant events (Vicari et al. 2006).

In this sense, the objective of this work was to study - through cytogenetic tech-
niques - a population of Hoplias malabaricus collected in a natural lagoon in the region of
Palmas, in the far south of the State of Parana — Brazil. This lagoon has no contact with
other aquatic environments and is isolated from other river systems, in order to better
understand the geographical distribution of the group in the basin of the Iguacu river.

Methods

Four specimens were collected (2 males and 2 females) of Hoplias malabaricus from iso-
lated lagoons in the region of Palmas of the Wildlife Refuge of Campos de Palmas, in
the Iguacu river basin, belonging to the State of Parana — Brazil (Fig. 1). This reduced
sample is duet to the collections being made on a conservation unit, and a major sam-
pling would be justified if intra- or interpopulational chromosomal polymorphisms
were observed. The samples were anesthetized and sacrificed by an overdose of clove oil
(Griffiths 2000) for the removal of the material for the cytogenetic study. The mitotic
chromosomes were obtained from a cell suspension using the anterior portion of the
kidney in accordance with the technique adapted by Bertollo et al. (1978) and Foresti
et al. (1993). Thirty metaphases spreads from each fish were analyzed and ten of the
best mitotic metaphases were used to measure karyotypes. For the AgNORs analysis,
the impregnation by silver nitrate has been used based on the methodology of Howell
and Black (1980), and to determine the distribution pattern of heterochromatin, C-
banding with barium hydroxide was used, following the proposal of Sumner (1972)
with modifications proposed by Lui et al. (2012). For the analysis of fluorescent in
situ hybridization (FISH) 5S rDNA probes of Leporinus elongatus Valenciennes, 1850
(Martins et al. 2000) and 18S rDNA of Prochilodus argenteus Spix & Agassiz, 1829
were used (Hatanaka and Galetti 2004). Each one of them was marked, respectively,
with digoxigenin-11-dUTP and biotin-16-dUTP (Roche). The detection and amplifi-
cation of the hybridization signal was performed using antidigoxigenin-rhodamine for
5S rDNA (Roche) and avidin-FITC and anti-avidin-biotin for 18S rDNA (Sigma).
FISH was performed according to Pinkel et al. (1986) and modifications suggested by

De Gisele Gemi et al. / Comparative Cytogenetics 8(3): 211-222 (2014)

Figure |. Map of sampling sites of Hoplias malabaricus populations in Iguacu river basin: a Piraquara

b Sao José dos Pinhais ¢ Palmeira d Poco Preto e Nova Prata do Iguacu and f Palmas (present paper).

Margarido and Moreira-Filho (2008). The best metaphases were captured in an Olym-
pus BX60 photomicroscope with a digital camera DP71 and DPcontroller 3.2.1.276
software (Olympus). The FISH slides were analyzed with an epifluorescence photomi-
croscope under an appropriate filter. The chromosomes were arranged in groups clas-
sified in metacentric, submetacentric, subtelocentric and acrocentric, according to the
calculation of arm ratio as proposed by Levan et al. (1964).

Results

The cytogenetic analysis observed diploid number of 42 chromosomes with 24 meta-
centric chromosomes and 18 submetacentric chromosomes, for male and female,
and without a sex chromosome system (Fig. 2). The impregnation by silver nitrate
showed multiple AgNORs, ranging from 4 to 6 NORs. The analyzed metaphases
with silver nitrate impregnation presented bi-telomeric labels in the metacentric pair
7 and telomeric labels on the short arm of the metacentric pair 10 (Fig. 2, in box),
coinciding with 18S rDNA, evidenced in FISH (Fig. 3). Five other pairs carrying
rDNA 18S were marked by FISH, the metacentric 4 in both telomeric regions, the
submetacentric pair 13 in the telomeric region of the short arm, pair 16 in the inter-
stitial region of the long arm, pair 20 in both telomeric regions, and pair 21 in the
terminal region of the long arm (Fig. 3). The C-banding revealed heterochromatin
in the centromeric and pericentromeric region in most chromosomes of the comple-
ment, as well as bitelomeric and terminal heterochromatin in some chromosomes,
these being coincident with the AgNORs (pairs 7 and 10) (Fig. 2). The FISH with
5S rDNA probe revealed two pairs of chromosomes, being interstitial on the long
arm of the metacentric 8 and on the short arm close to the centromere of the sub-
metacentric 14 (Fig. 3).

Basic cytogenetics and physical mapping of 5S and 18S ribosomal genes... 215

a
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Figure 2. Karyotypes of Hoplias malabaricus stained with Giemsa (a) and treated through the C-banding (b).
The AgNORs bearing chromosome pairs (7 and 10) are presented in box. Bar = 10 um.

216 Gisele Gemi et al. / Comparative Cytogenetics 8(3): 211-222 (2014)

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Discussion

Hoplias malabaricus comprises a complex of species due to its wide karyotype diversity,
and some karyomorphs are geographically widely distributed, while others have lower
distribution and are restricted to certain basins, and even sympatric karyomorphs may
occur without the detection of hybrids (Bertollo et al. 2000, Born and Bertollo 2006).
The specimens analyzed showed chromosomal characteristics related to a diploid num-
ber, absence of a system of sexual chromosomes and karyotype formula that fits them in
the karyomorph A of the 4. malabaricus group as designated by Bertollo et al. (2000).
Previous studies in populations of H. malabaricus from the Iguacu river showed that
this karyomorph is the most widely distributed in the basin (Fig. 1, Table 1). Accord-
ing to Blanco et al. (2010), who reviewed the chromosomal studies related to karyo-
morph A, a great part of South American river basins contain them, and multiple levels
of chromosomal differentiation can be observed among allopatric populations (i.e.,
karyotype formula, heterochromatic distribution, AgZNORs/18S rDNA, 5S rDNA and
5SHind III satellite DNA).

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218 Gisele Gemi et al. / Comparative Cytogenetics 8(3): 211-222 (2014)

For the populations of karyomorph A of Hoplias malabaricus from the Iguacu riv-
er, the karyotype formula does not show any clear marker to differentiate populations
throughout this basin (Vicari et al. 2006) or to distinguish them from populations in
neighboring basins (Ribera, Tibagi and Ivai) of the Iguacu river (Vicari et al. 2005),
which is different from what is observed for allopatric populations of several other re-
gions (Blanco et al. 2010). Despite the paper of Lemos et al. (2002), bringing a slightly
distinct formula (20m + 22sm), and the one from Bertollo et al. (2000) of not separating
meta- and submetacentric chromosomes, our observation of these karyotypes suggests
that they could be rearranged by 24 metacentrics and 18 submetacentrics, as detected
by Vicari et al. (2003, 2005, 2006) and for the population of this study. It is worth
noting that this conservation of the karyotype formula is not a common situation for
populations distributed along this basin, as was already observed for Astyanax altiparanae
Garutti & Britski, 2000, Oligosarcus longirostris Menezes & Géry, 1983, Corydoras palea-
tus Jenyns, 1842, Pimelodus ortmanni Haseman, 1911 and Glanidium ribeiroi Haseman,
1911 (Kantek et al. 2007). Furthermore, in relation to karyomorph A of 1. malabaricus,
this level of conservation was not observed with other chromosomal markers.

The distribution of heterochromatin in all the karyomorphs of the H. malabaricus
complex has often been described in the terminal and pericentromeric region of some
pairs of chromosomes (Dergam and Bertollo 1990, Haff et al. 1993, Bertollo et al.
1997a, 1997b, Born and Bertollo 2000, Vicari et al. 2005, Blanco et al. 2010), and
was also observed in the population of the present study. However, a small variation
in the amount and location of heterochromatin can be observed between the various
allopatric populations already studied (i.e., Blanco et al. 2010). When the distribution
of heterochromatin of the population in this study is compared to others of the Iguacu
river (Lemos et al. 2002, Vicari et al. 2006), or even with those that are present in the
basins next to the Iguacu (Vicari et al. 2005), a great similarity can be observed.

The analyses carried out by Vicari et al. (2006) in populations belonging to the
basin of the Iguacu river (Nova Prata do Iguacu and Palmeira), demonstrated a vari-
able number of AgNORs, usually located in the telomeric region, and bitelomeric
AgNORs were also found in both populations, as for the population analyzed in this
study. However, interstitial nucleolus organizing regions were observed on the long
arm of chromosome pair 16 of the Palmeira population, this characteristic being un-
common for H. malabaricus. Note that bitelomeric AgNORs have usually been found
in H. malabaricus (Bertollo 1996, Born and Bertollo 2001), being this characteristic
considered a probable synapomorphy for the group (Vicari et al. 2006). Up to now,
there is no evidence of populations belonging to karyomorph A of H. malabaricus that
do not have bitelomeric AgNORs (Blanco et al. 2010).

Only a population with hybridization data with 18S rDNA is described in litera-
ture regarding the Iguacu river, with four pairs being detected, one pair with bitelom-
eric marking, one interstitial pair and two pairs with terminal marking. All these pairs
of the previous study (Vicari et al. 2003, 2005) have chromosomes corresponding to
the population of this study. In addition, other pairs showed sites carrying 18S rDNA
(pair 4 and 20, bitelomeric; pair 13, terminal on short arm). More than one chromo-

Basic cytogenetics and physical mapping of 5S and 18S ribosomal genes... 219

some pair with 18S rDNA has already been detected for the karyomorph A of H.
malabaricus (Blanco et al. 2010). However, this is the first report of this last three pairs.

This study showed two pairs of 5S rDNA sites carrying chromosomes. Previous studies
showed that this marker varies in number of sites among populations of karyomorph A,
with a small metacentric pair with interstitial marking that seems to be conserved (Ferreira
et al. 2007, Blanco et al. 2010), and a second pair (large submetacentric) that can be de-
tected with interstitial marking on the short arm in a population of the Sao Francisco river
(Blanco et al. 2010). The two pairs detected in the population of the Iguacu River in this
paper seem to be corresponding to these pairs mentioned above. Ferreira et al. (2007) com-
pared the location of 5S rDNA sites in three karyomorphs of the H. malabaricus group (A,
D and F) and observed obvious differences between them, indicating that the number and
distribution of sites are good markers of the Erythrinidae family, which shows the need for
data for this marker in other populations of this basin, since the 5S rDNA data presented
in this paper are the first related to the Iguacu river basin.

With the uplift of the Iguacu Falls, an effective geographic isolation was created for
the ichthyofauna of the First and Second Plateaus in the largest part of the Iguacu river
(Maack 1981), resulting in a pronounced endemism of its ichthyofauna (Garavello et
al. 1997). This endemism is proposed for several groups of fish, therefore the occurrence
of H. malabaricus in the basin could be due to human introduction (Sampaio 1988,
Dergam et al. 1998). However, other explanations are considered as well relating to its
presence in the basin. The karyomorph A of 1. malabaricus features a wide distribution
throughout the southeast and south of Brazil, being present in several rivers in the State
of Parana, reaching Uruguay and Argentina (Bertollo et al. 2000). Due to the old shaping
of the Iguacu river basin and the broad distribution that has been detected for this karyo-
morph A, Vicari et al. (2006) proposed that this species may not have been introduced
as was previously believed. The analysis of this population present in the city of Palmas
reinforces this hypothesis, not only due to being another population of this karyomorph
in the basin, but mainly because this population comes from a natural lagoon located in
an isolated region that is part of the hydrographic system of the Iguacu river.

Therefore, the population analyzed in this study showed the same diploid number,
karyotype formula, lack of a differentiated sex chromosomes system when compared to
other populations of the Iguacu river, in addition to sharing some characteristics with
respect to the number and location of AgNORs, distribution of heterochromatin and
18S rDNA sites. These data confirm the hypothesis that H. malabaricus is natural to the
Iguacu River, and in spite of presenting some intrinsic characteristics of this population, it
represents the same evolutionary unit along the basin, which is in the process of allopatric
differentiation through the setting of small rearrangements in the microstructure.

Acknowledgements

The authors are grateful to Dr. Weferson Junio da Graga for the identification of the
specimens and to Instituto Chico Mendes de Conservagao da Biodiversidade (ICMBio)

220 Gisele Gemi et al. / Comparative Cytogenetics 8(3): 211-222 (2014)

for authorizing the capture of the fishes (License number: SISBIO 10522-1). ‘This stu-
dy was financed by Conselho Nacional de Desenvolvimento Cientifico e Tecnoldgico
(CNPq), by Fundagao Araucaria (Fundacgéo Araucaria de Apoio ao Desenvolvimento

Cientifico e Tecnolégico do Estado do Parana) and by Coordenag¢ao de Aperfeicoamento
de Pessoal de Nivel Superior (CAPES).

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