DNA Barcodes 2015; 3: 129–138
Research Article
Open Access
Camila da Silva de Souza, Claudio Oliveira, Luiz Henrique Garcia Pereira*
Knodus moenkhausii (Characiformes: Characidae):
one fish species, three hydrographic basinsa natural or anthropogenic phenomenon?
DOI 10.1515/dna-2015-0016
Received February 21, 2015; accepted July 6, 2015
Abstract: We used the DNA barcoding technique (COI
and CytB markers) combined with GMYC analysis to
characterize the genetics of the widely distributed
Neotropical fish species Knodus moenkhausii from three
different isolated hydrographic basins. Despite the fact
that most of the Neotropical hydrographic basins have
been isolated for millions of years, species could be shared
between basins due to natural events (stream capture) or
anthropogenic activities. Recent surveys, however, have
shown that many widely distributed species are actually
species complexes divided into previously unrecognized
cryptic species. In this study, we tested the hypothesis that
K. moenkhausii from three hydrographic basins represent
a single panmictic species and discuss the most likely
explanation of its present geographical distribution.
The GMYC analysis revealed that all specimens of K.
moenkhausii represent a single species: the intra- and
intergroup minimum K2P genetic distances for both
genes were zero and haplotypes were shared among the
three hydrographic basins. This suggests there has been
recent interchange of K. moenkhausii throughout the three
hydrographic basins. It is likely that this is due to recent
human activities, either the transposition of natural
barriers or intentional introduction or accidental escape
due to the ornamental fish trade.
Keywords: Neotropical, COI, CytB, Upper Paraná river,
São Francisco river, Paraíba do Sul river, Human activities,
species introduction, GMYC, ornamental fishes.
*Corresponding author: Luiz Henrique Garcia Pereira, Centro de
Ciências da Vida e da Natureza, Universidade Federal da Integração
Latino-Americana – UNILA, Foz do Iguaçu, Paraná, Brazil, 85866-000,
E-mail: [email protected]
Camila da Silva de Souza, Claudio Oliveira, Departamento de Morfologia, Universidade Estadual Paulista – UNESP, Botucatu, São Paulo,
Brazil, 18618-970
1 Introduction
The Neotropical region comprises 78 freshwater
ecoregions (hydrographic basins), of which 28 occur
in Brazil, including some of the major river basins of
the world, such as the Amazonas, Paraná and Uruguay
Rivers [1]. This region contains the most diversified
freshwater ichthyofauna of the world, with about 6,000
species to date [2]. The geographical distribution of this
fauna is highly complex, with some species occurring in
restricted areas, sometimes only known from the type
locality (e.g. Trichomycterus maracaya, Characidium
xanthopterum), while others have broad distributions and
occur even in multiple hydrographic basins (e.g. Hoplias
malabaricus, Astyanax paranae, and Knodus moenkhausii)
[2,3]. The latter phenomenon is especially interesting,
since most hydrographic basins have been isolated for
millions of years [4,5]. Nevertheless, shared species
among Neotropical hydrographic basins are a frequent
occurrence, such as between Paraíba do Sul and the Upper
Paraná River basins with 21 shared species and between
the Upper Paraná and São Francisco River basins with 47
shared species [6,7,8]. The occurrence of shared species
among hydrographic basins could be attributed either to
natural phenomena such as headwater stream capture
or to anthropogenic activities such as the accidental or
intentional introduction of nonnative species.
Headwater stream capture (also called stream capture
or stream piracy) occurs when the whole or a part of a
stream/river is shifted to the drainage of a neighboring
basin due to geomorphological processes, which then
allows the dispersal of species to the new basin [5,9].
Headwater stream capture has been used to explain the
occurrence of many shared species between neighboring
basins, such as in the case of Mimagoniates microleps
(Characidae) between the Iguaçu River and the Coastal
Eastern basins [10] and Cnesterodon brevirostratus
(Poeciliidae) between the Uruguai River and the South
Coastal drainages [4]. On the other hand, the introduction
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130 C. da Silva de Souza, et al.
of species by anthropogenic activities has become an
increasingly common event [11,12,13]. The number of
species found outside of their native region has doubled
in the last three decades [14]. In Brazil, the number of
introduced species is uncertain, due mostly to the lack of
surveys and because many regions of Brazilian watersheds
are poorly explored [3,6,15]. Nevertheless, the few surveys
show an extensive number of introduced species (either
exotic or allochthonous), such as in the Alto Paraná and
Paraíba do Sul River basins which contain 74 (23.9%) and
64 (49%) introduced species respectively [6,16,17].
An alternative explanation for apparently shared
species between basins is that these species represent
unrecognized cryptic species, which would only be
revealed by molecular studies. Indeed, some recent
studies have shown that many widely distributed species
are actually cases of cryptic species complexes which had
gone unrecognized [18-23].
In this study, we assess the genetic status of populations
of the freshwater fish species Knodus moenkhausii which
occurs in three isolated hydrographic basins (of the Upper
Paraná, São Francisco, and Paraíba do Sul Rivers). Knodus
moenkhausii (Eigenmann & Kennedy, 1903) is a small fish
(~5 cm) belonging to the family Characidae. This species
mainly inhabits small streams with structurally simple
habitats of sandy bottom, mild currents, shallow water
(~45 cm), and without cover and marginal vegetation;
often K. moenkhausii is the most abundant species in this
habitat [24,25]. Identifying this species can be difficult
due to its similarity to other species of the genus and the
related genera Creagrutus, Piabina, and Bryconamericus.
The species was first described from the Paraguay River
basin (Eigenmann & Kennedy, 1903), and later reported
from the Upper Paraná [6,24-27] and São Francisco
River basins [28]. Recently, our research group collected
specimens of K. moenkhausii in the Paraíba do Sul River
basin as well. These findings raise the question whether
K. moenkhausii represents a single panmictic species or
distinct genetic populations native to each hydrographic
basin in which it occurs.
To resolve this question, we used DNA barcoding
combined with GMYC analysis (Generalized Mixed Yule
Coalescent) for delimiting species. DNA barcoding was
proposed specifically to characterize species using
a standard, short (~650 pb), and universal sequence
fragment of the Cytochrome c Oxidase subunit I (COI)
gene [29]. The COI sequence can be considered a genetic
“barcode”, which ideally would be unique to each
species, since some divergence would be expected to
develop in this region during the evolutionary history of
different taxa. At present, the barcoding database (BOLD)
contains about 10,800 species of fishes (www.fishbol.
org), and in some studies the successful identification
rate can exceed 90% [30,31]. The GYMC analysis [32] uses
maximum-likelihood statistics and a time-calibrated
gene tree to delimit species using sequences from a single
locus. The method combine a speciation model (Yule)
with a population model (coalescent) to delimit species
by characterizing the transition between intra-specific
(population) and interspecific (species) events [32]. In
general, this method is more robust and less subjective
than traditional barcoding analysis methods. In this study,
we analyzed specimens of K. moenkhausii from the Upper
Paraná, São Francisco and Paraíba do Sul River basins to
test the hypothesis that these specimens belong to a single
panmictic species. Furthermore, we discuss the likely
determinants of the current geographical distribution of
this species.
2 Material and Methods
We analyzed 36 K. moenkhausii specimens from three
hydrographic basins (Upper Paraná, São Francisco and
Paraíba do Sul Rivers) (Fig. 1), available in the tissue
collection of the Laboratório de Biologia e Genética de
Peixes (LBP), São Paulo, Brazil (Supplementary table 1). A
single specimen each of Knodus borki, Knodus chapadae,
Knodus victoriae, and Bryconamericus sp., were used for
comparison (Supplementary table 1).
2.1 Extraction, PCR and Sequencing
Total genomic DNA was isolated from fin or muscle
tissue of each specimen using the DNeasy Blood and
Tissue Kit (Qiagen – California – USA) according to the
manufacturer’s instructions. The partial mitochondrial
COI gene, was amplified by the PCR using the primers:
C_FishF1t1/C_FishR1t1, 5’GTA AAA CGA CGG CCA GTC AAC
CAA CCA CAA AGA CAT TGG CAC-3’, 5’-TGT AAA ACG ACG
GCC AGT CGA CTA ATC ATA AAG ATA TCG GCA C-3’, 5’-CAG
GAA ACA GCT ATG ACA CTT CAG GGT GAC CGA AGA ATC
AGA A-3’, 5’-CAG GAA ACA GCT ATG ACA CCT CAG GGT
GTC CGA ARA AYC ARA A-3’ [33]. We also amplified the
mitochondrial gene Cytochrome B (CytB), amplified by PCR
using the primers LNF (5’-GAC TTGA AAA ACC AYC GTT GT)
and H08R2 (5’-GCT TTG GGA GTT AGD GGT GGG AGT TAG
AAT C) [34]. PCR was carried out on a thermocycler Veriti
96-well Fast (ABI-Applied Biosystems – California - USA),
with a final volume of 10.0 µl containing 5.0 µl Buffer 2X, 3.3
µl ultrapure water, 1.0 µl each primer (10µM ), 0.2 µl enzyme
Phire® Hot Start II DNA polymerase (Life Technologies –
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One fish species, three hydrographic basins: natural or anthropogenic? 131
Figure 1: South America Hydrographic Map: Hydrographic map showing the three hydrographic basins with K. moenkhausii sampled. Blue
= Upper Paraná River basin; green = São Francisco River basin; red = Paraíba do Sul River basin. Red dots = K. moenkhausii sample sites;
yellow dots = K. chapadae sample sites; green dots = K. victoriae sample sites; grey squares = Bryconamericus sp. sample sites; dark line =
Itaipu Dam.
California – USA) (5U) and 0.5 µl of DNA template (~50 ng).
The thermocycler conditions to amplify the COI gene were
initial denaturation at 98°C for 5 min followed by 30 cycles
denaturation at 98°C for 5 s, annealing at 56°C for 20 s and
extension at 72°C for 30 s, followed by a final extension
step at 72°C for 5 min. The thermocycler conditions to
amplify the CytB gene were initial denaturation at 98°C
for 5 min followed by 30 cycles of denaturation at 98° for
5 s, annealing at 50°C for 15 s and extension at 72°C for
45 s, followed by a final extension step at 72°C for 5 min.
Amplified products were checked on 1% agarose gels.
The PCR products were purified with ExoSap-IT® (USB
Corporation, Cleveland, OH, USA), and the purified PCR
product was used as template to sequencing both DNA
strands. Sequencing reactions were performed using
the “BigDye® Terminator v3.1 Cycle Sequencing Ready
Reaction” (Applied Biosystems – California - USA) and
sequencing was performed on the automatic sequencer ABI
3130 DNA Analyzer (Applied Biosystems – California - USA).
2.2 Data analysis
The sequences were edited using the software programs
ATGC (Genetyx Corporation – Tokio - Japan) and Bioedit
[35] to obtain the consensus sequences. For verification
of contaminants (exogenous DNA), the sequences
were submitted to the software BLAST available on the
NCBI site. The sequences were aligned by the editor
ClustalW [36] coupled with Dambe software [37]. The
genetic distances were calculated using the Kimura-2parameter (K2P) distance model [38] by the program
MEGA v.6.06 [39]. The K2P model was chosen due its
widespread application to calculate genetic distances
in barcoding surveys. To determine species boundaries,
we used GMYC analysis with single threshold as
described in Pons et al. 2006 [32]. For the analysis we
generated a uncalibrated ultrametric tree with the
Beast 1.5 program [40]. This tree was obtained with the
General Time Reversible (GTR) model and a relaxed
clock (uncorrelated lognormal). We used the speciation
birth-death process model. We started the search with
a UPGMA tree and then a Markov Chain Monte Carlo
(MCMC) model with 30 x106 generations. One tree was
sampled every 1000 generations. The tree with the
highest probability was selected in the TreeAnnotator
V1.5.3 [41] and used as an ultrametric tree for the GMYC
analysis. The GMYC analysis was carried out on the
GMYC web server available at http://species.h-its.org/
gmyc/.
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132 C. da Silva de Souza, et al.
3 Results
We obtained 36 COI gene sequences (~554 bp) and 24
CytB gene sequences (~850 bp) from our K. moenkhausii
samples as well as single COI and CytB sequences for the
congeneric species K. borki, K. chapadae, K. victoriae and
the related species Bryconamericus sp.. No sequences
showed insertions, deletions, or stop-codons, and the
analysis with the BLAST tool did not show contaminants.
The GMYC analysis suggest the existence of five ML
entities (independent coalescent group) for both genes
(confidence interval = 5-5 and 5-6 and, threshold time
= -0.00204447 and -0.003365575 for COI and CytB,
respectively), corresponding to Bryconamericus sp.,
K. borki, K. chapadae, K. victoriae and K. moenkhausii
(Fig. 2). All specimens of K. moenkhausii from the three
different hydrographic basins grouped in a single ML
cluster (Fig. 2). To calculate the genetic divergence values,
K. moenkhausii sequences were divided into three different
groups corresponding to each hydrographic basin (Upper
Paraná, São Francisco, and Paraíba do Sul Rivers) for
both genes. No genetic divergence was observed for the
COI sequences among and within K. moenkhausii groups,
except for the Upper Paraná group that showed a mean
intraspecific genetic divergence of 0.1% (Table 1). The COI
sequences of K. moenkhausii consist of four haplotypes,
with the most frequent haplotype (Hap1 – 86.1%) shared
across the three hydrographic basins. The three remaining
haplotypes (Hap2, Hap3 and Hap4) were exclusive to the
Upper Paraná River basin (Table 2). The genetic distance
between K. moenkhausii and the three other congeneric
species ranged from 8.6% to 11.0%, and from 14.8% to
Table 1: K2P genetic divergences for COI (below diagonal) and CytB (above diagonal) between K. moenkhausii groups and related species.
Red = K2P genetic divergences within groups; Blue = among three K. moenkhausii groups (groups = separate hydrographic basin samples).
1
2
3
4
5
6
7
1 - Upper Paraná river basin
0.1/0.1
0.1
0.1
9.3
9.5
10.6
13.8
2 - Paraíba do Sul river basin
0
0/-
0
9.7
9.7
11.0
14.0
3 - São Francisco river basin
0
0
0/0.1
9.7
9.8
11.0
14.0
4 - Knodus victoriae
8.7
8.6
8.6
-/-
10.2
9.4
15.0
5 - Knodus chapadae
8.9
8.7
8.7
7.9
-/-
10.1
15.6
6 - Knodus borki
10.9
11.0
11.0
11.5
10.0
-/-
15.2
7- Bryconamericus sp.
15.1
14.8
14.8
15.6
12.6
11.7
-/-
Table 2: Haplotypes of COI and CytB genes for K. moenkhausii. UP = Upper Paraná River basin; SF = São Francisco River basin; PS = Paraíba
do Sul River basin.
Nucleotide position
Absolute number
Frequency
Occurrence
123
351
477
COI
haplotypes
C T G
31
86.1%
UP, SF, PS
Hap2
C C G
3
8.3%
UP
Hap3
C C A
1
2.8%
UP
Hap4
A T G
1
2.8%
UP
CytB haplotypes
174
230
236
537
602
803
Hap1
Hap1
Hap2
G C T T A T
20
83.3%
UP, SF, PS
G T C C G T
2
8.3%
UP
Hap3
G C T T A C
1
4.2%
UP
Hap4
A C T T A T
1
4.2%
SF
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Figure 2: Generalized Mixed Yule Coalescent species delimitation tree showing the five independent coalescence groups (ML entities) found for both genes (COI and CytB). Blue = Upper Paraná
River basin, green = São Francisco River basin, red = Paraíba do Sul River basin.
One fish species, three hydrographic basins: natural or anthropogenic? 133
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134 C. da Silva de Souza, et al.
15.1% between K. moenkhausii and Bryconamericus sp.
(Table 1). For the CytB sequence, the genetic distance
ranged from 0 to 0.1% and 0 to 0.1% among and within the
three groups, respectively (Table 1). The CytB sequences of
K. moenkhausii comprise four haplotypes, with the most
frequent haplotype (Hap1 – 83.3%) shared across the three
hydrographic basins. The Hap2 and Hap3 haplotypes were
exclusive to the Upper Paraná River basin and Hap4 was
exclusive to the São Francisco River basin (Table 2). The
genetic distance in CytB between K. moenkhausii and its
three congeneric species ranged from 9.3% to 11.0% and
from 13.8% to 14.0% from Bryconamericus sp (Table 1).
4 Discussion
The results obtained in the present study indicate that
the K. moenkhausii specimens from the Upper Paraná,
São Francisco and Paraíba do Sul River basins represent a
panmictic genetic unit, likely representing a single species.
This finding is inconsistent with many studies that suggest a
pattern of limited dispersal in small Neotropical freshwater
fishes [42,43]. This limited dispersal ability should restrict
the geographical distribution of these fishes facilitating
the subdivision of populations and leading to speciation
by geographic isolation (allopatry) [43]. Additionally,
several surveys have shown that many species with wide
geographic distribution actually represent complexes
of distinct cryptic species. For example, a recent review
of Pseudoplatystoma using morphological characters
showed that the widely distributed species P. fasciatum
actually represents a complex of five species, one in
each hydrographic basin that they occupy [44]. Another
survey of different populations of Gymnotus pantherinus
from southeast Brazil showed that each population
was genetically distinct with few shared and many
exclusive haplotypes [45]. The authors suggested that
the development of geographic barriers resulting from
the formation of the Serra do Mar, resulted in a pattern of
allopatric speciation [45]. Two surveys analyzing Piabina
argentea from the Upper Paraná and São Francisco River
basins with molecular and cytogenetic tools showed that
the nominal species comprises a species complex with six
distinct putative species [18,19]. Interestingly, this species
shows a similar geographic distribution to K. moenkhausii
and is a similar small Neotropical fish [46].
In contrast to the above cases, we found that K.
moenkhausii presents a very different genetic profile. The
GMYC analyses conducted with both mitochondrial genes
(COI and CytB) showed that all samples of K. moenkhausii
form a single genetic unit. There was no genetic divergence
among the three hydrographic basins and the haplotypes
of both genes were shared among the three hydrographic
basins revealing a complete lack of population structure.
The genetic distance from congeners ranged from
8.6% to 11.0 and 9.3% to 11.0% for COI and CytB genes,
respectively: values consistent with those found in other
DNA barcoding surveys that showed average genetic
distances between congeners ranging from 7% to 11% in
more than 90% of comparisons [28,30,31,47,48]. Similarly,
these other studies report genetic divergences within
species of about 0.5%, a similar value to that found in
the present survey. Ward [30] analyzed the COI sequences
of 1088 fish species available in BOLD , and found a
minimum interspecific distance of 2% to be a useful
threshold to delimiting species, usually with an order of
magnitude lower variation within species. These findings
generally support the hypothesis that K. moenkhausii
represents a single panmictic species.
4.1 Geographical distribution of
K. moenkhausii
Headwater stream capture could explain the occurrence of
K. moenkhausii in the three different hydrographic basins
analyzed. However, the absence of genetic divergence
among the three groups for both genes and broadly
shared haplotypes indicates completely unstructured
populations. Considering that the three hydrographic
basins have been isolated for millions of years [4,5],
one would expect, at least, some degree of population
structure, considering that K. moenkahusii has a relatively
limited dispersal ability [42,43]. Based on this, we believe
headwater stream capture may be insufficient to explain
the genetic profile we found in the current geographical
distribution of K. moenkhausii.
On the other hand, the introduction of species by
human activities may better explain the pattern we
found. The numerous shared haplotypes among the
three hydrographic basins suggest a recent event. The
main anthropogenic pathways introducing nonnative
fish species across hydrographic basin boundaries
include the transposition of natural barriers through the
construction of canals, allowing communication of water
between isolated rivers or the inundation of areas due the
construction of hydroelectric power dams. In addition,
aquaculture includes the intentional introduction of fishes
using exotic or allochthonous species (stocking strategy),
fishing techniques using live bait introduce nonnative
fishes, and the trade in ornamental fishes includes
transferring species between locations [11,12,49]. In fact,
the number of recognized introduced species (either
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One fish species, three hydrographic basins: natural or anthropogenic? exotic and allochthonous) to the Upper Paraná, Paraíba
do Sul, and São Francisco River basins are extensive, with
74 (23.9%), 64 (49%), and 24 (9.8%) introduced species,
respectively [6,16,50].
Additonal evidence that anthropogenic activities
are responsible for the present distribution pattern of K.
moenkhausii is provided by the species list of Upper Paraná
River basin [6]. K. moenkhausii was described from the
Paraguay River, in the drainage of the Lower Paraná River
basin (Eigenmann and Kennedy 1903). K. moenkhausii
was recorded in the Upper Paraná River basin only after
the construction of the Itaipu hydroelectric power dam in
1982 [6]. The Itaipu Reservoir inundated an area of about
1350 km2 [51] that included the Sete Quedas Falls, a natural
and effective barrier that was the limit of the hydrographic
basins of the Lower and Upper Paraná Rivers [6,52]. The
inundation of Sete Quedas falls moved the barrier between
these two hydrographic basins to 150 Km below the Itaipu
Dam, allowing the mixing of species from the two basins.
At present, at least 33 species initially limited to the Lower
Paraná River basin have successfully colonized the Upper
Paraná River basin, including K. moenkhausii [52].
The lists of introduced species in the Paraíba do
Sul and São Francisco River basins do not include K.
moenkhausii [16,50]. The occurrence of K. moenhkausii
in the São Francisco river basin was first documented in
2011 by Carvalho [28] and its occurrence in the Paraíba
do Sul River is first reported here. We propose that this
recent spread is due to construction of hydroelectric
power dams or canals as occurred in the Upper Paraná
River basin. These three watersheds are located in the
most urbanized and exploited area of Brazil. Brazil has
1175 hydroelectric plants in operation (476 small, 498
medium and 201 large), most of them (45%) located
along these three hydrographic basins [53]. There are
also more 49 hydroelectric plants under construction and
173 planned [53]. It is likely that flooding behind dams
has connected isolated streams or rivers from different
hydrographic basins, such as in the aforementioned
case of the Itaipu Dam. A similar well-recorded case
occurred when the Piumhi River (with its 22 tributaries)
which belonged to the Upper Paraná River basin was
entirely transferred to São Francisco River basin after
construction of the Furnas hydroelectric power dam
[54]. This transposition introduced at list nine species
from Upper Paraná River basin to São Francisco River
basin [55,56,57,58,59]. However, in general, the impact
of transpositions of rivers due to hydroelectric power
dams are rarely recorded. The Paraíba do Sul River, the
most anthropogenically impacted river of Brazil [8], is a
well-documented case of the transposition of water by
135
construction of canals [54,60], however, there are no
studies about its impact on the fauna of the river basins.
An alternative or additional explanation is the
artificial spread of K. moenkhausii by the ornamental
fish trade. The ornamental fish trade was valued at about
US$330 million in 2009, selling approximately 2 billion
specimens of almost 2,000 species [61,62]. It is likely that
there are extensive intentional introductions or accidental
escapes of fishes into non-native basins [64]. It is a
plausible explanation for K. moenkhausii which is traded
as an ornamental fish due its small size and attractive
coloration in aquariums, although it is traded in relatively
low numbers (data from Ministry of Development,
Industry and Foreign Trade of Brazil – MDIC - 2012). The
ornamental fish trade has been reported as the cause of
many species introductions, for example in the Upper
Paraná River basin where eleven fishes species were
introduced [6,65] and the São Francisco river basin, where
seven species were introduced [7]. The Paraíba do Sul
River basin is documented to have 56 introduced species
[66]. This high level reflects the fact that it is the major
ornamental fish-farming center of Brazil with about 350
farms with many documented intentional and accidental
escapes of fishes to neighboring streams and rivers
[66,67]. Since K. moenkhausii is traded as an ornamental
fish, it is likely that K. moenkhausii species has also been
introduced into the São Francisco and Paraíba do Sul
Rivers basins in this way, especially since the species is
highly opportunistic, facilitating its adaptation to new
habitats [25] and accounting for its rapid spread into
different hydrographic basins.
5 Conclusions
The analysis of mitochondrial COI and CytB sequences
showed that K. moenkhausii represents a single
panmictic species occupying the three hydrographic
basins we studied (Upper Paraná, Paraíba do Sul, and
São Francisco Rivers). The results are most consistent
with the hypothesis that the presence of K. moenkhausii
in the three hydrographic basins is due its introduction
by human activities, probably due to either or both
the transposition of natural barriers to dispersal or to
intentional or accidental escapes caused by the trade
in ornamental fishes. Our results confirm the efficacy
and utility of the combination of DNA barcoding and
GYMC analysis for helping to delimit and confirm
species status and exploring the determinants of the
geographic distribution of Neotropical freshwater
fishes.
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136 C. da Silva de Souza, et al.
Acknowledgements: The research was supported by
the Brazilian agencies FAPESP (Fundação de Amparo à
Pesquisa do Estado de São Paulo) and CNPq (Conselho
Nacional de Desenvolvimento Científico e Tecnológico).
We also thank Katiane M. Ferreira and Mahmoud N.
Mehanna for help in species identification.
Conflict of interest: The authors declare no conflict of
interest.
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Supplemental Material: The online version of this article
(DOI: 10.1515/dna-2015-0016) offers supplementary material.
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