G C A T
T A C G
G C A T
genes
Article
An Insight into the Chromosomal Evolution of
Lebiasinidae (Teleostei, Characiformes)
Francisco de M. C. Sassi 1 , Terumi Hatanaka 1 , Renata Luiza R. de Moraes 1 ,
Gustavo A. Toma 1 , Ezequiel A. de Oliveira 2 , Thomas Liehr 3, * , Petr Rab 4 ,
Luiz A. C. Bertollo 1 , Patrik F. Viana 5 , Eliana Feldberg 5 , Mauro Nirchio 6 ,
Manoela Maria F. Marinho 7,8 , José Francisco de S. e Souza 5 and Marcelo de B. Cioffi 1
1
2
3
4
5
6
7
8
*
Laboratório de Citogenética de Peixes, Departamento de Genética e Evolução, Universidade Federal de São,
Carlos, SP 13565-905, Brazil; fmcsassi@estudante.ufscar.br (F.d.M.C.S.); hterumi@yahoo.com.br (T.H.);
rlrdm@hotmail.com (R.L.R.d.M.); gustavo_toma@hotmail.com (G.A.T.); bertollo@ufscar.br (L.A.C.B.);
mbcioffi@ufscar.br (M.d.B.C.)
Secretaria de Estado de Educação do Mato Grosso–SEDUC-MT, Cuiabá, MT 78049-909, Brazil;
ezekbio@gmail.com
Institute of Human Genetics, University Hospital Jena, Jena 07747, Germany
Laboratory of Fish Genetics, Institute of Animal Physiology and Genetics, Czech Academy of Sciences, 27721
Liběchov, Czech Republic; Rab@iapg.cas.cz
Laboratório de Genética Animal, Coordenação de Biodiversidade, Instituto Nacional de Pesquisas da
Amazônia, Manaus, AM 69067-375, Brazil; patrik.biologia@gmail.com (P.F.V.); feldberg@inpa.gov.br (E.F.);
sousa.josef@gmail.com (J.F.d.S.e.S.)
Facultad de Ciencias Agropecuarias, Universidad Técnica de Machala, Machala 070151, Ecuador;
mauro.nirchio@gmail.com
Museu de Zoologia da Universidade de São Paulo (MZUSP), São Paulo, SP 04263-000, Brazil;
manumfm@yahoo.com.br
Departamento de Sistemática e Ecologia, Universidade Federal da Paraíba, João Pessoa, PB 58033-455, Brazil
Correspondence: Thomas.liehr@med.uni-jena.de; Tel.: +49-3641-9396850; Fax: +49-3641-9396852
Received: 5 March 2020; Accepted: 26 March 2020; Published: 28 March 2020
Abstract: Lebiasinidae fishes have been historically neglected by cytogenetical studies. Here we
present a genomic comparison in eleven Lebiasinidae species, in addition to a review of the ribosomal
DNA sequences distribution in this family. With that, we develop ten sets of experiments in
order to hybridize the genomic DNA of representative species from the genus Copeina, Copella,
Nannostomus, and Pyrrhulina in metaphase plates of Lebiasina melanoguttata. Two major pathways on
the chromosomal evolution of these species can be recognized: (i) conservation of 2n = 36 bi-armed
chromosomes in Lebiasininae, as a basal condition, and (ii) high numeric and structural chromosomal
rearrangements in Pyrrhulininae, with a notable tendency towards acrocentrization. The ribosomal
DNA (rDNA) distribution also revealed a marked differentiation during the chromosomal evolution
of Lebiasinidae, since both single and multiple sites, in addition to a wide range of chromosomal
locations can be found. With some few exceptions, the terminal position of 18S rDNA appears as a
common feature in Lebiasinidae-analyzed species. Altogether with Ctenoluciidae, this pattern can
be considered a symplesiomorphism for both families. In addition to the specific repetitive DNA
content that characterizes the genome of each particular species, Lebiasina also keeps inter-specific
repetitive sequences, thus reinforcing its proposed basal condition in Lebiasinidae.
Keywords: comparative genomic hybridization; ribosomal DNA; Neotropical fishes; cytogenetics;
karyotype
Genes 2020, 11, 365; doi:10.3390/genes11040365
www.mdpi.com/journal/genes
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1. Introduction
Advanced molecular approaches have been widely applied in cytogenetic studies of many animal
groups, providing useful insights into their karyotype differentiation and genome evolution. Although
in fishes, such procedures have also improved investigations as a whole, chromosomal analysis of
several taxa is still emerging [1]. Obtaining good metaphases plates, both in quantity and quality,
stands out as the reason for such scenarios, mainly for small-sized and miniature fishes. Thus, dealing
with chromosomes of miniature species, which reach a maximum length of 26 mm in maturity [2], is
challenging, but possible [3–7].
Lebiasinidae is a freshwater characiform family comprising about 75 recognized species [8],
distributed throughout Central and South America except Chile, which experienced body
miniaturization along with their evolution [2]. Two distinguishable subfamilies are recognized:
(i) Lebiasininae, comprising Lebiasina, Piabucina and Derhamia, and (ii) Pyrrhulininae, including Copeina,
Copella, Nannostomus, and Pyrrhulina [8]. However, Netto-Ferreira [9] proposed the inclusion of
Derhamia in Pyrrhulininae, based on morphological characters.
The phylogenetic position of Lebiasinidae within the order Characiformes has been frequently
discussed [10–14], but without a conclusive solution. Recent analyses based on molecular data showed
that Ctenoluciidae emerged as the sister group of Lebiasinidae [15–17]. A further indication of such
close relationship was found using cytogenetic approaches where whole chromosome painting (WCP)
experiments with probes from the first chromosome pair of Lebiasina bimaculata (Lebiasinidae) and
Boulengerella lateristriga (Ctenoluciidae) revealed great similarity between them; a fact also extended to
other Ctenoluciidae species [6]. Additionally, a comparative genomic hybridization (CGH) experiment
showed co-localized scattered signals on L. bimaculata and B. lateristriga chromosomes, indicating that
shared syntenic regions remained conserved during the evolutionary process of these groups [6].
Lebiasina (Lebiasininae) is one of the most unexplored taxa among Lebiasinidae in terms of
cytogenetic data. It is considered a basal group within Lebiasinidae, with morphological [9,18]
and cytogenetic [6] features corroborating such position. This makes Lebiasina an interesting group
for evolutionary studies. For such purposes, CGH is a helpful methodology that has improved
the evolutionary cytogenetics field by comparing entire genomes. Although initially developed to
use in clinical approaches [19], CGH is now successfully used to trace evolutionary trends among
different metazoan groups. In fishes, distinctive evolutionary processes (including the differentiation
of sex chromosomes) have been highlighted among different species and groups using this advanced
technique [5,6,20–22].
This study is part of a series focusing on the chromosomal evolution of the Lebiasinidae. Here,
CGH experiments were used for the cross-species painting of 11 lebiasinid species and to revise the
distribution of ribosomal sequences across their genomes, thus providing additional insight into their
chromosomal evolution.
2. Materials and Methods
2.1. Samples
Eleven Lebiasinidae species from several Brazilian rivers were analyzed (Figure 1; Table 1).
Fieldwork had authorization from Brazilian Environmental Agencies ICMBIO/SISBIO (License number
48628-2) and SISGEN (A96FF09). Individuals were taxonomically identified and deposited at the
Museu de Zoologia da Universidade de São Paulo (MZUSP; Table 1)
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Figure 1. Map of the central portion of South America showing the Brazilian sample sites of Copeina
guttata, Copella nattereri, Lebiasina melanoguttata, Nannostomus eques, N. marginatus, N. trifasciatus,
N. unifasciatus, Pyrrhulina australis, Pyrrhulina aff. australis, P. brevis and P. semifasciata. The map
was produced using the software QGis 3.4.4 (https://qgis.org), Inkscape 0.92 (https://inkscape.org), and
Adobe Photoshop CC 2015 (San Jose, CA, USA).
Table 1. Collection sites and sample sizes (N) of the species examined. All from Brazil.
Species
Locality
Copeina guttata Steindachner,
1876
Copella nattereri Steindachner,
1876
Lebiasina melanoguttata
Netto-Ferreira, 2012
Nannostomus eques
Steindachner,1876
Nannostomus marginatus
Eigenmann, 1909
Nannostomus beckfordi Günther,
1872
Nannostomus trifasciatus
Steindachner, 1876
Nannostomus unifasciatus
Steindachner, 1876
Pyrrhulina australis Eigenmann &
Kennedy, 1903
Tefé, Amazonas
(S03◦ 23′ 07.7′′ , W64◦ 46′ 43.7′′ )
Manaus, Amazonas(S02◦ 35′ 42.9′′ ,
W60◦ 02′ 23.8′′ )
Cachoeira da Serra, Pará
(S08◦ 58′ 18,7′′ , W54◦ 58′ 18,7′′ )
Manaus, Amazonas
(S02◦ 47′ 58.1′′ , W60◦ 29′ 19.8′′ )
Manaus, Amazonas
(S02◦ 55′ 53.9′′ , W59◦ 58′ 30.7′′ )
Manaus, Amazonas
(S02◦ 55′ 53.9′′ , W59◦ 58′ 30.7′′ )
Manaus, Amazonas
(S02◦ 44′ 59.6′′ , W60◦ 01′ 37.9′′ )
Manaus, Amazonas
(S02◦ 47′ 58.1′′ , W60◦ 29′ 19.8′′ )
Santo Afonso, Mato Grosso
(S14◦ 27′ 25.2′′ , W57◦ 34′ 35.2′′ )
Deposit Number
N
11♀; 06♂ ♀
♂MZUSP 124915
04♀; 06♂ ♀
♂MZUSP 124923
22♀; 14♂ ♀
♂MZUSP 124457
02♀; 02♂ ♀
♂MZUSP 123084
03♀; 05♂ ♀
♂MZUSP 123079
09♀; 17♂ ♀
♂MZUSP 123071
07♀; 12♂ ♀
♂MZUSP 123071
05♀; 07♂ ♀
♂MZUSP 123083
30♀; 18♂ ♀
♂MZUSP 119079
♀
♂
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Table 1. Cont.
Species
Pyrrulina aff. australis
Pyrrulina brevis Steindachner,
1876
Pyrrulina semifasciata
Steindachner, 1876
Locality
Barra do Bugres, Mato Grosso
(S15◦ 04′ 27.5′′ , W57◦ 11′ 05.4′′ )
Manaus, Amazonas
(S02◦ 55′ 53.9′′ , W59◦ 58′ 30.7′′ )
Tefé, Amazonas
(S3◦ 39′ 45.8′′ , W64◦ 35′ 33.3′′ )
N
Deposit Number
22♀; 16♂
MZUSP 119077
13♀; 17♂
MZUSP 124916
07♀; 12♂
MZUSP 123073
2.2. Chromosome Preparations and Ideograms
Mitotic chromosomes were prepared by the direct conventional air-drying technique [23] from
kidney cells. All experiments followed the ethical/anesthesia conducts and were approved by the
Ethics Committee on Animal Experimentation of the Universidade Federal de São Carlos (Process
number CEUA 1853260315). Schematic representations, to demonstrate the chromosomal distribution
of the 5S and 18S rDNA sequences in respective representative Ctenoluciidae and Lebiasinidae, were
arranged using the Adobe Photoshop CC 2015 (San Jose, CA, USA), according to the data from [3–7,24].
Four genera were not included in our ideogram since there is no available data for the rDNA position
on chromosomes of Copella, Derhamia, Piabucina (Lebiasinidae), and Ctenolucius (Ctenoluciidae).
2.3. Probes for Comparative Genomic Hybridization (CGH)
Ten sets of experiments were undertaken to hybridize the genomic DNA (gDNA) of Copeina, Copella,
Nannostomus, and Pyrrhulina species under study onto metaphase plates of Lebiasina melanoguttata.
For this purpose, the female-derived gDNA of L. melanoguttata, C. guttata, C. nattereri, P. australis,
Pyrrhulina aff. australis, P. brevis, P. semifasciata, N. eques, N. marginatus, N. trifasciatus, and N. unifasciatus
were extracted from liver tissues by a standard phenol–chloroform–isoamyl alcohol method [25]. For
all assays, the female-derived gDNA of L. melanoguttata was directly labeled with Atto488 (green
fluorescence) using the Nick-translation labeling kit (Jena Bioscience, Jena, Germany), while the gDNA
of C. guttata, C. nattereri, P. australis, Pyrrhulina aff. australis, P. brevis, P. semifasciata, N. eques, N. marginatus,
N. trifasciatus, and N. unifasciatus were directly labeled with Atto550 (red fluorescence) also using
the Nick-translation labeling kit (Jena Bioscience, Jena, Germany). The final hybridization mixtures
contained 500 ng of L. melanoguttata gDNA plus 500 ng of gDNA from one of the above-described
species. In all experiments, repetitive sequences were blocked using 15 µg of C0t-1 female-derived DNA
from each species, prepared according to Zwick et al. [26], and dissolved in 20 µL of the hybridization
buffer (50% formamide, 2x SSC, 10% SDS, 10% dextran sulfate, and Denhardt’s buffer, pH 7.0). The
chosen ratio of probe vs. C0t-1 DNA amount was based on the experiments performed in previous
studies in several fish groups [5,6,20,27].
2.4. Fluorescence in Situ Hybridization (FISH) for CGH
CGH experiments were performed using the protocol of Symonová et al. [27]. Slides were first
aged for 1 to 2 h at 60 ◦ C and then treated with RNase A (20 µg/mL; 90 min at 37 ◦ C in a wet chamber),
and pepsin (50 µg/mL; 3 min at 37 ◦ C). Chromosomes were denatured in 75% formamide diluted in
2x SSC at 74 ◦ C for 3 min. At the same time, the probes were also denatured at 86 ◦ C for 10 min and
chilled on ice for 10 min. Then, the hybridization mix was applied to the slides, followed by a three-day
incubation in a wet chamber (37 ◦ C). The non-specific hybridization remnants were removed by a
stringent washing at 44 ◦ C, two washes in 50% formamide/2x SSC (10 min each), three washes in 1x
SSC (7 min each), and a final wash in 2x SSC at room temperature. Chromosomes were counterstained
with DAPI (1.2 µg/mL) and mounted in an antifade solution (Vector, Burlingame, CA, USA).
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3. Results
3.1. Chromosomal Distribution of the rDNA Sequences Across the Genome of Lebiasinidae and Ctenoluciidae
Species Understudy
Boulengerella (Ctenoluciidae) species (Figure 2a) had 5S rDNA sites located in the terminal and
pericentromeric regions of the first and the 10th chromosome pairs, respectively. The only exception
for this pattern occurred in B. lucius, which had the fourth chromosome pair, instead of the tenth one,
bearing these sites. As to the 18S rDNA, it was found only in the telomeric region of the 18th pair in
the karyotypes of all Boulengerella species [4].
a
c
d
b
e
Figure 2. Schematic representation of chromosomes of Lebiasinidae and Ctenoluciidae species,
highlighting the position of 5S rDNA (green) and 18S rDNA (red). The small box highlights a sex
chromosome system in Pyrrhulina semifasciata, while the bigger box highlights the Ctenoluciidae
members. FISH data were taken from [3–7,24]. Letters correspond to the investigated genera:
(a)—Boulengerella, (b)—Nannostomus, (c)—Lebiasina, (d)—Copeina, and (e)—Pyrrhulina.
Nannostomus species (Figure 2b) possessed 5S rDNA sequences in one chromosome pair only,
although with variable positions, i.e., (1) telomeric region of the short (p) arms of the pairs 03 of N.
eques and 04 of N. marginatus, (2) proximal region of the long (q) arms of the pair 07 of N. unifasciatus,
and (3) telomeric region of the pair 18 of N. beckfordi. However, the 18S rDNA sites were more varied
in distribution, both in number and location among species: (1) one signal in the telomeric region of
the short arms of the 2nd chromosome pair of N. beckfordi, (2) two signals, both in the interstitial region
of the q arms of the 2nd pair of N. unifasciatus, (3) one signal in the telomeric region of the p arms of
the chromosomes 02 and 18 in N. eques, and (4) one telomeric signal in the p arms of the pair 03 of N.
marginatus, with an additional pericentromeric signal in the q arms of pair 19 [24].
Lebiasina species (Figure 2c) also had distinct patterns of rDNA distribution. The Ecuadorian
species L. bimaculata had 5S sites in the interstitial position of the first chromosome pair and 18S sites in
the telomeric region of pair 03. On the other hand, the Brazilian species L. melanoguttata had multiple
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18S sites, with 12 telomeric ones in the chromosome pairs 01, 02, 03, 07, and 09, but also including sites
in both telomeric regions in pair 02. The 5S rDNA sequences were in the proximal region of the q arms
of chromosome 01, with a probable paracentric inversion, together with the 18S rDNA, and also in the
p arms of pair 13 [6].
Copeina guttata (Figure 2d) possessed 5S rDNA signals in the proximal region of the q arms of the
second chromosome pair, and also in the short arms of the 15th one. On the other hand, the 18S rDNA
has a single distribution, being located in the short arms of pair 04 [7].
Pyrrhulina species showed the most diversified rDNA distribution patterns than those of the other
Lebiasinidae, with multiple 5S and 18S rDNA chromosomal sites (Figure 2e). In P. semifasciata, the
p arms of the pairs 07, 08, 09, 15, and 21 possessed 5S rDNA sequences, while the 18S rDNA ones
were located in the chromosomes 01, 03, 06, and 11. Similarly, P. brevis also had five chromosomes
with 5S rDNA sequences in their short arms (pairs 03, 07, 08, 10, and 14). In the 7th pair, an additional
interstitial signal occurs on the long arms, as well as in chromosome 10, but the proximal region. The
5S and 18S rDNA sequences were located in syntenic sites in p arms of chromosome pairs 03 and 14, in
addition to pair 11 with 18S rDNA sites only. In P. australis, 18S rDNA sites were found in the p arms
of the pairs 01, 06, 11, and 19, in both telomeric regions of pair 04, and also in this same region of the
q arms of pair 07. The 5S and 18S sequences were in the p arms of pair 14 in the syntenic position,
together with other 5S sites in the p arms of the chromosomes 03, 07, 08, 09, 10, 15, and 16. Pyrrhulina
aff. australis possessed four chromosomes with 5S rDNA sites (pairs 03, 07, 15, and 16) in their p arms.
18S sequences were also in the 7th pair, but in the telomeric region of the q arms, besides an additional
site in the p arms of pair 06 [3,5].
3.2. Comparative Genomic Hybridization (CGH)
Comparative genomic hybridization (CGH) experiments revealed that a significant level of
genomic divergence occurs between L. melanoguttata and the other lebiasinid species (Figures 3–5).
A high level of species-specific genomic compartmentalization stood out, with distinct patterns of
repetitive DNA sequences both in amount and distribution in the chromosomes. Besides, some
inter-specific segments of repetitive DNAs were also highlighted as shared among species.
Figure 3. Comparative genomic hybridization using the gDNA of Lebiasina melanoguttata, Copeina
guttata, and Copella nattereri against the chromosomal background of Lebiasina melanoguttata. Genomic
probes from L. melanoguttata and Copeina guttata hybridized against L. melanoguttata chromosomes
(a–d). Genomic probes from L. melanoguttata and Copella nattereri hybridized against L. melanoguttata
chromosomes (e–h). The first column (a,e): DAPI images (blue); second column (b,f): hybridization
μ
patterns using gDNA probe from L. melanoguttata;
third column (c,g): hybridization patterns using
gDNA probes from Copeina guttata and Copella nattereri, respectively; fourth column (d,h) merged
images of both genomic probes and DAPI staining depicting the common regions in yellow. Scale
bar = 5 µm.
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Figure 4. Comparative genomic hybridization using the gDNA of Lebiasina melanoguttata and Pyrrhulina
species against a chromosomal background of Lebiasina melanoguttata. Genomic probes from L.
melanoguttata and P. australis hybridized against L. melanoguttata chromosomes (a–d). Genomic probes
from L. melanoguttata and Pyrrhulina aff. australis hybridized against L. melanoguttata chromosomes (e–h).
Genomic probes from L. melanoguttata and P. brevis hybridized against L. melanoguttata chromosomes
(i–l). Genome from L. melanoguttata and P. semifasciata hybridized against L. melanoguttata chromosomes
(m–p). The first column (a,e,I,m): DAPI images (blue); second column (b, f, j, and n): hybridization
patterns using gDNA probe from L. melanoguttata; third column (c,g,k,o): hybridization patterns using
gDNA probes from P. australis, Pyrrhulina aff. australis, P. brevis, and P. semifasciata, respectively; fourth
column (d,h,l,p) merged images of both genomic probes and DAPI staining, depicting the shared
regions in yellow. Scale bar = 5 µm.
μ
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Figure 5. Comparative genomic hybridization among Lebiasina melanoguttata and Nannostomus
species. Genomic probes from L. melanoguttata and N. unifasciatus hybridized against L. melanoguttata
chromosomes (a–d). Genomic probes from L. melanoguttata, and N. trifasciatus hybridized against L.
melanoguttata chromosomes (e–h). Genomic probes from L. melanoguttata, and N. beckfordi hybridized
against L. melanoguttata chromosomes (i–l). Genomic probes from L. melanoguttata and N. eques
hybridized against L. melanoguttata chromosomes (m–p). Genomic probes from L. melanoguttata and
N. marginatus hybridized against L. melanoguttata chromosomes (q–t). The first column (a,e,i,m,q):
DAPI images (blue); second column (b,f,j,n,r): hybridization patterns using gDNA probe from L.
melanoguttata; third column (c,g,k,o,s): hybridization patterns using gDNA probes from N. unifasciatus,
N. trifasciatus, N. beckfordi, N. eques, and N. marginatus, respectively; fourth column (d,h,l,p,t) merged
μ DAPI staining depicting the shared regions in yellow. Scale
images of both genomic probes and
bar = 5 µm.
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4. Discussion
Karyotype and Chromosomal Differentiation in Lebiasinidae
Extensive chromosomal rearrangements, both in 2n and karyotype morphology, which may be
probably linked to speciation processes, took place during the diversification of the Lebiasinidae.
Overall, two major pathways can be recognized in the chromosomal evolution of the family: (i)
conservation of 2n = 36 and karyotype composed of exclusively bi-armed chromosomes in the
Lebiasininae as a basal condition; (ii) high 2n and structural chromosomal rearrangements in the
Pyrrhulininae, with karyotypes prominently dominated by acrocentric chromosomes (Figure 2). These
findings fit with the hypothesis that several derived fish clades predominantly possess mono-armed
chromosomes, while basal ones have karyotypes dominated by bi-armed chromosomes [28].
Teleost fishes display varied modes of chromosomal evolution. It is noteworthy, for example,
that several groups within Characiformes have more conserved karyotypes, maintaining the 2n
very close or even equal to 54 and a relatively similar karyotype structure such as in Anostomidae,
Curimatidae, Prochilodontidae, Hemiodontidae, and Chilodontidae fishes [29]. Such characteristics
could be associated with the so-called karyotypic orthoselection [30], leading to the conservation of
bi-armed chromosomes among related groups. However, rapid and recent speciation events can also
create conserved karyotypes [31], a fact that cannot be ruled out for lebiasinid fishes since the only
phylogenetic analysis of the family does not make references to divergence time [9]. Certainly, although
Lebiasininae species possess a conserved karyotype macrostructure, interspecific genomic divergences
are extensively observed, as here highlighted [6]. However, other fish groups show considerable
divergences of the karyotype structure among its species, for example, the Erythrinidae [32,33], the
Characidae in the Characiformes [29], and the Loricariidae in the Siluriformes [34–36]. Remarkably,
both trends, i.e., (i) conservation of the basal condition 2n = 36 and karyotype composed exclusively
by bi-armed chromosomes in Lebiasininae, and (ii) predominance of acrocentric chromosomes in the
karyotype of Pyrrhulininae species with a high numeric and structural chromosomal variation are
found in Lebiasinidae, thus differentiating the evolutionary pathways of both subfamilies.
The divergent evolutionary pathways between the genomes of Lebiasininae and Pyrrhulininae
species are also demonstrated by our CGH experiments, where repetitive DNA sequences hybridized
in different positions in their genomes, thus showing a high degree of genomic divergence among
them. It is striking that divergent patterns of hybridization have occurred even among closely
related species, such as L. bimaculata and L. melanoguttata [6], and P. semifasciata and P. brevis [5],
revealed by species-specific CGH signals. In Lebiasina, this is a somewhat expected feature since L.
melanoguttata is endemic, remaining isolated from distribution areas of several other lebiasinids by
a distance of minimum 1500 km [37,38]. The presence of two other Lebiasina species (L. marilynae
and L. minuta) in this same isolated area suggests the occurrence of allopatric speciation events [38],
favoring the emergence of different patterns of genomic diversification. However, together with such
general genomic divergence, it is also evident that inter-specific hybridization of repetitive sequences
still occurs in Lebiasina chromosomes, in this way supporting the proposed basal position in the
Lebiasinidae [16,17].
The distribution of ribosomal DNA sites is also a characteristic that experienced an extensive
differentiation during the chromosomal evolution of Lebiasinidae species. Our review demonstrates
that these sequences are distributed from a single site in the karyotype (i.e., Lebiasina bimaculata) to
multiple ones (i.e., Pyrrhulina australis) and in a broad range of chromosomal locations. The evolution
of rDNA sequences follows the concept of concerted evolution, maintaining the functionality and
homogeneity of these genes [39,40]. However, since homologous and non-homologous recombinations
are processes that mediate the concerted evolution, unequal sister chromatid recombination or
retrotransposition may lead to favor a copy number variation of such sequences [41–44]. Indeed, this
copy number variation can generate some non-transcribed rDNA copies that have extreme importance
on genome integrity [45]. In fishes, copy number variation of ribosomal DNAs was extensively
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reported, since their gene regulation processes seem to be more relaxed than in higher vertebrates [42].
In turn, it is meaningful that Ctenoluciidae fishes possess a conserved pattern of rDNA distribution
since, in this family, a single site of 18S rDNA is found in all species [4]. Therefore, as the basal genus
Lebiasina shares this same pattern, this characteristic may have arisen before the split of Lebiasinidae
and Ctenoluciidae.
The terminal position of the 18S rDNA in chromosomes appears common for Nannostomus,
Pyrrhulina, Lebiasina, and Copeina. With the same pattern in the sister family Ctenoluciidae, this
trait can be considered as symplesiomorphic [24]. The terminal position of 45S rDNA is a common
characteristic for several groups, including fish, in contrast to the 5S loci that appear to have a more
frequent interstitial location along the chromosomes [43]. However, this later condition of 5S rDNA
does not apply to Lebiasinidae and even Ctenoluciidae, where both terminal and interstitial positions
are observed, but with a preferential location at the chromosome termini in Nannostomus and Pyrrhulina
chromosomes [3–5,24].
It is also noteworthy that genomes of Nannostomus unifasciatus and Pyrrhulina brevis exhibit
particular arrangements of ribosomal DNAs. To some extent, this is an expected trait for N. unifasciatus,
since this species has the lowest diploid number among lebiasinid fishes, with 2n = 22 and the karyotype
formed by Robertsonian fusions [46]. In turn, peri- and/or paracentric inversions appear to have had
an important role in the karyotype differentiation of P. brevis [5]. In this sense, besides the action
of possible transposable elements, rDNA sequences may have been shifted by such rearrangements
during the karyotype evolution. Furthermore, syntenic 5S and 18S sites in Lebiasina melanoguttata (pair
01), P. australis (pairs 07 and 14), Pyrrhulina aff. australis (pair 07), and P. brevis (pairs 03 and 14) were
detected, and this situation may increase the recombination frequency [43], and, in association with
heterochromatin, may act as recombination hotspots [47–49].
The evolutionary process may be highly influenced by chromosome rearrangements since
they might facilitate the creation or the break of linkage-groups and alter gene expression [50,
51]. Additionally, mechanisms for post-zygotic reproductive isolation may also be generated by
chromosome fusions, for example [52]. It is also noteworthy that the distribution of repetitive DNA
sequences could explain the genome dynamics from a chromosomal point of view, helping to untangle
taxonomic issues [33,53,54], patterns of sex chromosome differentiation [5,21,22] and even recognizing
hybridization events [55,56]. By that, in an evolutionary context, it is relevant that cytogenetical studies
deliver chromosomal data for repetitive DNA distribution and chromosome rearrangements.
5. Conclusions
The studies of Arcila et al. [16], and Betancur-R et al. [17], indicate the proximity of the Lebiasinidae
and Ctenoluciidae families, besides corroborating the monophyly of the two lebiasinid subfamilies,
Lebiasininae and Pyrrhulininae. This means conventional and molecular cytogenetic data, which
have been progressively improved for miniature fishes, actually corroborate and strengthen the
proposed proximity relationship between Lebiasinidae and Ctenoluciidae. Additionally, it is also
notorious as the evolutionary divergence that appears to differentiate both Lebiasinidae subfamilies.
The chromosomal diversity in Pyrrhulininae hugely contrasts with the apparent conservatism of
Lebiasininae. Furthermore, in addition to the specific repetitive DNA content that characterizes
the genome of each particular species, Lebiasina also keeps inter-specific repetitive sequences, thus
reinforcing its proposed basal condition within Lebiasinidae. The results now available provide
significant advances in understanding the chromosomal evolution of Lebiasinidae fishes, a historically
neglected group of the Neotropical Ichthyofauna in resolute cytogenetic investigations.
Author Contributions: Conceptualization, L.A.C.B.; Data curation, M.M.F.M.; Formal analysis, F.d.M.C.S., T.H.,
R.L.R.d.M., G.A.T., E.A.d.O., L.A.C.B., P.F.V., E.F., M.N., M.M.F.M., M.d.B.C., and P.R.; Funding acquisition,
L.A.C.B., M.d.B.C.; Investigation, F.d.M.C.S., T.H., T.L., P.F.V., E.F., M.N., M.M.F.M., J.F.d.S.e.S., and M.d.B.C.;
Methodology, F.d.M.C.S., T.H., R.L.R.d.M., G.A.T., E.A.d.O., T.L., and J.F.d.S.e.S.; Project administration, T.L.,
L.A.C.B. and M.d.B.C.; Resources, T.L.; Supervision, L.A.C.B., M.d.B.C.; Validation, T.L., M.N., and M.M.F.M.;
Visualization, F.d.M.C.S., R.L.R.d.M., G.A.T., E.A.d.O., L.A.C.B., P.F.V., E.F., J.F.d.S.e.S., and P.R.; Writing–original
Genes 2020, 11, 365
11 of 14
draft, F.d.M.C.S. and M.d.B.C.; Writing–review and editing, F.d.M.C.S., L.A.C.B, T.H., R.L.R.d.M., G.A.T., E.A.d.O.,
T.L., P.F.V., E.F., M.N., M.M.F.M., J.F.d.S.e.S., M.d.B.C., and P.R. All authors have read and agreed to the published
version of the manuscript.
Funding: M.d.B.C. was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
(Proc. nos 401962/2016-4 and 302449/2018-3), the Fundação de Amparo à Pesquisa do Estado de São Paulo
(FAPESP) (Proc. No 2018/22033-1), and CAPES/Alexander von Humboldt (Proc. No. 88881.136128/2017-01).
L.A.C.B. was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Proc. Nos
401575/2016-0 and 306896/2014-1), and the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
(Proc. No. 2018/24235-0). M.M.F.M. was supported by the Fundação de Amparo à Pesquisa do Estado de São
Paulo (FAPESP) (Proc. No. 2017/09321-5; 2018/114115). This study was financed in part by the Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior, Brasil (CAPES), Finance Code 001.
Acknowledgments: The authors are grateful for FAPESP, CNPq, CAPES, and Alexander von Humboldt for the
support. We also appreciate the text contributions of Karine Frehner Kavalco and Mara Zélia de Almeida.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
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