Cytogenet Genome Res
DOI: 10.1159/000354871
High Evolutionary Dynamism in 5S rDNA
of Fish: State of the Art
L. Rebordinos I. Cross A. Merlo
Area de Genética, Facultad de Ciencias del Mar y Ambientales, CEI-Mar, Universidad de Cádiz, Puerto Real, Spain
Key Words
Birth-and-death evolution · Concerted evolution ·
Horizontal transfer · Multigene family · 5S rDNA
Abstract
The 5S ribosomal DNA (rDNA) consists of one transcriptional
unit of about 120 base pairs, which is separated from the
next unit by a non-transcribed spacer (NTS). The coding se-
quence and the NTS together form a repeat unit which can
be found in hundreds to thousands of copies tandemly re-
peated in the genomes. The NTS regions seem to be subject
to rapid evolution. The first general model of evolution of
these multigene families was referred to as divergent evolu-
tion, based on studies using hemoglobin and myoglobin as
model systems. Later studies showed that nucleotide se-
quences of different multigene family members are more
closely related within species than between species. This ob-
servation led to a new model of multigene family evolution,
termed concerted evolution. Another model of evolution,
named the birth-and-death model, has been found to be
more suitable to explain the long-term evolution of these
multigene families. According to this model, new genes orig-
inate by successive duplications, and these new genes are
either maintained for a long time or are lost, or else degener-
ate into pseudogenes. In this review we describe different
sources of variability in the 5S rDNA genes observed in sev-
© 2013 S. Karger AG, Basel
1424–8581/13/0000–0000$38.00/0
E-Mail karger@karger.com
www.karger.com/cgr
Published online: September 24, 2013
eral distinct fish species. This variability is mainly referred
to NTSs and includes the presence of other multigene fami-
lies (mainly LINEs, SINEs, non-LTR retrotransposons, and U
snRNA families). Different types of microsatellites have also
been found to contribute to the increase of variability in this
region. Our recent results suggest that horizontal transfer
contributes to the increase of diversity in the NTSs of some
species. Variability in the 5S rDNA coding region affecting
the stability of the structure, but without effects on the func-
tion of the 5S rRNA, is also described. Retrotransposons seem
to be responsible for the high dynamism of 5S rDNA, while
microsatellites acting as recombination hot spots could sta-
bilize a wide variety of unusual DNA structures, affecting
DNA replication and enhancing or decreasing promoter ac-
tivity in gene expression. The relationship between the high
variability found at molecular level and the low variability
found at chromosomal level is also discussed.
© 2013 S. Karger AG, Basel
Ribosomal Multigene Families
In eukaryotes, ribosomal DNA (rDNA) is generally ar-
ranged in 2 different clusters (multigene families), each
composed of hundreds to thousands of gene copies. The
coding genes for ribosomal RNA (rRNA) are 45S rDNA
(major genes) and 5S rDNA (minor genes), which are or-
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Laureana Rebordinos
Area de Genética, Facultad de Ciencias del Mar y Ambientales
CEI-Mar, Universidad de Cádiz
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ES–11510 Puerto Real (Spain)
E-Mail laureana.rebordinos @ uca.es
ganized in tandem repeats. The secondary structure of
these genes, different evolutionary rates among regions
and the organization in tandem repeats make the rDNA
a good candidate for species and population characteriza-
tion and for studies of phylogenetic and evolutionary re-
lationships and genome structure [Messias et al., 2003;
Jansen et al., 2006]. These 2 rDNA families usually are not
linked in eukaryotes [Torres-Machorro et al., 2009], al-
though their linkage as an ancestral feature has been de-
scribed in terrestrial plants [Wicke et al., 2011].
In relation to the number of chromosomes bearing
these 2 kinds of rDNA, in invertebrates every multigene
family is usually localized in 1 chromosome pair [Gor-
nung et al., 2005; Vitturi et al., 2005]. In mammals, how-
ever, the 45S rDNA is often localized in several chromo-
some pairs, while the 5S rDNA is found in 1 chromosome
pair [Martins and Wasko, 2004]. In amphibians, the 2
ribosomal families may have multiple chromosome loca-
tions [Schmid et al., 1987; Lucchini et al., 1993].
At the present time, there is a lot of data available about
the chromosomal location of 45S and 5S rDNA in fishes.
This has been made possible by the fluorescence in situ
hybridization (FISH) technique. This technique com-
bines cytogenetic and molecular genetic methodologies,
and it has been shown to be very useful for establishing
cytogenetic maps and conducting genomic studies [Gal-
lardo-Escárate et al., 2005; Pérez-García et al., 2010]. Ow-
ing to organization in tandem repeats, which makes it
easier to determine their location by means of this tech-
nique, the rRNA genes have been very useful in genetic
mapping. In fishes, as in amphibians, the rDNA may be
found in more than one single chromosome pair. The
number of chromosomes bearing these genes depends on
family or species. Furthermore, the 2 multigene families
are usually not colocalized, which is similar to other ver-
tebrates [Martins and Wasko, 2004]. Only 20% of all the
species studied until now show colocation of the 2 rRNA
genes.
Molecular Description of the 5S rDNA
The 5S rDNA consists of 1 transcriptional unit of
about 120 base pairs, which is separated from the next
unit by a non-transcribed spacer (NTS) (fig.  1A). Al-
though the 5S rRNA gene is highly conserved, even be-
tween unrelated species, the NTSs are variable, both in
length and in sequence. These variations are due to dele-
tions, insertions, internal subrepeats, base substitutions,
and pseudogenes, and they have been used for evolution-
2 Cytogenet Genome Res
DOI: 10.1159/000354871
ary studies and as species-specific or population-specific
markers [e.g. Ferreira et al., 2007; Campo et al., 2009;
Merlo et al., 2013b] (fig. 1B). As a consequence of these
variations, some pseudogenes can appear. The NTS re-
gions seem to be subject to rapid evolution, which makes
them important for studies concerning the organization
and evolution of the 5S multigene family, and also as
markers for tracing recent evolutionary events [Pinhal et
al., 2009; Úbeda et al., 2010a, b].
Evolution of the 5S rDNA: Concerted Evolution
versus Birth-and-Death Model of Evolution
Studies of mechanisms governing evolution of multi-
gene families have generated some controversy. The first
efforts to resolve the controversy about the evolutionary
dynamics of gene families date back to the early 1960’s,
with studies using hemoglobin and myoglobin as model
systems [Ingram, 1961; Eirín-Lopez et al., 2012]. The first
general model of evolution of these multigene families
was referred to as divergent evolution. Development of
DNA sequencing techniques during the 1970’s helped re-
searchers to analyze the patterns of variation in coding
and non-coding regions. The studies showed that nucleo-
tide sequences of different multigene family members are
more closely related within species than between species.
This observation led to a new model of multigene family
evolution, termed concerted evolution. Given the success
of this model, all multigene families were considered to
follow the concerted evolution model [Hood et al., 1975;
Ohta, 1980].
Nevertheless, further studies have demonstrated that
concerted evolution does not explain the evolution of
some multigene families, because no apparent homogeni-
zation mechanisms could be detected among the different
units of the multigene family, and a high degree of vari-
ability could be observed [Hughes and Nei, 1989; Nei et
al., 2000; Eirín-López et al., 2004a]. Since then, another
model of evolution, named the birth-and-death model,
has been found to explain better the long-term evolution
of these multigene families. According to this model, new
genes originate by successive duplications, and these new
genes are either maintained for a long time or are lost, or
else degenerate into pseudogenes [Nei and Rooney, 2005].
Given the apparent homogeneity observed among the
various copies, 5S rRNA genes have been used to show
the archetypal example of a gene family subject to con-
certed evolution [Eirín-Lopez et al., 2012]. Moreover, giv-
en the high degree of homogeneity in the 5S rRNA genes
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 A
Fig. 1. A 5S rDNA structure showing the coding region (120 bp long) and NTSs. B Schematic representation
showing polymorphisms that can be found in the NTSs. This example shows different types of NTSs observed in
a highly polymorphic 5S rDNA of the fish D. sargus [Merlo et al., 2013b].
usually observed within species, several authors have
used them as species-specific markers [Manchado et al.,
2006a]. In other studies, however, intensive dynamism
has been observed in this gene, leading to phenomena
such as pseudogenes [Frederiksen et al., 1997; Martins et
al., 2002; Freire et al., 2010], different types of 5S rDNA
[Vierna et al., 2009; Freire et al., 2010; Úbeda-Manzana-
ro et al., 2010a] and linkage to other multigene families
[Drouin and Moniz de Sá, 1995; Manchado et al., 2006b;
Freire et al., 2010]. Recent studies have shown the pres-
ence of elements such as tRNA-derived short interspersed
nuclear elements (SINEs), long terminal repeats (LTRs)
and microsatellites, among others, in the NTSs of fishes
(fig. 1B) [Merlo et al., 2012a, 2013b], revealing a putative
main role for these elements in the dynamics of the 5S
rDNA, as will be discussed in this review.
All these phenomena give the 5S rDNA multigene
family a variability which is not known in other tandem-
ly arrayed multigene families. Therefore, an increasing
number of authors have rejected the exclusivity of con-
certed evolution as the model which explains the 5S
rDNA evolution [Rooney and Ward, 2005; Fujiwara et
al., 2009; Vierna et al., 2009; Freire et al., 2010; Úbeda-
Manzanaro et al., 2010a; Perina et al., 2011; Pinhal et al.,
High Evolutionary Dynamism in 5S
rDNA of Fish
2011]. However, they also observed a homogenization
process within each different type of 5S rDNA obtained,
and hence they concluded that a mixed model between
concerted and birth-and-death evolution is the most
probable for the 5S rDNA multigene family. In this sce-
nario, the successive duplications of the birth-and-death
model would act to generate new variants, and the mech-
anisms of the concerted model (unequal cross-over and
gene conversion) would homogenize each variant at lo-
cus level.
Sources of Variability in the 5S rDNA
Linkage between 5S rDNA and Other Multigene
Families
The 5S rDNA is known to be a multigene family which
can be found linked with a variety of other multigene
families. These linkages include major ribosomal genes,
histones, trans-spliced leader genes and small nuclear
RNA (snRNA) genes [e.g. Drouin and Moniz de Sá, 1995;
Eirín-López et al., 2004b; Manchado et al., 2006b]. A link-
age between 5S rDNA and transposable elements (TEs)
has also been demonstrated in fishes. For example, a
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LTR-Gypsy repetitive fragment and a non-LTR repetitive
element [long interspersed nuclear element (LINE)
CR1–79_HM] were found within a NTS variant in the
fish Diplodus sargus [Merlo et al., 2013b] (fig. 1B). The
role of these elements will be discussed in the following
sections. Recently, in the Haemulidae species Plectorhin-
chus mediterraneus, a tRNA-derived SINE was observed
for the first time in the NTS type α of the 5S rDNA mul-
tigene family [Merlo et al., 2012a]. The presence of ret-
rotransposable elements (non-LTR retrotransposons) has
been described in linkage with the 5S rDNA multigene
family in the fish Erythrinus erythrinus [Cioffi et al., 2010].
Interestingly, in the sole Solea senegalensis, one 2,166-bp
5S rDNA unit was composed of two 5S rRNA genes sepa-
rated by NTS-I and followed by a 1,721-bp spacer con-
taining the U2, U5, and U1 snRNA genes. They were in-
verted and arranged in the transcriptional direction op-
posite to that of the 5S rRNA gene. This simultaneous
linkage of 3 different snRNAs had never been observed
before [Manchado et al., 2006a]. The linkage between 5S
rDNA and snRNA genes had been described previously
in mollusks. In oysters from the Crassostrea genus, the U2
snRNA and 5S rRNA genes were closely linked in a gene
cluster in the genomes of C. angulata and C. gigas [Cross
and Rebordinos, 2005]. This linkage could be explained
by 5S rDNA retrotransposition mechanisms via an RNA
intermediate, as found in rats and mice [Drouin, 2000].
In crustaceans, a linkage between 5S rDNA and the U1
snRNA gene has also been described [Pelliccia et al.,
2001].
Taking these findings into account, the ability of the
5S rDNA to jump to other loci seems evident. Two pos-
sible mediators for these translocations are via extrachro-
mosomal circular DNA (eccDNA) and via retrotranspo-
son. The formation of eccDNA from arrays of tandem
repeats may cause deletions, and the possible re-integra-
tion of rolling circle replication products could expand
these arrays in the genome. Examples of eccDNA homol-
ogous to 5S rDNA have been detected in Xenopus, Dro-
sophila, Arabidopsis thaliana, and Brachycome dichromo-
somatica [see review by Cohen and Segal, 2009, and refer-
ences therein]. The role played by the retrotransposon
elements in the evolution and variability of the 5S rDNA
will be discussed next.
The Role of Retrotransposons in the 5S rDNA
Dynamism
A recent study concerning 5S rDNA variability in a
fish species belonging to the Sparidae family (D. sargus)
revealed the most intensive multi- and polymorphic-
4 Cytogenet Genome Res
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M 1 2 3
1,400
1,200
1,000
800
700
600
500
400
300
200
Fig. 2. PCR amplification polymorphisms of 5S rDNA found in 3
different individuals of D. sargus (lanes 1–3). M = 100-bp marker.
band PCR amplification pattern ever described [Merlo et
al., 2013b] (fig. 2). This multiband pattern was observed
in every sample studied, and the specific pattern varied
among individuals. Fourteen variants were obtained
from 15 bands of 10 different sizes in just 6 specimens
analyzed. Many types and variants were interrelated to
each other by their NTS sequences with different possible
rearrangements, such as inversion, insertion-deletion or
nucleotide substitution. Only 2 of the types were non-
functional genes or pseudogenes; and pseudogenes of the
5S rRNA gene were observed within the NTSs of 5 vari-
ants. Furthermore, microsatellite regions were found in
the NTS of 2 variants.
Although the presence of microsatellite repetitions is
not common in the 5S rDNA of fishes, several cases have
been described [Pasolini et al., 2006; Úbeda-Manzanaro
et al., 2010a].The presence of large microsatellite arrays is
a characteristic of the Rajidae family [Pasolini et al., 2006].
It has been proposed that microsatellites play an impor-
tant role in stabilizing a wide variety of DNA structures,
including the structure of the tandemly arranged multi-
gene families [Cross and Rebordinos, 2005], since the mi-
crosatellites can act as hot spots for recombinations, and
the homogenizing mechanisms could then be favored
[Chistiakov et al., 2006].
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 Finally, retrotransposon elements were found within
this highly polymorphic 5S rDNA of D. sargus. Four out
of the 14 types of NTSs showed a LTR-Gypsy (SZ-7A_I)
repetitive fragment. A non-LTR repetitive element (LINE
CR1-79_HM) was also found within 1 out of the 14 types
of NTSs (fig. 1B). The presence of so many variants has
never been described before in any other fish species. In
Amphychthys cryptocentrus, a high variability in NTSs
(indels, microsatellites, duplications) was observed, but
only 2 different variants were found [Úbeda-Manzanaro
et al., 2010a], while in D. sargus 4 completely different
variants were found [Merlo et al., 2013b]. In D. sargus, the
presence of residual fragments of some TEs, such as LTRs
and LINEs, suggests that the high variability observed in
the NTS is mediated by TEs [Merlo et al., 2013b]. This is
not the only species in which a TE is associated with 5S
rDNA. In the fish E. erythrinus, a non-LTR retrotranspo-
son is also colocalized with the 5S rDNA [Cioffi et al.,
2010], and those authors proposed this colocalization as
the reason for the high variability observed.
The 5S rDNA variability found in D. sargus is not ex-
plained under the concerted evolution model; however,
another model, the birth-and-death evolution model,
may do so. We have theorized that, at genome level, the
mechanism of the birth-and-death model generated the
new 5S types in this species; but at locus level, the mecha-
nisms responsible for concerted evolution are maintain-
ing the sequence homogeneity of each type, resulting in a
‘tandem model’ of birth-and-death and concerted evolu-
tion. This mixed model has been previously described by
other authors [Freire et al., 2010; Pinhal et al., 2011; Vi-
zoso et al., 2011], but it is not known why retrotranspo-
sons play this major role in generating high polymor-
phism and dynamism in this species. D. sargus includes
several subspecies inhabiting differentiated but adjacent
geographic areas [Bargelloni et al., 2005]. In the contact
zone of these subspecies, hybridization events may occur
because those zones increase the interspecies (and sub-
species) and interhybrid crossing [Fontdevila, 2005].
The scenario just described increases genetic variabil-
ity in such populations and in the breeding stock from the
region near the Strait of Gibraltar (Cadiz Bay, Spain) from
which the samples were obtained. This area is a contact
zone of the 2 subspecies D. sargus sargus and D. sargus
cadenati. According to Fontdevila [2005], a burst of trans-
positions follows hybridization and may contribute to the
genetic instability observed after that event. Therefore the
fragments of TEs found in the 5S rDNA of D. sargus sar-
gus could be evidence for such a hybridization process
[Merlo et al., 2013b]. Interestingly, when 5S rDNA was
High Evolutionary Dynamism in 5S
rDNA of Fish
localized in chromosomes of D. sargus by means of the
FISH technique [Merlo et al., 2013b], the huge variability
observed in its sequence was not related to the number of
loci observed in the chromosomes, since the 5S rDNA
probe hybridized with between 3 up to 6 chromosomes,
with 4 being the modal number (fig. 3). So, just 2 chromo-
some pairs explain up to 14 variants, grouped in 4 com-
pletely different groups. The use of probes containing
several variants could reveal more loci in the chromo-
somes of this species. However, with the results obtained,
the molecular variability observed in the 5S rDNA was
focused on just 2 loci in the genome.
The case of P. mediterraneus (Teleostei, Haemulidae)
is another interesting example of dynamism in the 5S
rDNA. Up to 4 variants of 5S rDNA were found in this
species [Merlo et al., 2012a]. However, in this species, the
localization of the 5S rRNA gene by means of the FISH
technique showed only a single pair of chromosomes
bearing this gene (fig. 3). Concerning the molecular anal-
ysis, the most abundant variant (α) showed 2 interesting
features related to its dynamism. The first consisted of a
region of 101 nucleotides at the 5′ end of the NTS which
was homologous with NTS regions of several different
fish species. The presence of this region could be ex-
plained by its role in regulating 5S rDNA expression or as
an enhancer element, like a D-box of mammals [Hallen-
berg and Frederiksen, 2001].
The second interesting characteristic was the presence
of a region similar to the glutamine tRNA gene in this α
NTS. This gene was probably not functional owing to
polymorphisms affecting the secondary structure of the
tRNA. The dynamics of the 5S rDNA suggests that a
transposition event occurred in the tRNA gene family.
However, the authors hypothesized that the observed
tRNA-like gene could be a SINE. The majority of SINEs
derive mainly from tRNA sequences and have some fea-
tures present in the NTS α of P. mediterraneus. Hence, it
is possible that a tRNA-derived SINE was linked with the
5S rDNA in this variant. From an evolutionary point of
view, it has been postulated that SINE integration into
new localizations has potential implications, since it can
disturb gene expressions or serve as a source of genomic
innovation and a factor of genome plasticity [Makalows-
ki, 2000; Merlo et al., 2012a].
Two of the 4 variants observed in P. mediterraneus
showed a high degree of homology with that of Raja as-
terias (Elasmobranchii, Rajidae). The putative implica-
tion of a horizontal transfer (HT) event and its conse-
quences for the organization, evolution and dynamisms
of the 5S rDNA will be discussed in the next section.
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 A1
1 μm
B1
1 μm
Fig. 3. Localization of 5S rDNA by FISH on chromosomes of P. mediterraneus (A1, A2) showing hybridization in
a single pair of chromosomes, and D. sargus (B1, B2) displaying multiple hybridization signals. Scale bars = 1 μm.
The Role of 5S rDNA in Horizontal Transfer
P. mediterraneus is, in fact, an interesting species to il-
lustrate the dynamics of 5S rDNA. Following the discov-
ery of a type of NTS (1 out of 4) with a tRNA-derived
SINE, another 2 NTS variants (named β and δ) found in
this species showed another relevant feature [Merlo et al.,
2012a]: these 2 variants presented high homology with
type I of the 5S rDNA from R. asterias [Pasolini et al.,
2006], in both coding and non-coding regions. P. medi-
terraneus and R. asterias belong to different classes: the
former to Actinopterygii and the latter to Chondrichthy-
es. The 2 classes separated 527 Mya [Hedges, 2009]. Two
hypotheses were examined by Merlo et al. [2012a]: in the
first, the 2 variants (β and δ) could have originated before
the separation of the 2 classes and could have been main-
tained only in some lineages. This hypothesis was rejected
because the NTSs are very dynamic regions and are con-
sidered almost neutral for natural selection, so they can
be either lost or fixed, causing differences between close-
ly related species. Moreover, the pseudogenization ob-
served in these 2 variants would accelerate the accumula-
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A2
1 μm
B2
1 μm
tion of mutations, so this first hypothesis was considered
unlikely. The second hypothesis proposed by Merlo et al.
[2012a] was the occurrence of an HT event. After an HT
event, the transferred 5S rRNA gene lost its functionality
and became a pseudogene. It has been postulated that HT
is a very important mechanism, the source for much bio-
logical innovation; indeed, some of the most significant
events in cell evolution have been associated with HT,
such as the origin of the primordial eukaryote cell, and
HT has contributed to the characteristics of multicellular-
ity [Schaack et al., 2010]. The novel results obtained by
Merlo et al. [2012a] have huge implications on the evolu-
tion of the species; and in fishes an HT event like this has
been described only in 1 other species: the toadfish Halo-
batrachus didactylus [Merlo et al., 2012b].
The Lusitanian toadfish H. didactylus is a member of
the Batrachoididae family and lives on the Eastern Atlan-
tic Coast. It has traditionally been used as a model animal
in toxicology experiments [Sarasquete et al., 1982] and in
hematology, reproduction and histophysiology studies
[Sarasquete, 1983; Palazón et al., 2001; Desantis et al.,
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2007]. A study of the 5S rDNA variability showed 2 vari-
ants, named α and β, in this species [Merlo et al., 2012b].
The NTSs from the 2 variants were distantly related to
those of another 4 species of the Batrachoididae family
from the Western Atlantic previously studied [Úbeda-
Manzanaro et al., 2010a]. This feature indicates that H.
didactylus has evolved independently from the Western
Atlantic species, which could have contributed to the
greater divergence found between Eastern and Western
Atlantic species. The most relevant result of the 5S rDNA
study obtained by Merlo et al. [2012b] was the high degree
of homology between the β-type NTS of H. didactylus and
the NTS of Sparus aurata described previously by Sola et
al. [2003]. The analysis with the β-type demonstrated that
this type was present in all specimens of H. didactylus
studied, as well as in other species analyzed in that study
belonging to the Batrachoidiformes and Perciformes or-
ders, but it was not present in either Pleuronectiformes or
Cupleiformes orders. S. aurata and H. didactylus belong
to different superorders (Acanthopterygii and Paracan-
thopterygii, respectively), which diverged 55 Mya [Finn
and Kristoffersen, 2007]. The same hypotheses explained
for P. mediterraneus have been proposed in this case.
Taking into account the high degree of dynamics to which
the NTSs are subjected, and the time elapsed since the 2
superorders began to diverge, the theory of HT, as in the
case of P. mediterraneus, was proposed [Merlo et al.,
2012a, b].
HT events have been documented extensively in pro-
karyotes, and it is a well-known mechanism of gene ex-
change [Frost et al., 2005]; but the occurrence of HT
among eukaryote organisms is scarcely documented.
Some of the most significant events in cell evolution have
been associated with HT [Schaak et al., 2010]. All docu-
mented cases of HT between eukaryotes involve TEs,
since their ability to mobilize and integrate within a ge-
nome makes them susceptible to horizontal transposable
transference [Kidwell, 1992]. Although the transfer of
host genes by TEs between different eukaryote species has
not yet been observed, it is known that TEs are able to
capture and transduce sequences with considerable fre-
quency within a species [Morgante et al., 2005]. Therefore
HT could be the mechanism responsible for a lateral
movement of genes between eukaryotes [Merlo et al.,
2012a, b]. The 5S rDNA family is an excellent candidate
for this kind of HT, since interactions between retrotrans-
posons and 5S rDNA have been demonstrated [Kapi-
tonov and Jurka, 2003; da Silva et al., 2011], and this in-
teraction could open the way for a 5S rRNA gene lateral
transfer [Merlo et al., 2012a, b].
High Evolutionary Dynamism in 5S
rDNA of Fish
Another possible mechanism that has been considered
is sperm-mediated gene transference, since it is well-
known that sperm cells are able to capture exogenous
DNA and to transfer it to the oocyte on fertilization
[Spadafora, 2008]. Furthermore, exogenous DNA is ac-
cumulated in marine sediments [Dell’Anno and Cori-
naldesi, 2004] and its content is 3 or 4 orders of magni-
tude higher than in the water column [Smith, 2002;
Dell’Anno and Corinaldesi, 2004]. In the case of H. didac-
tylus the benthic behavior of the majority of species of the
Batrachoidiformes order makes the possibility of sperm-
mediated gene transference more plausible [Merlo et al.,
2012b].
Microsatellites as a Source of NTS Variability
The size polymorphisms of the 5S rDNA repeats are
due to the presence of indels, microsatellite elements,
duplicated and modified multi-residues, among others
[Gornung et al., 2007]. Among these elements, microsat-
ellites are of special interest because of their putative role
in the evolution of the NTSs in the 5S rRNA genes.
In a recent work [Merlo et al., 2010], a study on the
variability of the 5S rDNA in 2 fish species belonging to
the Moronidae family, Dicentrarchus punctatus and D.
labrax, revealed a size variability in PCR products, main-
ly due to the presence of a trinucleotide microsatellite in
the NTSs and to hexanucleotide duplication which was
located immediately before the microsatellite region.
In the fish D. sargus, out of the 14 variants described
[Merlo et al., 2013b], two showed a microsatellite region
in the NTS, too (fig. 1B). In the Batrachoididae family, the
molecular characterization of 5S rDNA revealed dinucle-
otide motifs in some NTSs of A. cryptocentrus and Batra-
choides manglae, and different types of microsatellites
were found: a composite microsatellite, an imperfect
composite microsatellite, a pure microsatellite, and, in
the Thalassophryne maculosa species, an imperfect
poly(A) region. Within Rajidae, 2 different NTSs (named
I and II) have been described [Pasolini et al., 2006]. The
NTS-II length showed great size polymorphism, mostly
related to the length of 3 regions that were constituted by
the aggregation of numerous simple sequence repeats
(SSRs). Di-, tri-, tetra-, and hexanucleotide motifs were
found and formed perfect, imperfect, and composite mi-
crosatellite sequences, as in the species of the Batrachoidi-
dae family described previously.
The number and the localization of motifs in the 3 SSR
regions are highly conserved across Rajidae and Dasyati-
dae NTS-II sequences. The total length of the SSRs rang-
es from 27 to 43% of the NTSs observed. The unusually
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Cytogenet Genome Res 7
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 Fig. 4. Secondary structure prediction of 5S rRNA type α (in the box) found in H. didactylus and structural chang-
es produced in 5 additional haplotypes. The free energy (ΔG) of the structures is shown; it is calculated consider-
ing a temperature of 15 ° C (mean seawater temperature).
high frequency of SSRs in the NTS-II of Rajiformes (but
not in the NTS-I nor in the NTSs of most bony fish stud-
ied) seems to exclude a general function of these elements
in the 5S rDNA units of fishes [Pasolini et al., 2006].
The functional relevance of a significant number of
microsatellites includes stabilizing a wide variety of un-
usual DNA structures, acting as hot spots for recombina-
tion, influencing DNA replication, and enhancing or
decreasing promoter activity in gene expression [Chis-
tiakov et al., 2006; Úbeda-Manzanaro et al., 2010a]. Mi-
crosatellites have also been found at chromosomal fragile
sites, which are associated with some pathological states
in humans [Richard et al., 2008]. Cross and Rebordinos
[2005] suggested that the presence of microsatellites
could also play a role in maintaining tandem arrays. In
any case, the direct correlation linking NTS length and
SSR extension speaks in favor of a structural role of SSRs
in promoting easier maintenance and enhanced dynam-
ics of one of the 2 functional 5S rRNA gene clusters [Pa-
solini et al., 2006].
Variability in the 5S rDNA Coding Region: Effects on
the Secondary Structure
The secondary structure of the 5S rRNA is highly con-
served among species. This structure consists of 3 do-
mains named α, β and γ, joined by a hinge structure
named A-loop (fig. 4). There are 5 loop (A–E) and 5 helix
(I–V) structures [Smirnov et al., 2008]. Variations in the
5S rDNA coding region can affect the secondary structure
and make it more or less stable. Depending on the site in
the coding region where the polymorphism occurs, the
modification in the secondary structure will have a major
or minor effect. As a consequence, this modified second-
8 Cytogenet Genome Res
DOI: 10.1159/000354871
ary structure could have effects on the rRNA function. In
the meagre (Argyrosomus regius), sequence analysis re-
vealed that 1 monomer unit in the dimer had a deletion
inside the coding region, which extends from nucleotide
+22 to +25. The secondary structure of the 5S rRNA with
the deletion affects the B-loop size and makes the struc-
ture less stable [Merlo et al., 2013a]. Similarly, in the Ba-
trachoididae family, deletions have also been found in-
side the 5S coding region, which affect the C-loop size,
and no variation has been observed either in the coding
region or in surrounding NTSs [Úbeda-Manzanaro et al.,
2010a]. Some studies indicate that minor mutations in B-
loop, helix III and C-loop regions might not affect the
functionality of 5S rRNA [Searles et al., 2000].
Zheng and Gerstein [2007] stated that pseudogenes
are not necessarily non-functional. Therefore Merlo et al.
[2013a] excluded the hypothesis that the deletion form
of the 5S rRNA gene of A. regius was a pseudogene. Dif-
ferent deletions have been described in S. aurata [Sola et
al., 2003], the Acipenseridae family [Robles et al., 2005]
and the Cyprinidae family [Fujiwara et al., 2009], but
these authors kept the view that deletion forms are pseu-
dogenes, despite the low variability found in their coding
regions and NTSs [Merlo et al., 2013b]. In D. punctatus,
changes in the 5S rDNA coding sequence affecting the 3
domains of the secondary structure have been reported
[Merlo et al., 2010]. The stability of this structure was
lower, but it did not affect the function of the 5S rRNA.
In H. didactylus, several changes (up to 10 haplotypes) in
the sequence of the coding regions have been described
[Merlo et al., 2012b]. These changes modified the sec-
ondary structure in the γ domain and in the helix III, A-
loop and helix II (fig. 4). In P. mediterraneus, the NTS
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Rebordinos/Cross/Merlo
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that showed high homology with the NTS of S. aurata,
presented in 1 case (NTS δ) an anomalous structure in
the helix III, but the stability was high [Merlo et al.,
2012a].
Conclusions
Given the apparent homogeneity observed among var-
ious copies, 5S rRNA genes have been used as a showcase
of the archetypal example of a gene family subject to con-
certed evolution. In several studies, however, intensive
dynamics has been observed in this gene, leading to the
emergence of pseudogenes, inclusion of different types of
5S rDNA and linking to other multigenes. Recent studies
have shown the presence of elements such as tRNA-de-
rived SINEs, LTRs and microsatellites, among others, in
the NTSs of fishes, revealing a putative main role in the
dynamics of the 5S rDNA of fishes. Interestingly, events
of HT involving 5S rRNA genes have recently been re-
ported in 2 species of fish. The contribution of all these
elements to the maintenance and evolution of 5S rDNA
has been discussed in the review.
Acknowledgements
This work was supported by grants from the Junta de Andalu-
cia (group BIO-219) and from the Ministerio de Ciencia e Innova-
cion of Spain – MICINN (AGL2011-25596).

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