CompCytogen 6(4): 359-369 (2012) COMPARATIVE A veerrerewet open-access over

doi: 10.3897/CompCytogen.v6i4.3062 Kas Cyto genetics

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

Karyosystematics, and Molecular Systematics

Discriminatory profile of rDNA sites and trend for
acrocentric chromosome formation in the genus
Trachinotus Lacépeéde, | 801 (Perciformes, Carangidae)

Uedson Pereira Jacobina', Marcelo Ricardo Vicari’,
Luiz Antonio Carlos Bertollo?, Wagner Franco Molina!

| Department of Cell Biology and Genetics, Centro de Biociéncias, Universidade Federal do Rio Grande do
Norte, Campus Universitario, 59078 — 970, Natal, RN, Brazi 2 Department of Structural, Molecular Biology
and Genetics, Universidade Estadual de Ponta Grossa, Ponta Grossa, PR, Brazil 3 Department of Genetics and
Evolution, Universidade Federal de Sto Carlos, Via Washington Luiz, Km 235, 13565 — 905, Sao Carlos, Sto
Paulo, Brazil

Corresponding author: Wagner Franco Molina (molinawf@yahoo.com.br)

Academic editor: V. Gokhman | Received 9 March 2012 | Accepted 29 May 2012 | Published 31 October 2012

Citation: Jacobina UP, Vicari MR, Bertollo LAC, Molina WF (2012) Discriminatory profile of rDNA sites and trend
for acrocentric chromosome formation in the genus Trachinotus Lacépéde, 1801 (Perciformes, Carangidae). Comparative

Cytogenetics 6(4): 359-369. doi: 10.3897/CompCytogen.v6i4.3062

Abstract

Chromosomal traits have provided valuable information for phylogeny and taxonomy of several fish
groups. Three Atlantic Carangidae species of the genus Trachinotus Lacépéde, 1801 (T° goodei Jordan et
Evermann, 1896, 7’ carolinus (Linnaeus, 1766) and 7. falcatus (Linnaeus, 1758)) were investigated, hav-
ing 2n=48 chromosomes but different chromosomal arms (FN number), i.e., 52, 56 and 58, respectively,
in view of the different number of two-armed chromosomes found in their karyotypes. Thus, 7’ goodei,
T. carolinus and T. falcatus present a progressive distance from the probable basal karyotype proposed for
Perciformes (2n=48 acrocentrics, FN=48). At first sight, these findings do not agree with the phylogenetic
hypothesis based on mitochondrial sequences, where 7’ goodei appear as the most derived species, followed
by 7. falcatus and T: carolinus, respectively. However, the chromosomal mapping of ribosomal DNAs
was informative for clarifying this apparent conflict. Indeed, the multiple 5S and 18S rDNA sites found
in 7) goodei corroborate the most derived condition for this species. In this sense, the occurrence of the
unexpected number of two-armed chromosomes and FN value for this species, as well as for 7’ carolinus,
must be due to additional rounds of acrocentric formation in these species, modifying the macrostructure

of their karyotypes.

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

360 Uedson Pereira Jacobina et al. / Comparative Cytogenetics 6(4): 359-369 (2012)

Keywords
Carangidae, 18S rDNA, 5S rDNA, cytotaxonomic markers, evolutionary pathways

Introduction

The genus Trachinotus Lacépéde, 1801, also known as pompanos, encompasses 20
species distributed in tropical and subtropical oceans (Cunha 1981). In the Eastern
Atlantic, the species Trachinotus carolinus (Linnaeus, 1766), popular for both sport
and commercial fishing, 7’ falcatus (Linnaeus, 1758), a game fish species, and 7’ goodei
Jordan et Evermann, 1896, a species with a high potential for aquaculture and sport
fishing, are the most widely distributed, occurring from the Southern United States to
Northern Argentina (McMaster 1988, Lazo et al. 1998, Heilman and Spieler 1999).
Recent data identified population differentiations in the number and positions of the
ribosomal sites among the extensively distributed species, 7’ falcatus and T. goodei (Ac-
cioly et al. in press). Indeed, there is growing evidence that cytotaxonomic markers,
particularly ribosomal sites, may reveal the genetic structure of marine fish populations
(Motta-Neto et al. 201 1a, Lima-Filho et al. in press).

In addition to their biological significance in commercial and sport fishing, repre-
sentatives of the genus 77achinotus are considered potentially suitable for pisciculture pur-
poses (Watanabe 1995, Weirich et al. 2006). Trachinotus species have very desirable bio-
logical characteristics, such as fast adaptation to confined environments, good tolerance to
extreme environmental conditions and rapid growth (Jory et al. 1985). Nevertheless, ge-
netic and cytogenetic foundations supporting their cultivation remain largely unknown.

Most species of the marine Perciformes exhibit a basal karyotype composed of 2n=48
acrocentric chromosomes, extensively conserved in several families (Molina 2007). Giv-
en the large number of species, most cytogenetic studies have focused on mapping bio-
diversity in this order, the largest of all living vertebrates. Among the family Carangidae,
cytogenetic data have already been reported for a total of 27 species in 13 genera (e.g.
Caputo et al. 1996, Sola et al. 1997, Rodrigues et al. 2007, Chai et al. 2009). Of these,
few species occur exclusively in the Atlantic. The present cytogenetic study characterizes
the species Trachinotus carolinus, T. falcatus and T. goodei through conventional staining,
Ag-NOR detection, C-banding, CMA,/DAPI fluorochrome staining, and mapping of
the 18S and 5S rDNA sequences by dual-color FISH. Useful phylogenetic information
was provided by ribosomal sequences mapping, indicating an intriguing scenario with
additional acrocentrics formation in 7: goodei and T. carolinus.

Material and methods

Samples of the species Trachinotus carolinus (N=5; 3 males. one female, one imma-
ture), 7! falcatus (N=10; 4 males, 3 females, 3 immatures) and T’ goodei (N=10; 6
males, 4 females) were obtained on the coast of Rio Grande do Norte state (05°05'26"S,

Karyotype evolution in Trachinotus genus 361

36°16'31"W), in Northeast Brazil. Prior to chromosomal preparations, specimens were
submitted to im vivo mitotic stimulation for 24 hours, through intramuscular and in-
traperitoneal injection of complex antigens (Molina et al. 2010). Individuals were an-
esthetized with clove oil (Griffiths 2000) and sacrificed. Mitotic chromosomes were ac-
quired from cell suspensions of anterior kidney fragments according to in vitro mitotic
block (Gold et al. 1990). Cell suspensions were dripped onto slides coated with a film
of distilled water heated to 60°C, and stained with 5% Giemsa diluted in a phosphate
buffer pH 6.8. The material was analyzed under 1000x magnification and the best
metaphases were photographed under an Olympus BX50° epifluorescence microscope,
with an Olympus DP70° digital image capturing system. About 30 metaphases were
analyzed for each individual in order to determine the diploid number for every species.

Chromosome nomenclature

Chromosomes were classified as metacentric (m), submetacentric (sm), subtelocentric
(st) and acrocentric (a), based on the system proposed by Levan et al. (1964).

Chromosome banding

The heterochromatic and nucleolar organizer regions (Ag-NORs) were identified using
techniques developed by Sumner (1972) and Howell and Black (1980) respectively.
CMA,/DAPI staining was applied in accordance with Barros-e-Silva and Guerra (2010).

Cytogenetic mapping protocols

Two probes were used: an 18S rDNA probe obtained from the nuclear DNA of Prochil-
odus argenteus Spix et Agassiz, 1829 (Hatanaka and Galetti 2004); a 5S rDNA probe
isolated from the genomic DNA of Leporinus elongatus Valenciennes, 1850 (Martins
and Galetti 1999); probes were labeled by polymerase chain reaction (PCR), using
biotin-16-dUTP (Roche Applied Science*) for 18S rDNA or digoxigenin-11-dUTP
(Roche Applied Science®) for 5S rDNA. PCR labeling for rDNA clones was performed
with specific primers, using 20 ng of template DNA, 1X Taq reaction buffer (200 mM
Tris pH 8.4, 500 mM KCl), 40 uM dATP, dGTP and dCTP, 28 uM of dTTP, 12 uM
biotin-16-dUTP or digoxigenin-11-dUTP, 1 uM primers, 2 mM MgCl, and 2 U of
Tag DNA Polymerase (Invitrogen*) under the following conditions: 5 min at 94°C; 35
cycles: 1 min at 90°C, 1 min 30s at 52°C and 1 min 30 s at 72°C; and a final extension
step at 72°C for 5 min.

The overall hybridization procedure followed the protocol described by Pinkel et
al. (1986), under high stringency conditions (2.5 ng/uL from each probe, 50% deion-
ized formamide, 10% dextran sulphate, 2XSSC, pH 7.0 — 7.2, at 37°C overnight).

362 Uedson Pereira Jacobina et al. / Comparative Cytogenetics 6(4): 359-369 (2012)

After hybridization, slides were rinsed in 15% formamide/0.2XSSC at 42°C for 20 min,
0.1XSSC at 60°C for 15 min, and 4XSSC/0.05% Tween at room temperature for 10
min (two times for 5 min each). Signal detection was performed using streptavidin-alexa
fluor 488 (Molecular Probes®) for the 18S rDNA probe; and anti-digoxigenin-rhoda-
mine (Roche Applied Science®) for 5S rDNA, which were detected by dual color FISH.

Results

All species analyzed exhibited 2n=48 chromosomes, however with a notable difference
in the number of two-armed (bibrachial) elements.

The karyotype of Trachinotus goodei (Figure 1a, d, g) is composed of 4 m/sm and
44a (FN=52). The heterochromatic regions in this species are very reduced and restrict-
ed to small blocks in the chromosomal pericentromeric regions. The Ag-NORs/18S
rDNA sites were identified near the centromeric region of two acrocentric pairs, ten-
tatively No. 5 and 11 of the karyotype. These sites proved to be rich in GC base
composition (CMA*/DAPI) (Figure 1d). Hybridization signals with 5S rDNA probes
were also identified on the terminal regions of the short arms of three acrocentric pairs,
tentatively numbered as 9, 12 and 22 (Figure 1g).

The 7: carolinus karyotype (Figures 1 b, e, h) consists of 8m/sm and 40a (FN=56).
The content of heterochromatin is also poorly distributed in the pericentromeric re-
gions of some chromosome pairs. Ag-NORs/18S rDNA sites were located on the short
arm of only one acrocentric pair, identified as number 5. These sites are clearly hetero-

Figure |. Karyotypes of Trachinotus goodei (a, d, g), T: carolinus (b, e, h) and T: falcatus (c, f, i). Conven-

tional staining (a, b, c) highlighting the chromosomal pairs carrying Ag-NOR sites; C-banding (d, e, f);
nucleolar organizer pairs are highlighted by staining with CMA,*/DAPI. Dual-color FISH (f, g, hh) show-
ing the chromosomal mapping of the 18S rDNA (green) and 5S rDNA (red) sites. Bar = 5 um.

Karyotype evolution in Trachinotus genus 363

chromatic, with a CMA*/DAPT pattern. The 5S rDNA sites were mapped only on the
short arm of the acrocentric chromosome 9.

The karyotype of T: falcatus (Figure Ic, f, i) has the largest number of bibrachial
elements if compared to the other species, i-e., 10 m/sm and 38a (FN=58). As in the
two previous species, small heterochromatic blocks are present in pericentromeric re-
gions of the chromosomes. Ag-NORs/18S rDNA sites were situated in the terminal
region of the short arm of the submetacentric chromosome pair 3, which also appears
heterochromatic after C-banding, with a CMA*/DAPI pattern. The 5S rDNA sites

were mapped exclusively on the short arms of the acrocentric pair 9.

Discussion

As in many species of Perciformes, the species analyzed displayed 2n=48 and large
numbers of acrocentric chromosomes, although there were notable differences in kary-
otype macrostructure. This is particularly evident for the number of chromosome arms
(FN) that varies between species. Thus, 7’ goodei exhibits FN=52, 7’ carolinus FN=56
and T’ falcatus FN=58. Karyotypes similar to those presented here for 7’ goodei and 7:
falcatus were previously identified in other populations of this species on the Southeast
and Northeast coasts of Brazil (Rodrigues et al. 2007, Accioly et al. in press).

Evolutionary karyotype modifications resulting from pericentric inversions are com-
mon in Perciformes. In fact, two-armed chromosomes have been found in approxi-
mately 30% of Carangidae species karyotyped to date (Chai et al. 2009). Furthermore,
other kinds of chromosomal diversification have been identified for this family including
Robertsonian translocations, transient in Seriola Cuvier, 1817 (Vitturi et al. 1986, Sola
et al. 1997) or already established in Selene setapinnis (Mitchill, 1815) Jacobina 2012).

Basing on morphological and molecular evidences, the genus Trachinotus is in-
cluded in the tribe Trachinotini, which is considered one of the least diverse groups
among carangids (Smith-Vaniz 1984, Gushiken 1988). Phylogenetic hypotheses based
on mitochondrial sequences (Reed et al. 2002) suggest 7: carolinus as the most basal
species, followed by more derived 7. falcatus and T. goodei, respectively. However,
these phylogenetic relationships do not agree with the karyotypic characteristics pre-
sented by these species (Figure 2a).

Whereas the fully acrocentric karyotype with 2n=48 (FN=48) is considered basal
for Perciformes, variations of this karyotypic formula can be interpreted as derived
conditions. Thus, an increase in the number of two-armed chromosomes, as sequen-
tially found in T° carolinus (eight two-armed chromosomes) and in 7. falcatus (ten
two-armed chromosomes), would be expected to represent derived cytogenetic char-
acteristics. As such, T: goodei, showing only four two-armed chromosomes and, con-
sequently, the largest number of acrocentric chromosomes, would be representing the
species with the karyotype closer to the basal one.

Many closely related species of Perciformes show poorly varied or cryptic cytoge-
netic characteristics, hampering their application in phylogenetic inferences (Molina

364 Uedson Pereira Jacobina et al. / Comparative Cytogenetics 6(4): 359-369 (2012)

P| T. goreensis
T. ovatus
T. goodei — (FN=52)
T. rhodopus
T. falcatus — (FN = 58)
T. carolinus > (FN = 56)
T. teraia

b Basal

Maaaa it it sci aie

2n=48 18S rDNA 5S rDNA

Figure 2. Phylogenetic tree from molecular data of some species of Trachinotini tribe (a), adapted
from Reed et al. (2002). The molecular relationship is confronted with the chromosomal formula of the
Trachinotus species analyzed. Schematic illustration shows the role of additional pericentric inversions
leading to new acrocentric chromosomes and modification of the FN value (b), and the derived condition

of multiple sites of 18S and 5S rDNAs in 7. goodei (Cc).

2007, Motta-Neto et al. 201 1a, b, c). Indeed, this is observed in the similar karyotype
macrostructure or heterochromatic patterns, such as those found in Trachinotus spe-
cies, where C-bands are inconspicuous and similarly located in the pericentromeric
region of the chromosomes. A reduced amount of heterochromatin is also a common
feature in other Perciformes, possibly resulting in lower karyotype evolution dynamics
(Molina and Galetti 2004, Molina 2007). On the other hand, NORs were promi-
nent characteristics, in lines with considerable karyotype variation between species.
Trachinotus carolinus and T. falcatus displayed only one pair of chromosomes carry-
ing ribosomal sites (Ag-NOR/18SrDNA/CMA‘*/DAPI). This condition is considered
basal and the most common for Carangidae (Caputo et al. 1996, Sola et al. 1997). As
previously confirmed (Accioly et al. in press), the 7: goodei population from Brazilian
Northeastern coast exhibits a more derived condition, with two chromosomal pairs
carrying ribosomal sites (pairs 5 and 11). Although multiple sites have not been iden-
tified in populations from the Southeastern coast (Rodrigues et al. 2007), the occur-
rence of more than one chromosome pair carrying NORs in 7! goodei indicates some
level of derivation in this species in relation to the others. Greater dynamic evolution
of the ribosomal sites in this species is corroborated by the presence of three chromo-
somal pairs carrying 5S rDNA sequences (pairs 9, 12, 22), a condition not present in
T. carolinus and T: falcatus, where these sites were mapped only in pair 9 (Figs 1, 2c).
In addition, dual-color FISH showed no synteny between 18S and 5S rDNA sites in
all the three species of Trachinotus analyzed here.

Karyotype evolution in Trachinotus genus 365

Simple ribosomal sites are considered an ancestral condition, most frequently found
in carangids (Caputo et al. 1996, Sola et al. 1997), as well as among marine Perciformes
(Galetti et al. 2000). Their location in distinct chromosomal pairs is an efficient cyto-
taxonomic marker of species and populations of Trachinotus (Accioly et al. in press).
Indeed, Southeastern populations of T: falcatus and T. goodei are characterized by hav-
ing simple Ag-NOR sites on the short arms of pair 18 and on the short arms of pair
3, respectively. The greater dynamic evolution of the 18S and 5S ribosomal sequences
in T: goodei corroborates its more derived condition in relation to the other species
(Figure 2), as suggested by molecular data (Reed et al. 2002). In turn, sharing of 5S
rDNA sequences by a same chromosome pair, tentatively identified as no. 9, probably
indicates homeologous chromosomes with similar syntenic content. The occurrence of
three pairs carrying 5S rDNA sequences (pairs 9, 12 and 22) in T° goodei is uncommon
among fish (Martins and Galetti 2000). The location of 5S and 18S rDNA sites in dif-
ferent chromosomes, and the functional divergence between 18S rDNA (transcribed by
RNA polymerase I) and 5S rRNA genes (transcribed by RNA polymerase I) (Martins
and Galetti 2000), supports the independent evolution of these multigene families due
to specific selection pressures (Amarasinghe and Carlson 1998).

Variations in the number and location of NORs in some cases, are likely to be
favored by a high and heterogeneous heterochromatic content, whereas the inverse
seems to reduce the evolutionary dynamism of these regions (Molina 2007). Besides
increasing the NORs’ dynamics, there are also indications that heterochromatin may
act as hotspots for chromosomal rearrangements (Almeida-Toledo et al. 1996; Jaco-
bina 2012). However, there is currently no information that the heterochromatin may
be exerting some role in the evolutionary dynamics of the rDNA in T° goodei. Disper-
sion of these sequences in the karyotype may occur via transposition events by mobile
elements in the carrier genome, with subsequent amplification and formation of new
repetitive DNA sites (Eickbush and Eickbush 1995; Almeida-Toledo et al. 1996).
Indeed, a surprising chromosome spreading of associated transposable elements and
ribosomal DNA (Rex3/5S rDNA) was demonstrated to occur in the freshwater fish
Erythrinus erythrinus (Bloch et Schneider, 1801) (Erythrinidae), increasing the num-
ber of such rDNA sequences from 2 to 22 between distinct populations (Cioffi et
al. 2010). Growing knowledge on the organization of repetitive DNAs also indicates
that their evolution may be mediated by unequal crossover, transposition mediated by
RNA and gene conversion (Dover 1986, Martins et al. 2006). ‘Thus, different events
may be associated with the serial repetition of the 5S rDNA multigene family in the
genome of 7. goodei, characterizing its more derived condition in relation to the other
species, 7) falcatus and T. carolinus.

The existing set of cytogenetic data for Carangidae suggests karyotype evolution
strongly mediated by pericentric inversion events. Based on the basal karyotype for
Perciformes (2n=48 acrocentrics, FN=48), the increase of FN indicates a derived con-
dition. Thus, if 7° goodei is the most derived species in respect to T! falcatus and T:
carolinus, as indicated by mitochondrial sequences (Reed et al. 2002), and supported
by the apomorphic features of its karyotype (multiple 18S and 5S rDNA sites), a par-

366 Uedson Pereira Jacobina et al. / Comparative Cytogenetics 6(4): 359-369 (2012)

ticular evolutionary pathway provided by pericentric inversions must be considered for
this species. Thus, the smaller number of two-armed chromosomes in T: goodei may
indicate additional rounds of pericentric inversions on two-armed chromosomes, in-
creasing the number of acrocentric chromosomes in the karyotype and, consequently,
decreasing the FN value (Fig. 2b). The same could be also considered for T: carolinus,
considering its more basal position in the phylogeny proposed for Trachinotus (Fig. 2a).
Our understanding of the karyotype evolution of Carangidae (including rDNA) was
improved by the present findings. Our data demonstrate that, in addition to structural
changes by pericentric inversions, rDNA sequences also acted as an important evolution-
ary indicator in Trachinotus karyotype. In this sense, the combined mapping of 18S and
5S rDNA sequences proved to be useful to clarify the relationships in this fish group.

Acknowledgements

We are grateful to the Coordination for the Improvement of Higher Education Teach-
ing Personnel (CAPES) and the National Council for Scientific and Technological
Development (CNPq) (Project No. 556793/2009-9) for their financial support. We
also thank IBAMA (Process No. 19135/1) and José Garcia Junior for the taxonomic

identification of the species.

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