DNA barcoding reveals hidden diversity in the Neotropical freshwater fish Piabina argentea (Characiformes: Characidae) from the Upper Paraná Basin of Brazil

Article (PDF Available)inMitochondrial DNA 22 Suppl 1(S1):87-96 · June 2011with122 Reads
DOI: 10.3109/19401736.2011.588213 · Source: PubMed
Abstract
We analyzed a small and wide geographically distributed Neotropical freshwater fish, the Piabina argentea from the Upper Paraná Basin, to check the hypothesis that this species is composed of more than one biological unit, since it has a limited dispersion, through the DNA barcode technique. Partial mitochondrial COI and CytB gene sequences were obtained for 58 specimens drawn from 13 localities. Phylogenetic analysis revealed six major clusters of P. argentea. Kimura-two-parameter (K2P) genetic divergences among these six P. argentea clusters ranged from 2 to 5.6% and from 2.3 to 5.4% for COI and CytB genes, respectively, and these values were on average approximately nine times greater than intra-cluster K2P divergences. The fixation index (F(ST)) among clusters showed very high values and the haplotype network analysis displayed seven unconnected units. These results reinforce the hypothesis that the widely distributed P. argentea species concept as currently conceived actually represents more than one species (possibly six). These results demonstrate the efficacy of DNA barcoding for the discovery of hidden diversity in Neotropical freshwater fishes, and we conclude that barcoding is a useful tool for alpha taxonomy.
RESEARCH PAPER
DNA barcoding reveals hidden diversity in the Neotropical freshwater
fish Piabina argentea (Characiformes: Characidae) from the Upper
Parana
´
Basin of Brazil
LUIZ H. G. PEREIRA
1
, MARLON F. PAZIAN
1
, ROBERT HANNER
2
, FAUSTO FORESTI
1
,
& CLAUDIO OLIVEIRA
1
1
Laborato
´
rio de Biologia e Gene
´
tica de Peixes, Instituto de Biocie
ˆ
ncias, Universidade Estadual Paulista (UNESP), Botucatu,
Sa˜o Paulo, Brazil, and
2
Department of Integrative Biology, Biodiversity Institute of Ontario, University of Guelph, Guelph,
Ont., Canada
(Received 2 September 2010; revised 29 October 2010; accepted 28 March 2011)
Abstract
Background and aims. We analyzed a small and wide geographically distributed Neotropical freshwater fish, the Piabina
argentea from the Upper Parana
´
Basin, to check the hypothesis that this species is composed of more than one biological unit,
since it has a limited dispersion, through the DNA barcode technique. Materials and methods. Partial mitochondrial COI and
CytB gene sequences were obtained for 58 specimens drawn from 13 localities. Results. Phylogenetic analysis revealed six
major clusters of P. argentea. Kimura-two-parameter (K2P) genetic divergences among these six P. argentea clusters ranged
from 2 to 5.6% and from 2.3 to 5.4% for COI and CytB genes, respectively, and these values were on average approximately
nine times greater than intra-cluster K2P divergences. The fixation index (F
ST
) among clusters showed very high values and
the haplotype network analysis displayed seven unconnected units. Conclusion. These results reinforce the hypothesis that the
widely distributed P. argentea species concept as currently conceived actually represents more than one species (possibly six).
These results demonstrate the efficacy of DNA barcoding for the discovery of hidden diversity in Neotropical freshwater
fishes, and we conclude that barcoding is a useful tool for alpha taxonomy.
Keywords: DNA barcode, Neotropical region, freshwater fishes, COI, CytB, mitochondrial DNA
Introduction
The Neotropical freshwater fish fauna is one of the
richest in the world (Schaefer 1998), with about 6000
species recognized in this region, out of which 4475
are actually considered valid and 1550 are recognized
but not yet described (Reis et al. 2003). In Brazil,
there are about 2587 valid species and many others to
be described (Buckup et al. 2007), but, even so, the
sampling of species is insufficient and many regions
remain almost unexplored (Langeani et al. 2006;
Junk 2007). Schaefer (1998) estimates that there may
be as many as 8000 species in the Neotropical region.
For example, in a recent study of the fish fauna from
the Upper Parana
´
Basin, the best studied region in
Neotropical area, Langeani et al. (2006) made an
inventory, which revealed that about 15% (, 50)
represent new species. Many other works pointed that
the number of fish species tends to increase mainly
among those fish belonging to small-sized groups and
that inhabit headwaters streams (Schaefer 1998; Vari
and Malabarba 1998; Castro et al. 2003, 2004, 2005;
Langeani et al. 2006). Additionally, the geographic
distribution pattern of the Neotropical fish species
is very complex, with some species having a very
restricted distribution (e.g. Trichomycterus maracaya,
Characidium xanthopterum) occurring mostly in
ISSN 1940-1736 print/ISSN 1940-1744 online q 2011 Informa UK, Ltd.
DOI: 10.3109/19401736.2011.588213
Correspondence: L. H. G. Pereira, Departamento de Morfologia, Instituto de Biocie
ˆ
ncias, UNESP, Distrito de Rubia
˜
o Junior S/N, Rubia
˜
o
Ju
´
nior, CEP 18.618-000, Botucatu, SP, Brazil. Tel/Fax: þ 55 14 3811 6264. E-mail: luizhgp@ibb.unesp.br
Mitochondrial DNA, 2011; Early Online: 1–10
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headwaters, and others having a wide distribution
(e.g. Hoplias malabaricus, Astyanax paranae) some-
times occurring in more than one hydrographic basin
(Reis et al. 2003; Junk 2007).
The genus Piabina, composed by small fishes
(, 50 mm), belongs to the family Characidae but its
relationship with the remaining characids is uncertain
(Lima et al. 2003). Two species are assigned to
Piabina: Piabina argentea Reinhardt, 1867 and Piabina
anhembi da Silva and Kaefer, 2003. P. argentea has a
wide geographic distribution occurring in the Upper
Parana
´
Basin (the same region of this work); in the Sa
˜
o
Francisco Basin (type-locality); and in the Itapirucu,
Paraı
´
ba do Sul, and Itapemirim rivers (eastern
Brazilian basins) (Vari and Harold 2001). P. anhembi
is restricted to its type-locality (Upper Tiete
ˆ
River,
Saleso
´
polis, Sa
˜
o Paulo, Brazil) (da Silva and Kaefer
2003). These two species differ from each other by
the teeth position, head size, and mouth proportions
(da Silva and Kaefer 2003). Piabina differs from its
putative sister group, Creagrutus, only by two subtle
characters: the fourth infraorbital bone morphology
and the teeth position (Vari and Harold 2001).
Creagrutus and Piabina were allopatric (Vari and
Harold 2001) until the discovery of a new Creagrutus
species in the Upper Parana
´
River Basin (Ribeiro et al.
2004). The Piabina species populations have a limited
dispersion, usually living in a restricted hydrographic
region (Lowe-Mcconnell 1999). Castro (1999)
suggests a limited dispersion to small fishes, which
restricts their geographical distribution and may
facilitate the population geographical subdivision
enabling the possible creation of new species by
geographic isolation (allopatry).
The advance of molecular techniques has proven
a useful tool in biodiversity studies, mainly in those
cases where the traditional tools are insufficient
or unable to identify species. The use of genetic
techniques has revealed that some species are actually
species complexes (Agostinho et al. 2007). Bickford
et al. (2006) showed that there has been an increased
recognition of cryptic species from different groups
of animals and plants in the past two decades due to
the use of molecular methods. Hebert et al. (2003)
proposed the DNA barcoding technique as a useful
molecular tool for the identification of species, and
many published works have shown the efficacy of this
methodology for the identification of several organ-
isms (Hebert et al. 2004a; Ward et al. 2005; Clare et al.
2007; Kelly et al. 2007; Hubert et al. 2008; Valdez-
Moreno et al. 2009). Hebert et al. (2004b) proposed a
threshold to delimit species that are 10 £ larger than
the intraspecific average values. New species have
been proposed with DNA barcoding data and some
of these species have been formally described later
(Smith et al. 2005; Witt et al. 2006; Ward 2007;
Nguyen and Seifert 2008; Ward et al. 2008; Yassin
et al. 2008).
Considering the wide distribution and limited
dispersion of small fish P. argentea and the promising
use of DNA barcodes for flagging new species, the
present work assessed samples of P. argentea from the
Upper Parana
´
and Sa
˜
o Francisco basins to check
the hypotheses that this species could represent more
than one biological unit.
Materials and methods
Specimen collection
Fifty-three P. argentea specimens from 12 sites located
in the Upper Parana
´
Basin and one in the Sa
˜
o
Francisco Basin and five P. anhembi specimens from the
Upper Parana
´
River Basin were collected (Table I and
Figure 1). The Velhas River in the Sa
˜
o Francisco Basin
was sampled because this is the type locality of
P. argentea. Additionally, two Creagrutus specimens
(Creagrutus meridionalis and Creagrutus paraguayensis)
from the Paraguay River Basin were used as
outgroup (Table I). All specimens had a fresh fragment
tissue removed and preserved in absolute ethanol
at 2 208C. Voucher specimens were deposited in the
collection of Laborato
´
rio de Biologia e Gene
´
tica
de Peixes, Departamento de Morfologia, Instituto de
Biocie
ˆ
ncias, UNESP, Botucatu, Sa
˜
o Paulo, Brazil. The
specimens’ provenance data were deposited in BOLD
Project EFUPR (Ratnasingham and Hebert 2007).
Extraction, PCR, and sequencing
Total genomic DNA was isolated from fin or muscle
tissue of each specimen using the DNeasy Tissue Kit
(Qiagen, Hilden, Germany) according to the manu-
facturer’s instructions. The partial mitochondrial
cytochrome c oxidase subunit I gene (COI, 648 bp)
was amplified by the PCR using two sets of primers:
FishF1, 5
0
-TCAACCAACCACAAAGACATTG-
GCAC-3
0
; FishF2, 5
0
-TCGACTAATCATAAAGAT-
ATCGGCAC-3
0
; FishR1, 5
0
-TAGACTTCTGGGT-
GGCCAAAGAATCA-3
0
; and FishR2, 5
0
-ACTTCA-
GGGTGACCGAAGAATCAGAA-3
0
(Ward et al.
2005). The whole cytochrome b (CytB, 1118 bp)
mitochondrial gene was amplified by PCR using the
CytB-F, 5
0
-GACTTGAAAAACCAYCGTTGT-3
0
,
and CytB-R, 5
0
-GCTTTGGGAGTTAGDGGTGG-
GAGTTAGAATC-3
0
(C. Oliveira, pers. comm.).
PCR was carried out on a thermocycler (Veriti
w
96-
Well Thermal Cycler; Applied Biosystems, Foster
City, California, USA) with a final volume of 12.5 ml
containing 0.3 ml dNTP (2 mM), 1.25 ml10£ Ta q
buffer (50 mM KCl, 10 mM TrisHCl, 0.1% Triton
X-100, and 1.5 mM MgCl
2
), 0.3 ml each primer
(10 mM), 0.7 ml MgCl
2
(50 mM), 0.05 ml Taq-Pht
DNA polymerase (5 U), 1 ml template DNA (10
20 ng), and ultrapure water. The thermocycler
conditions to amplify the COI were initial denatura-
tion at 958C for 5 min followed by 30 cycles of
L. H. G. Pereira et al.2
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Table I. Specimen data.
GPS GenBank accession number
Cluster Species name Voucher number Collection code Locality state Latitude Longitude BOLD process ID COI CytB
A P. argentea 36233 LBP 7329 Parana
´
2 25.092 2 52.495 FUPR120-09 HM144073 GU908189
A P. argentea 34659 LBP 7111 Parana
´
2 23.938 2 50.729 FUPR181-09 HM144065 GU908182
A P. argentea 36235 LBP 7329 Parana
´
2 25.092 2 52.495 FUPR122-09 HM144071 GU908187
A P. argentea 36234 LBP 7329 Parana
´
2 25.092 2 52.495 FUPR121-09 HM144072 GU908188
A P. argentea 17229 LBP 2594 Sa
˜
o Paulo 2 21.013 2 49.690 FUPR089-09 HM144104 GU908219
A P. argentea 25091 LBP 6745 Sa
˜
o Paulo 2 22.341 2 48.935 FUPR102-09 HM144091 GU908207
A P. argentea 14154 LBP 1996 Sa
˜
o Paulo 2 22.917 2 48.500 FUPR062-09 HM144107 GU908222
A P. argentea 21305 LBP 3509 Sa
˜
o Paulo 2 22.941 2 48.584 FUPR091-09 HM144102 GU908217
A P. argentea 25050 LBP 6741 Sa
˜
o Paulo 2 23.231 2 48.533 FUPR101-09 HM144092 GU908208
A P. argentea 23459 LBP 6741 Sa
˜
o Paulo 2 23.024 2 48.826 FUPR092-09 HM144101 GU908216
A P. argentea 20764 LBP 6741 Sa
˜
o Paulo 2 23.024 2 48.826 FUPR090-09 HM144103 GU908218
A P. argentea 25410 LBP 6745 Sa
˜
o Paulo 2 22.341 2 48.935 FUPR108-09 HM144085 GU908201
A P. argentea 17228 LBP 2594 Sa
˜
o Paulo 2 21.013 2 49.690 FUPR088-09 HM144105 GU908220
A P. argentea 17227 LBP 2594 Sa
˜
o Paulo 2 21.013 2 49.690 FUPR087-09 HM144106 GU908221
A P. argentea 29280 LBP 6268 Minas Gerais 2 21.285 2 46.493 FUPR201-09 HM144056 GU908174
A P. argentea 29282 LBP 6268 Minas Gerais 2 21.285 2 46.493 FUPR203-09 HM144054 GU908172
B P. argentea 25214 LBP 6743 Sa
˜
o Paulo 2 22.786 2 48.481 FUPR238-09 HM144052 GU908170
B P. argentea 23222 LBP 6743 Sa
˜
o Paulo 2 22.786 2 48.481 FUPR237-09 HM144053 GU908171
B P. argentea 25219 LBP 6743 Sa
˜
o Paulo 2 22.786 2 48.481 FUPR107-09 HM144086 GU908202
B P. argentea 22884 LBP 4032 Distrito Federal 2 15.115 2 47.046 FUPR189-09 HM144063 GU908181
B P. argentea 23220 LBP 6743 Sa
˜
o Paulo 2 22.786 2 48.481 FUPR109-09 HM144084 GU908200
B P. argentea 35870 LBP 7280 Goia
´
s 2 17.120 2 48.740 FUPR192-09 HM144060 GU908178
B P. argentea 35871 LBP 7280 Goia
´
s 2 17.120 2 48.740 FUPR193-09 HM144059 GU908177
B P. argentea 35849 LBP 7680 Goia
´
s 2 17.099 2 48.762 FUPR191-09 HM144061 GU908179
B P. argentea 35904 LBP 7292 Goia
´
s 2 17.801 2 48.372 FUPR115-09 HM144078 GU908194
B P. argentea 35906 LBP 7292 Goia
´
s 2 17.801 2 48.372 FUPR117-09 HM144076 GU908192
B P. argentea 35905 LBP 7292 Goia
´
s 2 17.801 2 48.372 FUPR116-09 HM144077 GU908193
B P. argentea 35848 LBP 7680 Goia
´
s 2 17.099 2 48.762 FUPR190-09 HM144062 GU908180
C P. argentea 31608 LBP 6513 Minas Gerais 2 19.385 2 43.659 FUPR113-09 HM144080 GU908196
C P. argentea 31605 LBP 6513 Minas Gerais 2 19.385 2 43.659 FUPR110-09 HM144083 GU908199
C P. argentea 31609 LBP 6513 Minas Gerais 2 19.385 2 43.659 FUPR114-09 HM144079 GU908195
C P. argentea 31606 LBP 6513 Minas Gerais 2 19.385 2 43.659 FUPR111-09 HM144082 GU908198
C P. argentea 31607 LBP 6513 Minas Gerais 2 19.385 2 43.659 FUPR112-09 HM144081 GU908197
D P. argentea 25162 LBP 6744 Sa
˜
o Paulo 2 23.231 2 48.533 FUPR103-09 HM144090 GU908206
D P. argentea 25156 LBP 6744 Sa
˜
o Paulo 2 23.231 2 48.533 FUPR097-09 HM144096 GU908211
D P. argentea 25030 LBP 6744 Sa
˜
o Paulo 2 23.231 2 48.533 FUPR093-09 HM144100 GU908215
D P. argentea 25031 LBP 6744 Sa
˜
o Paulo 2 23.231 2 48.533 FUPR094-09 HM144099 GU908214
D P. argentea 25167 LBP 6744 Sa
˜
o Paulo 2 23.231 2 48.533 FUPR106-09 HM144087 GU908203
D P. argentea 25165 LBP 6744 Sa
˜
o Paulo 2 23.231 2 48.533 FUPR104-09 HM144089 GU908205
D P. argentea 25166 LBP 6744 Sa
˜
o Paulo 2 23.231 2 48.533 FUPR105-09 HM144088 GU908204
D P. argentea 25161 LBP 6744 Sa
˜
o Paulo 2 23.231 2 48.533 FUPR099-09 HM144094 GU908209
D P. argentea 25157 LBP 6744 Sa
˜
o Paulo 2 23.231 2 48.533 FUPR098-09 HM144095 GU908210
D P. argentea 25155 LBP 6744 Sa
˜
o Paulo 2 23.231 2 48.533 FUPR096-09 HM144097 GU908212
Hidden diversity in Piabina argentea revealed by barcoding 3
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denaturation at 958C for 45 s, annealing at 558C for
30 s, and extension at 728 C for 60 s. A final extension
at 728C for 10 min was performed. The thermocycler
conditions to amplify the CytB were initial denatura-
tion at 958C for 5 min followed by two cycles of
denaturation at 958C for 30 s, annealing at 558C
for 45 s, and extension at 728C for 60 s; two cycles of
denaturation at 958C for 30 s, annealing at 508C
for 45 s, and extension at 728C for 60 s; two cycles of
denaturation at 958C for 30 s, annealing at 488C
for 45 s, and extension at 728C for 60 s; 25 cycles
of denaturation at 958C for 30 s, annealing at 508C for
45 s, and extension at 728C for 60 s; and a final
extension at 728C for 5 min. Amplified products
were checked on 1% agarose gels stained with Blue
Green Loading Dye I (LGC Biotecnologia, Cotia, Sa
˜
o
Paulo, Brazil). The PCR products were purified with
ExoSAP-IT
w
(USB Corporation, Cleveland, OH,
USA) following the manufacturer’s protocol. The
purified PCR product was used as template to
sequence both DNA strands. The cycle sequencing
reaction was carried out using a BigDyee Terminator
v3.1 Cycle Sequencing Ready Reaction kit (Applied
Biosystems) in a final volume of 7 ml containing 1.4 ml
template, 0.35 ml primer (10 mM), 1.05 ml buffer 5 £ ,
0.7 ml BigDye mix, and water. The cycle sequencing
conditions were initial denaturation at 968 C for 2 min
followed by 30 cycles of denaturation at 968C for 45 s,
annealing at 508C for 60 s, and extension at 608C for
4 min. The PCR sequencing products were purified
with ethylenediamine tetraacetic acid/sodium aceta-
te/alcohol following the protocol suggested in the
BigDyee Terminator v3.1 Cycle Sequencing kit’s
manual (Applied Biosystems). All samples were
sequenced on an ABI 3130 Genetic Analyzer (Applied
Biosystems) following the manufacturer’s instruc-
tions. All sequences were deposited in the GenBank
and in the Barcode of Life Data Systems (Project
EFUPR) (Table I).
Data analysis
All sequences were analyzed using SeqScape
w
software
v2.6 (Applied Biosystems) to obtain the consensus
sequences and check the occurrence of deletions,
insertions, and stop codons. The sequences were
aligned using the online version of MUSCLE (Edgar
2004). The genetic distance among and within
observed clusters was calculated using the Kimura-
two-parameter (K2P) distance model (Kimura 1980)
for both genes separately. A neighbor-joining (NJ) tree
of K2P distances using the combined COI and CytB
sequences was created to provide a graphic represen-
tation of the relationships among specimens and
clusters with the software MEGA 4.0 (Tamura et al.
2007). Bootstrap resampling (Felsenstein 1985) was
applied to assess the support for individual nodes using
1000 pseudo-replicates.
TABLE I continued
GPS GenBank accession number
Cluster Species name Voucher number Collection code Locality state Latitude Longitude BOLD process ID COI CytB
E P. argentea 29240 LBP 6226 Minas Gerais 2 21.321 2 46.511 FUPR200-09 HM144057 GU908175
E P. argentea 29239 LBP 6226 Minas Gerais 2 21.321 2 46.511 FUPR199-09 HM144058 GU908176
E P. argentea 29281 LBP 6268 Minas Gerais 2 21.285 2 46.493 FUPR202-09 HM144055 GU908173
F P. argentea 35934 LBP 7301 Goia
´
s 2 18.110 2 48.504 FUPR119-09 HM144074 GU908190
F P. argentea 35933 LBP 7301 Goia
´
s 2 18.110 2 48.504 FUPR118-09 HM144075 GU908191
F P. argentea 28396 LBP 5994 Minas Gerais 2 21.732 2 46.423 FUPR129-09 HM144067 GU908184
F P. argentea 28394 LBP 5994 Minas Gerais 2 21.732 2 46.423 FUPR127-09 HM144069 GU908185
F P. argentea 28393 LBP 5994 Minas Gerais 2 21.732 2 46.423 FUPR126-09 HM144070 GU908186
P. anhembi 23373 LBP 6742 Sa
˜
o Paulo 2 23.524 2 45.890 FUPR236-09 HM144047 GU908165
P. anhembi 23360 LBP 6742 Sa
˜
o Paulo 2 23.524 2 45.890 FUPR235-09 HM144048 GU908166
P. anhembi 20821 LBP 6742 Sa
˜
o Paulo 2 23.524 2 45.890 FUPR125-09 HM144049 GU908167
P. anhembi 23372 LBP 6742 Sa
˜
o Paulo 2 23.524 2 45.890 FUPR124-09 HM144050 GU908168
C. meridionalis 35631 LBP 7557 Mato Grosso do Sul 2 20.343 255.726 FUPR188-09 HM144042 GU908160
C. paraguayensis 35628 LBP 7557 Mato Grosso do Sul 2 20.343 2 55.726 FUPR185-09 HM144045 GU908163
LBP, Laborato
´
rio de Biologia e Gene
´
tica de Peixes.
L. H. G. Pereira et al.4
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Phylogenetic analyses using maximum parsimony
were performed using PAUP
*
version 4.0b10
(Swofford 2002) with heuristic searches, random
addition of sequences, and tree bisection and
reconnection algorithms. The ACCTRAN optimiz-
ation method was utilized. The parsimony trees were
constructed using a 1:1 transition transversion ratio.
Cluster robustness was assessed using 1000 bootstrap
pseudo-replicates (Felsenstein 1985) with the same
parameters cited above.
The seven major clusters obtained were considered
as different units for the fixation index (F
ST
) calcula-
tion using Arlequin 3.11 (Excoffier et al. 2005). A
statistical parsimony network was constructed using
TCS 1.21 (Clement et al. 2000), which employs the
method of Templeton et al. (1992) with a statistical
confidence interval of 90%. The analyses were carried
out in TCS using the “fix connection limit” option to
obtain the mutational steps necessary to connect the
seven observed haplotype networks. The ancestral
haplotype was also identified using TCS according to
the method of Castelloe and Templeton (1994).
Results
Sequence data
Sequence data for a 648 bp fragment of COI and
1118 bp of CytB were obtained for a total of 58
Piabina specimens (53 P. argentea and 5 P. anhembi).
We also obtained the COI and CytB sequences from
two specimens of Creagrutus (C. meridionalis and
C. paraguayensis) used as an outgroup. No sequences
showed insertions, deletions, or stop-codons, and the
global transition transversion ratio was 4.4. A total of
233 nucleotides (72 in COI and 161 in CytB) were
variable in the data set of Piabina specimens (, 13%—
outgroup not considered) and 209 of them were infor-
mative in the parsimony analyses. These variations
defined a total of 42 haplotypes (COI and CytB dis-
played 28 and 39 haplotypes, respectively). The two
methods of tree construction (NJ and maximum
parsimony) resulted in the same topology (except for
some internal taxa in the subclusters; data not shown),
which showed seven major clusters with high support
values (Figure 2). P. anhembi samples formed one
cluster and P. argentea samples were divided into
six clusters, one corresponding to the sample from the
Sa
˜
o Francisco River Basin (Cluster C) and five
clusters representing P. argentea samples from the
Upper Parana
´
Basin (Figure 2). These seven clusters
are divided into two major groups, one containing
Cluster A and a second group with the other clusters
(Figure 2). We use two different methodologies of tree
construction to check the robustness of the data.
The inter-cluster K2P genetic distance values
ranged from 2% (Clusters D £ E) to 5.6% (Clusters
A £ C) and from 2.3% (Clusters B £ C) to 5.4%
(Clusters A £ E) for COI and CytB, respectively
(Table II). The average intra-cluster K2P distance
ranged from 0 to 0.9% (average ¼ 0.36%) for COI
and from 0.1 to 1% (average ¼ 0.5%) for CytB
(Table II).
Cluster comparisons
The pairwise F
ST
index among the seven clusters
identified showed values from 0.77 to 0.98 for COI
and from 0.66 to 0.96 for CytB, all highly significant
( p , 0.001) (Table III).
The haplotype network based on Templeton’s
method (Templeton et al. 1992) with the combined
data set (COI/CytB) displayed seven unconnected
networks, one representing P. anhembi and the other
six representing P. argentea (Figure 3). This result is
consistent with the seven clusters identified through
the phylogenetic analysis (Figure 2). The number
of haplotypes present in each network range from 3
(Cluster E) to 11 (Cluster A) (Figure 3), and the
number of mutational steps necessary to connect the
independent P. argentea networks ranged from 45 to
110 (Figure 3, dashed lines). The haplotype network
was constructed for each separate gene to check
whether the same seven unconnected networks would
be obtained. Both genes displayed the same result,
with 14-34 (COI) and 25-71 (CytB) mutational steps
necessary to connect the independent networks
(networks not shown).
Discussion
The specimens of Piabina were divided into seven
clusters in the phylogenetic analysis, one cluster
representing P. anhembi and the six other representing
P. argentea (Figure 2). The data showed the absence of
genetic flow among local samples and permit one
to suggest that P. argentea represents six different
Figure 1. Map showing the distribution of samples of Piabina.
Letters correspond to P. argentea clusters. Black square represents
P. anhembi species.
Hidden diversity in Piabina argentea revealed by barcoding 5
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biological units (meaning a minimum of five new
species). These seven clusters were confirmed by
haplotype network (Figure 3) and are divided into two
major groups (Figure 2). The first group contains
the Cluster A and is the sister group of the second
group, composed by the remaining clusters, including
P. anhembi (Figure 2). The average inter-cluster K2P
distance values among P. argentea were about nine
times greater than the average intra-cluster values
found for the COI (from 5.6 to 15.6 £ ) and CytB
genes (from 4.6 to 10.8 £ ) (Table II) and the inter-
cluster values among the P. argentea units were similar
to the values between P. argentea clusters and their
congeners P. anhembi (average ¼ 3.0 and 3.8%
for COI and CytB, respectively), reinforcing the
hypothesis of the existence of more than one biological
Figure 2. NJ tree of COI/CytB showing the seven major clusters obtained among Piabina specimens (A F represent P. argentea). Node
values represent statistic support: upper values, NJ bootstrap (1000 pseudo-replicates); lower values, maximum parsimony bootstrap (1000
pseudo-replicates). Numbers on fishes represent voucher number and size of photographed specimens (left and right, respectively).
L. H. G. Pereira et al.6
Mitochondrial DNA Downloaded from informahealthcare.com by University of Guelph on 09/28/11
For personal use only.
unit for P. argentea (Table II). These results corro-
borate the hypothesis of limited dispersion for Piabina
species (Lowe-Mcconnell 1999) and other small
fishes (Castro 1999), which facilitates the population
geographical subdivision enabling the possible
creation of new species by geographic isolation
(allopatry).
Hebert et al. (2004b) suggested a threshold to
delimit species with DNA barcode data. These values
should be at least 10 £ the average intraspecific
values. The average intra-cluster values of the six
P. argentea clusters were 0.4% and 0.56% for COI and
CytB, respectively, and some inter-cluster divergences
within P. argentea are slightly below this limit (see
Table II). However, a recent review of “barcoded”
fishes (Ward 2009) noted that about 17% of the
genetic divergence values among congeneric species
were less than 3% divergent and that a further 3.7% of
congeners are less than 1% divergent. The author
suggests that if the unknown specimen is more than
2% divergent from the known specimen, it is very
likely that this is a different species with a probability
greater than 95%. Hence, the threshold limit proposed
by Hebert et al. (2004b) as an indicator of cryptic
speciation should be carefully analyzed for each group.
Ward et al. (2007), working with sharks of the genus
Squalus, observed the formation of two clusters in the
species Squalus acanthias, which showed a genetic
divergence of just 0.76% between them. Interestingly,
these two groups had been considered as two species
until the decade of 1960: S. acanthias from the
Atlantic and South Pacific Oceans and Squalus
suckkeyi from the North Pacific Ocean (see Jensen
1966). The authors suggested the revalidation of the
second species. The comparison with values among
congener species may be useful for the delimitation
of a threshold. Ornelas-Garcia et al. (2008), working
with species of the genus Astyanax from Mesoamerica,
found that some specimens formed separate clusters
and suggested the occurrence of a species complex in
this genus, assigning provisional names to each cluster
obtained. Ward et al. (2008), working with Asian sea
bass Lates calcarifer specimens from different localities
(Australia and Myanmar), found genetic distance
values of 9.5% between two groups for COI (DNA
barcode region) and 11.3% for CytB. The authors
suggested the existence of two species. The average
divergence value of “barcoded” congeneric fishes is
about 8.4% (Ward 2009). Values smaller than this
average, such as those observed in the present work
and in the above-cited papers, can be explained in two
ways: the rate of evolution can vary among different
higher taxa and, consequently, the accumulation of
substitutions can vary. In fact, it has been observed
that different teleost orders have different evolutionary
rates (Krieger and Fuerst 2002). Another possible
explanation could relate to species ages, where
evolutionarily “young” species may not have had
sufficient time to accumulate many mutations in their
barcodes. In fact, Montoya-Burgos (2003), working
with species of Hypostomus from South America,
suggested that the process of divergence and radiation
in this genus dates back to sometime between 12 and
4 million years ago. Hubert et al. (2007), working with
Serrasalmus and Pygocentrus from South America,
encountered similar values suggesting that species
separation dates back to sometime between 8 and 2
million years ago. Both authors suggested that this
Table II. K2P genetic distance obtained among the seven major Piabina clusters.
ABCDEFP. anhembi
A 0.009/0.010 0.047 0.047 0.042 0.054 0.052 0.048
B 0.042 0.007/0.010 0.023 0.030 0.047 0.050 0.027
C 0.056 0.025 0.003/0.004 0.031 0.053 0.053 0.030
D 0.035 0.021 0.029 0.002/0.002 0.041 0.049 0.025
E 0.048 0.025 0.031 0.020 0/0.005 0.049 0.049
F 0.047 0.039 0.045 0.032 0.036 0.003/0.003 0.051
P. anhembi 0.040 0.025 0.034 0.022 0.028 0.034 0.001/0.001
Note: COI below diagonal and CytB above diagonal. The average values of intra-K2P distance represented in bold on the diagonal
(COI/CytB).
Table III. Pairwise F
ST
index obtained among the seven major Piabina clusters.
ABCDEFP. anhembi
A 0.79254 0.84386 0.85336 0.85659 0.86067 0.86193
B 0.80038 0.65594 0.77328 0.78754 0.82325 0.70487
C 0.85826 0.76602 0.92187 0.91004 0.92957 0.91288
D 0.82645 0.78760 0.92876 0.94973 0.96113 0.95335
E 0.83305 0.77467 0.93190 0.93346 0.93750 0.95991
F 0.83610 0.84631 0.92541 0.92958 0.93352 0.96486
P. anhembi 0.81920 0.79240 0.93925 0.93510 0.97734 0.93127
Note: COI gene below diagonal, CytB gene above diagonal.
Hidden diversity in Piabina argentea revealed by barcoding 7
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pattern is valid for most Neotropical freshwater fishes.
In their studies of Rhamdia and Synbranchus fish
species, Perdices et al. (2002, 2005) proposed similar
patterns for Mesoamerica and Ornelas-Gacia et al.
(2008) corroborated the same patterns for Astyanax.
With increasing recognition that mitochondrial DNA
is under strong selection, some authors caution
against the use of mitochondrial DNA data for dating
divergence events, but, this caveat notwithstanding,
recognize that selective sweeps can be beneficial for
barcoding (Galtier et al. 2009). Molecular clock
approaches that infer age of the most recent common
ancestor tend to be overestimated using mitochondrial
DNA unless they correct for apparent rate differences
between short and long time frames (Rand 2008).
The intercluster analysis performed confirmed the
presence of seven dissimilar barcode sequence clusters
among the Piabina specimens examined. The haplo-
type networks obtained using the combined data set
(Figure 3) and those for each gene separately (data not
shown) displayed seven unconnected networks with
high numbers of mutational steps (ranged from 45 to
110; Figure 3) necessary to connect these independent
networks. This situation is not expected when the
specimens represent a single species (Hart and Sunday
2007), even when there is very strong structure among
populations. Some pairs of P. argentea clusters need
more mutational steps than others to connect with
their congener P. anhembi species (Figure 3). Thus,
these results support the hypothesis that P. argentea
comprises more than one biological species. Kon et al.
(2007), working with the gobioid fish Schindleria,
obtained an unconnected haplotype network with
seven independent clusters and suggested that Schin-
dleria represents a species complex, as imparted here.
The F
ST
index showed very high values among the
seven clusters obtained (Table III), with similar values
among P. argentea and P. anhembi clusters. Consider-
ing that F
ST
values between 0 and 0.05 indicate a
low genetic structure, values between 0.05 and 0.15
a moderate genetic structure, values between 0.15 and
0.25 a high genetic structure, values above 0.25 a
strong genetic structure, and values close to 1 are
usually found among different species (Wright 1978;
Hartl and Clark 1997); the values presented in
Table III strongly suggest that our seven clusters
represent different species.
Many species have been discovered with the use
of molecular data and some have been formally
described later (Smith et al. 2005; Witt et al. 2006;
Kon et al. 2007; Ward et al. 2007, 2008; Nguyen
and Seifert 2008; Yassin et al. 2008), and the DNA
barcode has also been utilized as part of the validation
and formal description of new fish species such as
Coryphopterus kuna (Victor 2007); Urolophus kapalen-
sis (Yearsley and Last 2006); Brachionichthys autralis
(Last et al. 2007); five new species of Chromis genus
(Pyle et al. 2008), Dipturus argentinensis (Diaz de
Astarloa et al. 2008), and Moenkhausia forestii (Benine
et al. 2009). Our data suggest that the widely
distributed P. argentea species represent more than
one biological unit in the Upper Parana
´
Basin, and
probably this hypothesis is valid all over the area of
occurrence of this species. Interestingly, some clusters
Figure 3. Seven unconnected haplotype networks among Piabina specimens. P. anhembi is represented in gray. Numbers inside the figures
represent specimens that share the same haplotype. Numbers on lines represent the mutational steps between haplotypes. Dashed lines
represent the necessary steps to connect the independent networks.
L. H. G. Pereira et al.8
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were found only in a single locality (Clusters C F, and
P. anhembi) while others are widely dispersed (Clusters
A and B) (Figure 1). The fact that Clusters A and B
are widely dispersed could be a cause of no prior
recognition of these possible species, since the area of
overlap between them could impede its recognition.
Thus, we suggest that a detailed review of Piabina be
conducted to validate these new species (sensu Padial
et al. 2010). On the other hand, we believe that the
analysis of many other widely distributed fish species
may also disclose new species.
Conclusions
Our data demonstrate the efficacy of DNA barcoding
for discriminating known species and to flag new ones,
alone or associated with other genes. Despite the
concerns of Hickerson et al. (2006) to the contrary,
DNA barcoding revealed the existence of separate
taxa with low divergence rate or recent radiation.
We also substantiate the use of DNA barcode
sequences as part of the formal description of species.
These data can be useful when morphological charac-
ters are insufficient or too weak to define a species
and, importantly, because they apply to any sex or life
stage, can help to disambiguate the application of
names in future studies.
Acknowledgements
The authors are grateful to Renato Devide
´
and
Ricardo Teixeira for their help with the fish collection.
Financial support for the present study was provided
by CNPq and FAPESP.
Declaration of interest : Financial support for this
study was provided by Fundac¸a
˜
o de Amparo a
Pesquisa do Estado de Sa
˜
o Paulo (FAPESP) and
Conselho Nacional de Desenvolvimento Cientifico e
Tecnolo
´
gico (CNPq).
References
Agostinho AA, Pelicice FM, Petry AC, Gomes LC, Ju
´
lio HF, Jr.
2007. Fish diversity in the Upper Parana
´
River Basin: Habitats,
fisheries, management and conservation. Aquat Ecosyst Health
Manag 10(2):174 186.
Benine RC, Mariguela TC, Oliveira C. 2009. Nem species of
Moenkhausia Eigenmann, 1903 (Characiformes: Characidae)
with comments on the Moenkhausiaoligolepis species complex.
Neotrop Ichthyol 7(2):161 168.
Bickford D, Lohman DJ, Sodhi NS, Ng PKL, Meier R, Winker K,
et al. 2006. Cryptic species as a window on diversity and
conservation. Trends Ecol Evol 22(3):148155.
Buckup PA, Menzes NA, Ghazzi MS. 2007. Cata
´
logo das espe
´
cies
de peixes de a
´
gua doce do Brazil. Rio de Janeiro, Brazil: Museu
Nacional.
Castelloe J, Templeton AR. 1994. Root probabilities for intraspecific
gene trees under neutral coalescent theory. Mol Phylogenet Evol
3:102 113.
Castro RMC. 1999. Evoluc¸a
˜
o da ictiofauna de riachos sul-
americanos: Padro
˜
es gerais e possı
´
veis processos causais. In:
Caramashi EP, Mazzoni R, Peres-Neto PR, editors. Ecologia de
Peixes de Riacho: Se
´
rie Oecologia Brasiliensis. Rio de Janeiro,
Brazil: PPGE-UFRJ. p 139155.
Castro RMC, Casatti L, Santos HF, Ferreira KM, Ribeiro AC,
Benine RC, Dardis GZP, Melo ALA, Stopligia R, Abreu TX,
Bockmann FA, Carvalho M, Gibran FZ, Lima FCT. 2003.
Estrutura e composic¸a
˜
o da ictiofauna de riachos do Rio
Paranapanema, sudeste e sul do Brazil. Biota Neotropica 3(1).
Available at: http://www.biotaneotropica.org.br/v3n1/pt/
abstract?article þ BN0170301. Accessed on 15 July 2010.
Castro RMC, Casatti L, Santos HF, Melo ALA, Martins LSF,
Ferreira KM, Gibran FZ, Benine RC, Carvalho M, Ribeiro AC,
Abreu TX, Bockmann FA, Pelic¸a
˜
o GZ, Stopligia R,
Langeani F. 2004. Estrutura e composic¸a
˜
o da ictiofauna de
riachos da bacia do Rio Grande, no Estado de Sa
˜
o Paulo,
Sudeste do Brazil. Biota Neotropica 4(1). Available at: http://
www.biotaneotropica.org.br/v4n1/pt/
abstract?article þ BN0170402004. Accessed on 15 July 2010.
Castro RMC, Casatti L, Santos HF, Vari RP, Melo ALA, Martins
LSF, et al. 2005. Structure and composition of the stream
ichthyofauna of four tributary rivers of the Upper Rio Parana
´
Basin, Brazil. Ichthyol Explor Freshwaters 16(3):193214.
Clare EL, Lim BK, Engstrom MD, Eger JL, Hebert PDN. 2007.
DNA barcoding of Neotropical bats: Species identification and
discovery within Guyana. Mol Ecol Notes 7:184 190.
Clement M, Posada D, Crandall KA. 2000. TCS: A computer-
program to estimate gene genealogies. Mol Ecol 9:16571659.
da Silva JFP, Kaefer CC. 2003. Uma nova espe
´
cie de Piabina
Reinhardt, 1867 (Teleostei: Ostariophysi: Characidae) para o
alto Rio Tiete
ˆ
,Sa
˜
o Paulo, Brazil. Comun Mus Cie
ˆ
nc Technol
PUCRS Se
´
r Zool Porto Alegre 16(1):5365.
Diaz de Astarloa JM, Mabragana E, Hanner R, Figueroa DE. 2008.
Morphological and molecular evidence for a new species of
longnose skate (Rajiformes: Rajidae: Dipturus) from Argentinean
waters based on DNA barcoding. Zootaxa 1921:3546.
Edgar RC. 2004. MUSCLE: A multiple sequence alignment
method with reduced time and space complexity. BMC
Bioinformatics 5:113.
Excoffier L, Laval G, Schneider S. 2005. Arlequin ver. 3.0: An
integrated software package for population genetics data
analysis. Evol Bioinformatics Online 1:47 50.
Felsenstein J. 1985. Confidence limits on phylogenies: An approach
using the bootstrap. Evolution 39:783791.
Galtier N, Nabholz S, Gle
´
min S, Hurst GDD. 2009. Mitochondrial
DNA as a marker of molecular diversity: a reappraisal.
Molecular Ecology 18:4541 4550.
Hart MW, Sunday J. 2007. Things fall apart: Biological species form
unconnected parsimony networks. Biol Lett 3:509512.
Hartl DL, Clark AG. 1997. Principles of population genetics.
Sunderland, MA: Sinauer Associates.
Hebert PDN, Cywinska A, Ball SL, deWaard JR. 2003. Biological
identifications through DNA barcodes. Proc R Soc Lond Ser B
Biol Sci 270:313 321.
Hebert PDN, Stoeckle MY, Zemlak TS, Francis CM. 2004a.
Identification of birds through DNA barcodes. PLoS Biol 2:
1657 1663.
Hebert PDN, Penton EH, Burns JM, Janzen DH, Hallwachs W.
2004b. Ten species in one: DNA barcoding reveals cryptic
species in the Neotropical skipper butterfly Astrapesfulgerator.
Proc Natl Acad Sci U S A 101(41):1481214817.
Hickerson MJ, Meyer CP, Moritz C. 2006. DNA barcoding
willoften fail to discover new animal species over broad
parameter space. Syst Biol 55:729 739.
Hubert N, Duponchelle F, Nun
˜
ez J, Garcia-Davila C, Paugy D,
Renno JF. 2007. Phylogeography of the piranha genera
Serrasalmus and Pygocentrus: Implications for the diversification
of the Neotropical ichthyofauna. Mol Ecol 16:21152136.
Hidden diversity in Piabina argentea revealed by barcoding 9
Mitochondrial DNA Downloaded from informahealthcare.com by University of Guelph on 09/28/11
For personal use only.
Hubert N, Hanner R, Holm E, Mandrak NE, Taylor E, Burridge M,
et al. 2008. Identifying Canadian freshwater fishes through DNA
barcodes. PLoS ONE 3(6):e2490.
Jensen AC. 1966. Life history of the spiny dogfish. Fish Bull 65:
527 554.
Junk W. 2007. Freshwater fishes of South America: Their
biodiversity, fisheries, and habitats: A synthesis. Aquat Ecosyst
Health Manag 10(2):228242.
Kelly RP, Sarkar IN, Eernisse DJ, DeSalle R. 2007. DNA barcoding
using chitons (genus Mopalia). Mol Ecol Notes 7:177 183.
Kimura M. 1980. A simple method for estimating evolutionary rate
of base substitutions through comparative studies of nucleotide
sequences. J Mol Evol 16:111 120.
Kon T, Yoshino T, Mukai T, Nishida M. 2007. DNA sequences
identify numerous cryptic species of the vertebrate: A lesson
from the gobioid fish Schindleria. Mol Phylogenet Evol 44:
53 62.
Krieger J, Fuerst PA. 2002. Evidence for a slowed rate of molecular
evolution in the order Acipenseriformes. Mol Biol Evol 19:
891 897.
Langeani F, Castro RMC, Oyakawa OT, Shibatta OA, Pavanelli CS,
Casatti L. 2006. Diversidade da fauna do Alto Rio Parana
´
:
Composic¸a
˜
o atual e perspectivas futuras. Biota Neotrop 7(3):
181 197.
Last PR, Gledhill DC, Holmes BH. 2007. A new handfish,
Brachionichthysaustralis sp. nov. (Lophiiformes: Brachionichthyi-
dae), with a redescription of the critically endangered spotted
handfish, B. hirsutus (Lace
´
pe
`
de). Zootaxa 1666:53 68.
Lima FCT, Malabarba LR, Buckup PA, Pezzi da Silva JF, Vari RP,
Harold A, et al. 2003. Genera Incertae Sedis in Characidae. In:
Reis RE, Kullander SO, Ferraris CJJr, editors. Checklist of the
freshwater fishes of South and Central America. Porto Alegre,
Brazil: Edipucrs. p 106168.
Lowe-Mcconnell RH. 1999. Estudos ecolo
´
gicos de comunidades
de peixes tropicais. Sa
˜
o Paulo, Brazil: EDUSP.
Montoya-Burgos JI. 2003. Historical biogeography of the catfish
genus Hypostomus (Siluriformes: Loricariidae), with implications
on the diversification of Neotropical ichthyofauna. Mol Ecol 12:
1855 1867.
Nguyen HDT, Seifert KA. 2008. Description and DNA barcoding
of three new species of Leohumicola from South Africa and the
United States. Personia 21:5798.
Ornelas-Garcia CP, Dominguez-Dominguez O, Doadrio I. 2008.
Evolutionary history of the fish genus Astyanax Baird & Girard
(1854) (Actynopterigii, Characidae) in Mesoamerica reveals
multiple morphological homoplasies. BMC Evol Biol 8:340.
Padial JM, Miralles A, De la Riva I, Vences M. 2010. The integrative
future of taxonomy. Front Zool 7:16.
Perdices A, Bermingham E, Montilla A, Doadrio I. 2002.
Evolutionary history of the genus Rhamdia (Teleostei: Pimelo-
didae) in Central America. Mol Phylogenet Evol 25:172 189.
Perdices A, Doadrio I, Bermingham E. 2005. Evolutionary history
of the synbranchid eels (Teleostei: Synbranchidae) in Central
America and the Caribbean islands inferred from their molecular
phylogeny. Mol Phylogenet Evol 37:460 473.
Pyle RL, Earle JL, Greene BD. 2008. Five new species of the
damselfish genus Chromis (Perciformes: Labroidei: Pomacen-
tridae) from deep coral reefs in the tropical western Pacific.
Zootaxa 1671:3 31.
Rand DM. 2008. Mitigating mutational meltdown in mammalian
mitochondria. PLoS Biology 6(2):e35.
Ratnasingham S, Hebert PDN. 2007. BOLD: The Barcode of
Life Data System (www.barcodinglife.org). Mol Ecol Notes 7:
355 364.
Reis RE, Kullander SO, Ferraris C. 2003. Check list of freshwater
fishes of South and Central America. Porto Alegre, Brazil:
Edipucrs.
Ribeiro AC, Benine RC, Figueiredo CA. 2004. A new species of
Creagrutus Gu
¨
nther (Teleostei: Ostariophysi: Characiformes),
from Upper Rio Parana
´
Basin, central Brazil. J Fish Biol 64:
597 611.
Schaefer SA. 1998. Conflict and resolution impact of new taxa on
philogenetic studies of Neotropicalcascudinhos (Siluriformes:
Loricariidae). In: Malabarba LR, Reis RE, Vari RP, Lucena
ZMS, Lucena CAS, editors. Phylogeny and classification of
Neotropical fishes. Porto Alegre, Brazil: Edipucrs. p 375 400.
Smith MA, Woodley NE, Janzen DH, Hallwachs W, Hebert PDN.
2005. DNA barcodes reveal cryptic host-specificity within the
presumed polyphagus members of a genus of parasitoid flies
(Diptera: Tachinidae). Proc Natl Acad Sci U S A 103(10):
3657 3662.
Swofford DL. 2002. PAUP*—Phylogenetic analysis using parsi-
mony (*and Other Methods) Version 4.0b10. Sunderland, MA:
Sinauer Associates.
Tamura K, Dudley J, Nei M, Kumar S. 2007. MEGA 4: Molecular
evolutionary genetics analysis (MEGA) software version 4.0.
Mol Biol Evol 24:1596 1599.
Templeton AR, Crandall KA, Sing CF. 1992. A cladistic-analysis of
phenotypic associations with haplotypes inferred from restriction
endonuclease mapping and DNA-sequence data. 3. Cladogra-
mestimation. Genetics 132:619 633.
Valdez-Moreno M, Ivanova NV, Elı
´
as-Guitie
´
rrez M, Contreras-
Balderas S, Hebert PDN. 2009. Probing diversity in freshwater
fishes from Mexico and Guatemala with DNA barcodes. J Fish
Biol 74:377402.
Vari RP, Harold AS. 2001. Phylogenetic Study of Neotropical Ish
Genera Creagrutus Gu
¨
nther and Piabina Reinhardt (Teleostei:
Ostariophysi: Characiformes), with a Revision of Cis-Andean
Species. Smithsonian Contributions to Zoology Number 613.
Washington, DC: Smithsonian Institution Press.
Vari RP, Malabarba LR. 1998. Neotropical ichthyology: An
overview. In: Malabarba LR, Reis RE, Vari RP, Lucena ZMS,
Lucena CAS, editors. Phylogeny and classification of Neotro-
pical fishes. Porto Alegre, Brazil: Edipucrs. p 111.
Victor BC. 2007. Coryphopteruskuna, a new goby (Perciformes:
Gobiidae: Gobinae) from the western Caribbean, with the
identification of the late larval stage and an estimate of the
pelagic larval duration. Zootaxa 1526:5161.
Ward RD. 2009. DNA barcode divergence among species and
genera of birds and fishes. Mol Ecol Resour 9:10771085.
Ward RD, Zemlak TS, Innes BH, Last PR, Hebert PDN. 2005.
DNA barcoding Australia’s fish species. Philos Trans R Soc
Lond Ser B Biol Sci 360:18471857.
Ward RD, Holmes BH, Zemlak TS, Smith PJ. 2007. DNA
barcoding discriminates spurdogs of the genus Squalus. In:
Last PR, White WT, Pogonoski JJ, editors. Descriptions of
new dogfishies of the genus Squalus (Squaloidea: Squalidae).
Hobart, Australia: CSIRO Marine and Atmospheric Research,
p 117 130.
Ward RD, Holmes BH, Yearsley GK. 2008. DNA barcoding reveals
a likely second species of Asian sea bass (barramundi) (Lates
calcarifer). J Fish Biol 72:458463.
Witt JDS, Threloff DL, Hebert PDN. 2006. DNA barcoding reveals
extraordinary cryptic diversity in an amphipod genus: Impli-
cations for desert spring conservation. Mol Ecol 15:3073 3082.
Wright S. 1978. Evolution and the genetics of populations 4:
Variability within and among natural populations. Chicago, IL:
University of Chicago Press.
Yassin A, Capy P, Madi-Ravazzi L, Ogereau D, David JR. 2008.
DNA barcode discovers two cryptic species and two geographi-
cal radiations in the invasive drosophilid Zaprionus indianus.
Mol Ecol Resour 8:491501.
Yearsley GK, Last PR. 2006. Urolophus kapalensis sp nov., a new
stingaree (Myliobatiformes: Urolophidae) off eastern Australia.
Zootaxa 1176:4152.
L. H. G. Pereira et al.10
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    • "Molecular identification of species using DNA sequences of a short standardized region from an unknown species, in order to compare with sequences of a particular species in databases is known as 'DNA barcoding'. This barcoding technique has been determined for many groups of organisms to assess biodiversity, as well as for ecological applications , forensic wildlife, and pathogenic animals (Wong et al., 2004; Pook and McEwing, 2005; Carvalho et al., 2011; Dubey et al., 2011; Feng et al., 2011; Pereira et al., 2011; Gaur et al., 2012; Nagy et al., 2012; Martin et al., 2013; Pramual and Adler, 2014). Barcodes are retained in reference sequence libraries, and constructed from reliably identified reference specimens. "
    [Show abstract] [Hide abstract] ABSTRACT: DNA barcodes of mitochondrial cytochrome c oxidase I (COI), cytochrome b (Cytb) genes, and their combined data sets were constructed from 35 snake species in Thailand. No barcoding gap was detected in either of the two genes from the observed intra- and interspecific sequence divergences. Intra- and interspecific sequence divergences of the COI gene differed 14 times, with barcode cut-off scores ranging over 2%–4% for threshold values differentiated among most of the different species; the Cytb gene differed 6 times with cut-off scores ranging over 2%–6%. Thirty-five specific nucleotide mutations were also found at interspecific level in the COI gene, identifying 18 snake species, but no specific nucleotide mutation was observed for Cytb in any single species. This suggests that COI barcoding was a better marker than Cytb. Phylogenetic clustering analysis indicated that most species were represented by monophyletic clusters, suggesting that these snake species could be clearly differentiated using COI barcodes. However, the two-marker combination of both COI and Cytb was more effective, differentiating snake species by over 2%–4%, and reducing species numbers in the overlap value between intra- and interspecific divergences. Three species delimitation algorithms (general mixed Yule-coalescent, automatic barcoding gap detection, and statistical parsimony network analysis) were extensively applied to a wide range of snakes based on both barcodes. This revealed cryptic diversity for eleven snake species in Thailand. In addition, eleven accessions from the database previously grouped under the same species were represented at different species level, suggesting either high genetic diversity, or the misidentification of these sequences in the database as a consequence of cryptic species.
    Full-text · Article · Sep 2016
    • "In a study of Piabina argentea in the basin of the Paraná River, DNA barcoding revealed the presence of six clades, diverging by 3.0-5.6%, indicating the presence of at least five distinct species (Pereira et al., 2011). The results of the present study confirmed the effectiveness of the COI barcode for the identification and discrimination of fish species analyzed in the Itapecuru Basin, with more than 90% of the species being determined. "
    [Show abstract] [Hide abstract] ABSTRACT: DNA barcoding is a useful complementary tool for use in traditional taxonomic studies due to its ability to detect cryptic species, and may be particularly efficient in the identification of fish species. The fish fauna of the Itapecuru River represents an important fishery resource in the Brazilian State of Maranhão, although it is currently suffering increasing degradation as a result of anthropogenic impacts. Therefore, DNA barcoding was used in the present study to identify fish species and establish a database of the rich freshwater fish fauna of Maranhão. A total of 440 specimens were analyzed, corresponding to 64 species belonging to 59 genera, 31 families, and 10 orders. Overall, 92.19% of these species could be identified by DNA barcoding, and were characterized by low levels (average 0.80%) of intra-specific divergence. However, five species (Anableps anableps, Gymnotus carapo, Sciades couma, Pseudauchenipterus nodosus, and Leporinus piau) presented values of mean genetic divergence above 3%, indicating the existence of cryptic diversity in these fishes. The DNA barcoding approach permitted the analysis of a large number of specimens and facilitated the discrimination and identification of closely related fish species in the Itapecuru Basin.
    Article · Aug 2016
    • "Concisely, it uses a short (~650 bp) gene fragment from the mitochondrial 5 0 region of the cytochrome c oxidase subunit I (COI) to discriminate species based on sequence differences (Hebert et al. 2003 ). The method has been applied to fishes of multiple genera/families in specific Neotropical river systems (Carvalho et al. 2011; Rosso et al. 2012; Pereira et al. 2013; Escobar-Camacho et al. 2015) as well as studies centred on specific fish families, including the Tetraodontidae (Amaral et al. 2013), Loricariidae (Roxo et al. 2012; Costa-Silva et al. 2015), Lebiasinidae (Benzaquem et al. 2015 ), Parodontidae (Bellafronte et al. 2013) and Characidae (Benine et al. 2009; Melo et al. 2011; Pereira et al. 2011; Silva et al. 2013; Castro Paz et al. 2014). The resultant progress is impressive, yet a challenging number of Neotropical fish clades remain to be explored via barcoding. "
    [Show abstract] [Hide abstract] ABSTRACT: Detritivores of the fish family Curimatidae are assigned to eight genera, one of which, the Curimatopsis, with only five species, is the least speciose genus and sister to other seven genera in the family. Ongoing morphological investigations reveal, however, the likely existence of additional species. In this study, fifty-one specimens of Curimatopsis from multiple rivers of the Amazon, Paraguay and Suriname drainages were identified morphologically according to the present species concepts and then barcoded using the universal cytochrome c oxidase subunit I (COI) mitochondrial marker. Species delimitation analyses were conducted using Bayesian methods through the general mixed Yule-coalescent analysis combined with conventional likelihood, genetic distance and haplotypic diversity approaches. We found eleven well-supported clusters that represent four of the named species and seven cryptic, undescribed species of Curimatopsis. Our results show a clear delimitation of species boundaries constrained by distinct Amazonian river ecotones that may have promoted intrageneric lineage diversification. This is the first of a series of genetic studies applicable to future taxonomic, phylogenetic and evolutionary studies across the Curimatidae.
    Full-text · Article · Mar 2016
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