Content uploaded by Victor A Tagliacollo
Author content
All content in this area was uploaded by Victor A Tagliacollo on Oct 14, 2017
Content may be subject to copyright.
This article appeared in a journal published by Elsevier. The attached
copy is furnished to the author for internal non-commercial research
and education use, including for instruction at the authors institution
and sharing with colleagues.
Other uses, including reproduction and distribution, or selling or
licensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of the
article (e.g. in Word or Tex form) to their personal website or
institutional repository. Authors requiring further information
regarding Elsevier’s archiving and manuscript policies are
encouraged to visit:
http://www.elsevier.com/copyright
Author's personal copy
Molecular phylogeny of Aphyocharacinae (Characiformes, Characidae)
with morphological diagnoses for the subfamily and recognized genera
Victor A. Tagliacollo
a,
⇑
, Rosana Souza-Lima
b
, Ricardo C. Benine
c
, Claudio Oliveira
a
a
Universidade Estadual Paulista, UNESP, Instituto de Biociências, Departamento de Morfologia, Distrito de Rubião Jr., s/no., CEP 18618–970, Botucatu, SP, Brazil
b
Universidade do Estado do Rio de Janeiro, UERJ, Faculdade de Formação de Professores, Departamento de Ciências, Rua Dr. Francisco Portela, No. 1470, Patronato,
CEP 24435-005, São Gonçalo, RJ, Brazil
c
Universidade do Sagrado Coração, USC, Centro de Ciências da Saúde, Setor de Zoologia, Laboratório de Ictiologia, Rua Irmã Arminda, No. 10-50, CEP 17011-160, Bauru, SP, Brazil
article info
Article history:
Received 25 August 2011
Revised 1 April 2012
Accepted 5 April 2012
Available online 19 April 2012
Keywords:
Neotropical fishes
Evolutionary relationships
South America
Aphyocharax
abstract
The subfamily Aphyocharacinae was recently redefined to comprise eight genera: Aphyocharax,
Prionobrama,Paragoniates,Phenagoniates,Leptagoniates,Xenagoniates,Rachoviscus and Inpaichthys. This
new composition, however, is partially incongruent with published results of molecular studies especially
concerning the positions of Rachoviscus and Inpaichthys. Our goal was to investigate the monophyly of
Aphyocharacinae and its interrelationships using three distinct phylogenetic methodologies:
Maximum-likelihood and Bayesian analyses of molecular data, and also Parsimony analysis of a concate-
nated molecular and morphological dataset. All tree topologies recovered herein suggest that Rachoviscus,
Inpaichthys and Leptagoniates pi do not belong to the Aphyocharacinae. The remaining aphyocharacin taxa
analyzed do form a monophyletic group, which is itself composed of two subgroups being one comprised
of Paragoniates,Phenagoniates,Leptagoniates and Xenagoniates, and the other comprised of Aphyocharax
and Prionobrama. Internal relationships among these genera are statistically well supported and
morphological synapomorphies are presented at the generic level. All tree topologies also indicate that
Aphyocharacidium is closely related to Aphyocharacinae suggesting that it should be included in this sub-
family. As recognized in the present study, the Aphyocharacinae is diagnosed by a single morphological
synapomorphy: two dorsal-fin rays articulating with the first dorsal pterygiophore.
Ó2012 Elsevier Inc. All rights reserved.
1. Introduction
Characidae is the largest and most diverse family of Characifor-
mes comprising around 200 genera and more than 1200 valid spe-
cies geographically widespread from southern USA to northern
Argentina (Reis et al., 2003; Eschmeyer, 2010). Phylogenetic rela-
tionships among characids, popularly known as ‘‘tetras’’, have been
the subject of intense phylogenetic studies in recent years
(Mirande, 2009, 2010; Javonillo et al., 2010; Oliveira et al., 2011).
While these studies have generated many novels and well sup-
ported hypotheses of relationships, some of the results from the
morphological and molecular analyses are incongruent, especially
in relation to the composition of the subfamilies (e.g., Mirande,
2009; Oliveira et al., 2011).
Günther (1864) was the first author to propose a division of Fam-
ily Characidae in 10 infra-families. In 1868, Günther described
Aphyocharax as a new genus in infra-family Tetragonopterina. In a
series of papers published around a century ago, Eigenmann
(1909, 1910, 1912) included the blood-fin tetra Aphiocharax [sic]
and some other genera of small-bodied characids (Cheirodon,Coelu-
richthys,Holoprion,Holoshestes,Odontostilbe, Probolodus, and Aphyo-
dite) in the subfamily Aphiocharacinae [sic], based on similarities in
the shape of the gill membranes, nares, fontanels, adipose fin, and
maxillary teeth. Eigenmann (1915) later subsumed most of these
species within a newly-recognized subfamily, the Cheirodontinae,
a taxonomic arrangement that served as the basis of classification
for many decades (e.g. Gregory and Conrad, 1938; Géry, 1960;
Géry and Boutiére, 1964).
In his revision of the family Characidae, Géry (1977) recognized
Aphyocharax as a member of a distinct subfamily, based on a
laterally compressed body, anal fin of intermediate length, midbody
position of dorsal fin, incomplete lateral line, and arrangement and
shape of teeth in the oral jaws. Although he placed Aphyocharax in
the monotypical subfamily Aphyocharacinae, Géry (1977) did note
similarities between Aphyocharax and a newly created subfamily
1055-7903/$ - see front matter Ó2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ympev.2012.04.007
⇑
Corresponding author. Present address: University of Louisiana at Lafayette,
Department of Biology, Lafayette, LA 70504-2451, USA.
E-mail addresses: victor_tagliacollo@yahoo.com.br (V.A. Tagliacollo), rosanasl@
yahoo.com.br (R. Souza-Lima), ricardo.benine@usc.br (R.C. Benine), claudio@ibb.
unesp.br (C. Oliveira).
Molecular Phylogenetics and Evolution 64 (2012) 297–307
Contents lists available at SciVerse ScienceDirect
Molecular Phylogenetics and Evolution
journal homepage: www.elsevier.com/locate/ympev
Author's personal copy
Paragoniatinae, in which he placed Rachoviscus,Paragoniates,
Phenagoniates,Leptagoniates,Xenagoniates and Prionobrama.Géry
and Junk (1977) described a new genus and species, Inpaichthys
kerri, indicating its general resemblance with Rachoviscus crassiceps
and ‘‘Paragoniates et al.’’ (‘‘et al.’’ possibly referring to the other spe-
cies of Paragoniatinae).
In the most complete osteological survey of Characidae to date,
Mirande (2009, 2010) recognized eight genera in the Aphyocharac-
inae: Paragoniates,Phenagoniates,Xenagoniates,Inpaichthys, Leptag-
oniates,Rachoviscus,Aphyocharax and Prionobrama. The genera
Rachoviscus and Leptagoniates were provisionally placed in the sub-
family following previous reports published in the literature (e.g.
Géry, 1977; Géry and Junk, 1977). Three synapomorphies
supported the monophyly of Aphyocharacinae: (1) presence of
synchondral articulation between lateral ethmoid and anterodor-
sal border of orbitosphenoid; (2) fourth infraorbital absent or much
reduced and bordered posteriorly by third and fifth infraorbitals;
and (3) six or less branched pelvic-fin rays (Mirande, 2010).
Mirande’s composition of Aphyocharacinae is partly incongru-
ent with two recently published molecular phylogenies of Characi-
dae (Javonillo et al., 2010; Thomaz et al., 2010). Although not
including many characid taxa, these studies indicated that
Inpaichthys and Rachoviscus do not belong to the Aphyocharacinae.
Javonillo et al. (2010) recovered Aphyocharax as the sister group of a
clade comprised of Exodon,Phenacogaster, Roeboides, Galeocharax,
Cynopotamus and Tetragonopterus. The authors additionally
indicated that Inpaichthys and Ctenobrycon are sister taxa, while
Rachoviscus is closer to a group including Hollandichthys.Thomaz
et al. (2010) also recovered Rachoviscus as the sister group of
Hollandichthys, however these genera were not close related to
Aphyocharax.Oliveira et al. (2011) in the broadest molecular analy-
sis of Characidae included all Aphyocharacinae genera proposed by
Mirande (2010) in their study and again Rachoviscus and Inpaichthys
do not appear as close related to the remaining Aphyochracinae.
Due to the extraordinary diversity and complexity of the
Characidae, with large amounts of morphological homoplasy and
character state reversals (Malabarba, 1998; Zanata and Vari,
2005; Toledo-Piza, 2007; Mirande, 2010), previous phylogenetic
studies of the group have been forced to sample a relatively small
proportion of all known taxa (Malabarba and Weitzman, 2003;
Mirande, 2009, 2010; Javonillo et al., 2010; Thomaz et al., 2010;
Oliveira et al., 2011). This strategy is called the ‘‘basal exemplar ap-
proach’’, which selects representative species from among what
are perceived to be the major distinct clades (Albert et al., 2009).
Here we build on the results of these previous phylogenetic stud-
ies, which allowed us to concentrate efforts on a far less-inclusive
efforts set of species. Thus this is the first analysis of morphological
and molecular data for all aphyocharacin genera, with data for
most species. Our aims were to investigate the monophyly and
interrelationships of Aphyocharacinae (sensu Mirande, 2010) using
model-based phylogenetic analyses of molecular data, and also do
total evidence analysis by parsimony.
1.1. Ingroup and outgroup criteria selection
Ingroup taxa were selected based on phylogenies proposed
by Mirande (2009, 2010). Following these hypotheses,
Aphyocharacinae comprises eight genera: Aphyocharax,Inpaichthys,
Leptagoniates, Paragoniates, Phenagoniates, Prionobrama,Rachoviscus
and Xenagoniates.
Outgroup taxa were selected based on phylogenies proposed by
Oliveira et al. (2011). Following their hypotheses, Characidae (node
37) is a well supported clade comprised of four monophyletic
units. All species of these four clades were selected as a distinct
outgroups and, in addition, two species of Salminus were included
as extra outgroups.
1.2. DNA extraction and sequencing
Total DNA was extracted from muscle tissue preserved in etha-
nol with DNeasy Tissue Kit following manufacturer’s instructions.
Partial sequences of the genes 16S rRNA (16S, 700 pb) and cyto-
chrome b (CytB, 900 pb) were amplified using one round of poly-
merase chain reaction (PCR). PCR amplifications were performed
in 50
l
l reactions consisting of 5
l
l 10 x reaction buffers, 1
l
l dNTP
mix at 10 mM each, 1
l
l of each primer at 10
l
M, 0.2
l
l Taq DNA
Polymerase 1 U of Polymerase per reaction, 1
l
l DNA, and 40.8
l
l
of double-distilled water. Cycles of amplification were pro-
grammed with the following profile: (1) 3 min at 94 °C (initial
denaturation), (2) 30 s at 94 °C, (3) 45 s at 48–54 °C, (4) 80 s at
72 °C, and 5 min at 72 °C (final elongation). Steps 2–4 were re-
peated 35 times. Additionally, sequences of myosin heavy chain
6 gene (Myh6, 750 pb), recombination activating gene 1 (RAG 1,
1250 pb) and recombination activating gene 2 (RAG 2, 950 pb)
were amplified through two rounds of PCR. The first was con-
ducted using external primers while the second was conducted
using internal primers (Supplementary material A). PCR amplifica-
tions were performed in 50
l
l reactions consisting of 5
l
l10
reaction buffers, 1
l
l dNTP mix at 10 mM each, 1
l
l of each primer
at 10
l
M, 0.2
l
l Taq DNA Polymerase 1 U of Polymerase per reac-
tion, 1
l
l DNA, and 40.8
l
l of double-distillated water. Cycles of
amplification were programmed with the following profile: (1)
3 min at 94 °C (initial denaturation), (2) 30 s at 94 °C, (3) 45 s at
50–54 °C (4) 80 s at 72 °C, and 5 min at 72 °C (final elongation).
Steps 2–4 were repeated 37–40 times. Products of all amplification
were identified on a 1% agarose gel. PCR products were purified
with the ExoSap-IT
Ò
. Sequencing reactions were performed with
the Big Dye Terminator Cycle Sequencing Ready Reaction 3.1 Kit
following instructions of the manufacturer, and were loaded on
an automatic sequencer 3130-Genetic Analyzer in the Instituto
de Biociências, Universidade Estadual Paulista, Botucatu, São Paulo.
Consensus sequences were assembled and edited in BioEdit 7.0.9.0
(Hall, 1999). Where uncertainty of nucleotide identity was de-
tected, IUPAC ambiguity codes were applied.
1.3. Sequencing alignment and phylogenetic analyses
1.3.1. Sequence data
Consensus sequences of each gene were independently aligned
using MAFFT v. 5.3 (Katoh et al., 2002, 2005) and, then, alignments
were inspected by eye for any obvious misreading. To evaluate the
occurrence of substitution saturation, the index of substitution sat-
uration (Iss) was estimated in DAMBE (Xia and Xie, 2001) as out-
line by Xia et al. (2003) and Xia and Lemey (2009). Overall
genetic distances (Tamura 3-parameter) among sequences were
calculated in Mega 5.04 (Tamura et al., 2011) and appropriate evo-
lutionary models were estimated by jModelTest under default
parameters (Posada, 2008).
1.3.2. Maximum-likelihood (ML)
ML was conducted in RAxML (Stamatakis, 2006) using the web
servers RaxML BlackBox (Stamatakis et al., 2008). Random starting
trees were applied for ML tree search and all other parameters
were set on default values. ML analyses were conducted under
GTR + G given that RAxML only applies such a model (Stamatakis
et al., 2008). Topological robustness was investigated using 1000
non-parametric bootstrap replicates. Branches with bootstrap val-
ues higher than 70% were considered well supported (see Hillis and
Bull (1993) for justification).
1.3.3. Bayesian inference (BI)
BI was conducted using MrBayes 3.1.2 (Huelsenbeck and
Ronquist, 2001; Ronquist and Huelsenbeck, 2003). Because
298 V.A. Tagliacollo et al. / Molecular Phylogenetics and Evolution 64 (2012) 297–307
Author's personal copy
MrBayes 3.1.2 only implements 1, 2 and 6 substitution rate models,
it was not possible to implement some of the models chosen by
jModelTest (Posada, 2008). Thus, correspondent models were se-
lected as indicated by MrModelTest 3.2 (Nylander, 2004). Two runs
of four independent MCMC chains were run with 50 million repli-
cates each, sampling one tree every 1000 generation. The distribu-
tion of log likelihood scores was examined using Tracer 1.4
(Rambaut and Drummond, 2004) in order to determine stationa-
rity for each search and to decide if extra runs were required to
achieve convergence. The first 5 million generation (10%) were dis-
carded as burn-in. The remaining trees were used to calculate a
50% majority-rule consensus topology. Branches with Bayesian
posterior probabilities P95% were considered well supported.
1.3.4. Total evidence analysis based on parsimony (TE)
TE analysis was composed of molecular and morphological
characters. Molecular data consisted of 4423 characters while mor-
phological data consisted of 373 characters, of which 365 were
published by Mirande (2010) and eight additional are proposed
herein (supplementary material B). These additional characters
were mainly based on osteological traits that were examined on
cleared-and-stained (c&s) specimens prepared using the method
outlined by Taylor and Van Dyke (1985). The resulting concate-
nated matrix was composed of 125 specimens (115 species) and
51 species coded also for morphological characters. Further infor-
mation can be seen in Supplementary material B.
TE was conducted using TNT 1.1 (Goloboff et al., 2008). No dif-
ferentially weighting or ordering of character states was adopted.
Gaps and question marks were treated as a missing data. Phyloge-
nies were obtained through searches using ‘sectorial searches’,
‘ratchet’, ‘drift’, and ‘tree fusing’ with their default values and
employing a driven search with initial level set at 100 and checking
level every three hits. Each of these searches was performed from
100 random addition sequences and TBR branch swapping. Consis-
tency and retention indexes were calculated with the script ‘stats’.
Bootstrap was calculated from 1000 pseudoreplicates. Branches
with bootstrap values higher than 70% were considerate well sup-
ported (see Hillis and Bull (1993) for justification).
2. Results
2.1. Dataset
Most of partial sequences of two mitochondrial (16S rRNA,
CytB) and three nuclear (Myh6, RAG1, RAG2) genes used herein
were published by Oliveira et al. (2011) while few others were
published by Javonillo et al. (2010). From the species Aphyocharax
alburnus,A.anisitsi A. dentatus,A.nattereri,A. sp, A.pusillus,
A. rathbuni,Prionobrama filigera,P.paraguayensis, and Leptagoniates
pi, sequences were generated in the present study. All sequences
were deposited in GenBank under the accession number shown
in Supplementary material A. Data matrix is deposited in the Dryad
Repository at http://dx.doi.org/10.5061/dryad.v24r8753.
The resulting molecular dataset was comprised of 4423 charac-
ters. This dataset was partitioned into five blocks, each one repre-
senting a different gene. The overall mean of genetic distance
ranged from 0.0695 ± 0.005 (Myh6) to 0.2295 ± 0.008 (CytB)
suggesting that the analyzed sequences have enough genetic vari-
ation for phylogenetic studies. Genes and also their respective co-
don position were tested to investigate the occurrence of
substitution saturation. The Iss index indicated that there are not
saturations in the genes, however significant saturation was found
for the 3rd codon position of the gene CytB in both asymmetrical
(Iss.cA) and symmetrical (Iss.cS) topologies (data not shown).
Although Iss is greater than Iss.cS, no statistical difference was
found, which means that this codon position can be used in phylo-
genetic analyses (Xia et al., 2003; Xia and Lemey, 2009). Appropri-
ate evolutionary models for the genes were investigated under the
Akaike information criterion (AIC) and Bayesian information crite-
ria (BIC). For 16S rRNA and CytB genes, both AIC and BIC selected
the GTR + I + G as the best fit-model while to the other genes each
criteria selected a different model. Therefore, the choice was to
adopt models selected according to AIC (Posada and Buckley,
2004 for justification). For each gene partition, information content
and characteristics such as: number of base pairs (bp) after align-
ment, base pair composition, overall mean genetic distance, substi-
tution saturation (Iss index), nucleotide substitution models,
a
(shape) parameter of
C
distribution and proportion of invariant
sites are shown in Table 1.
2.2. Model-based phylogenetic reconstructions
ML and BI show that excluding Rachoviscus,Inpaichthys, and
Leptagoniates pi, Aphyocharacinae as suggested by Mirande
(2010) constitute a statistically well supported group (Fig. 1). Addi-
tionally, molecular model-based hypotheses presume that Aphyo-
characinae has two major clades being one comprised of
Paragoniates,Phenagoniates,Leptagoniates, and Xenagoniates and,
another comprised of Aphyocharax and Prionobrama. Evolutionary
relationships among them are also statistically well supported
and most genera are monophyletic, except Leptagoniates given that
L.pi is the sister group of representatives of the subfamily Che-
irodontinae (Fig. 1). At species level, interrelationships among
some species of Aphyocharax are statistically poor supported under
ML (mainly the relationships among A.anisitsi,A. rathbuni,A.den-
tatus, and A. sp.), however well support under BI analysis. Trees
based on BI and ML analyses, bootstrap and posterior probabilities
values are shown in Fig. 1.
Table 1
Information content and characteristics of each gene partition.
GENES
16S rRNA CytB Myh6 RAG1 RAG2
Bp after alignment 540 918 700 1275 1051
P
A
0.3873 0.3377 0.2817 0.2866 0.2737
P
C
0.1939 0.3368 0.2261 0.2230 0.2185
P
G
0.1863 0.0612 0.2250 0.2244 0.2242
P
T
0.2324 0.2643 0.2673 0.2660 0.2837
Overall mean genetic distance (p-distance) 0.1183 ± 0.009 0.2295 ± 0.008 0.0695 ± 0.005 0.0904 ± 0.004 0.0802 ± 0.005
Substitution saturation Iss < Iss.c Iss < Iss.c Iss < Iss.c Iss < Iss.c Iss < Iss.c
Nucleotide substitution model GTR GTR TrN TPM2uf TPM2uf
a
(shape) parameter of
C
distribution 0.4130 0.3650 0.2760 0.6810 0.6480
Proportion of invariant (I) sites 0.5330 0.4890 – 0.2510 0.2550
V.A. Tagliacollo et al. / Molecular Phylogenetics and Evolution 64 (2012) 297–307 299
Author's personal copy
2.3. Total evidence phylogenetic reconstruction
As with the ML and BI phylogenies, TE parsimony-based analy-
sis suggests that Rachoviscus, Inpaichthys, and Leptagoniates pi do
not belong to Aphyocharacinae (Fig. 2). Furthermore, it presumes
that the subfamily has two statistically well supported clades
being one comprised of Paragoniates,Phenagoniates,Leptagoniates,
and Xenagoniates and, another comprised of Aphyocharax and
Fig. 1. ML (black topology) and BI (grey topology) analyses showing similar relationships among characid species. In this study results indicate that Aphyocharacinae (sensu
Mirande, 2010) is not monophyletic since Inpaichthys is more closely related to Nematobrycon and Carlana. Molecular data also suggest that Rachoviscus and Hollandichthys are
sister groups while Leptagoniates pi is the sister group of the Cheirodontinae. Dashed square limits Aphyocharacinae as proposed herein. Black numbers (above) correspond to
bootstrap values and gray numbers (below) to posterior probability values. ‘‘’’ indicates species that do not belong to the Aphyocharacinae as recognized herein.
300 V.A. Tagliacollo et al. / Molecular Phylogenetics and Evolution 64 (2012) 297–307
Author's personal copy
Prionobrama (Fig. 2). At species level, only a single discrepancy is
observed between model-based and parsimony-based phylogenies
(Figs. 1 and 2). Although statistically poor supported under ML
analysis, model-based hypotheses suggest that A. anisisti and A.
rathbuni are together the sister group of A.dentatus and A. sp.
(Fig. 1). Alternatively, TE analysis indicates that A.dentatus and A.
sp are both the sister group to A.rathbuni, and all three species to-
gether are the sister group of A.anisitsi (Fig. 2). Strict consensus
among 16 most parsimonious trees, length, number of informative
characters, consistency and retention indexes, and bootstrap val-
ues are presented in Fig 2. Further details can be seen in the
Fig. 3 and in Supplementary material B.
3. Discussion
3.1. Monophyly of Aphyocharacinae (sensu Mirande, 2010)
Recent morphological hypotheses on the taxonomic composi-
tion of Aphyocharacinae propose that the subfamily is comprised
of eight genera: Aphyocharax,Inpaichthys,Leptagoniates,Paragoni-
ates,Phenagoniates,Prionobrama,Rachoviscus, and Xenagoniates
(Mirande, 2009, 2010). This arrangement, however, is incongruent
with some recently published molecular phylogenies of the Char-
acidae (Javonillo et al., 2010; Thomaz et al., 2010; Oliveira et al.,
2011). The main disagreement refers to the position of the mono-
typic Inpaichthys, and the two known species of Rachoviscus.As
shown in previously published phylogenies (Javonillo et al.,
2010; Thomaz et al., 2010; Oliveira et al., 2011), molecular mod-
el-based hypotheses recovered herein suggest that Rachoviscus
constitutes the immediate sister group of Hollandichthys (Fig. 1)
and that Inpaichthys belongs to a clade comprised of Nematobrycon
plus Carlana (Fig. 1). Despite some uncertainty as to which species
is the sister taxon to Inpaichthys, the molecular phylogenies concur
that this species does not belong to Aphyocharacinae (Javonillo
et al., 2010; Oliveira et al., 2011).
As reported by Mirande (2010),Rachoviscus was provisionally
included within Aphyocharacinae based on general similarity be-
tween R. crassiceps and Inpaichthys kerri in overall body shape, col-
oration and, as previously noted by Géry and Junk (1977), the
presence of non-aligned premaxillary teeth. This latter trait, in
particular, is notable as it is also present in Hollandichthys
multifasciatus, and may therefore represent evidence for close
Fig. 1 (continued)
V.A. Tagliacollo et al. / Molecular Phylogenetics and Evolution 64 (2012) 297–307 301
Author's personal copy
relationship between Rachoviscus and Hollandichthys as already
proposed by other authors (Javonillo et al., 2010; Thomaz et al.,
2010; Oliveira et al., 2011).
Mirande (2010) also suggested that Inpaichthys be included
within Aphyocharacinae based on three morphological
synapomorphies. Nevertheless, the TE parsimony-based analysis
Fig. 2. Strict consensus of 16 most parsimony trees (18338 steps; 2006 parsimony-informative, CI: 0.2296; RI: 0.5832) based on TE analysis of 4423 molecular and 373
morphological characters concatenated. As with the model-based phylogenies, this cladogram suggests that Inpaichthys,Rachoviscus and Leptagoniates pi do not belong to
Aphyocharacinae. Dashed square limits Aphyocharacinae as proposed herein. This arrangement is supported by a single morphological synapomorphy (ch. 266: 1 > 0; RI: 0.62
RC: 0.43). Numbers in front of the branches correspond to bootstrap values. Numbers on the black bars indicate morphological synapomorphies. Further information can be
seen in Supplementary material B.
302 V.A. Tagliacollo et al. / Molecular Phylogenetics and Evolution 64 (2012) 297–307
Author's personal copy
recovered in the present study shows that Inpaichthys belongs to a
clade comprised of Nematobrycon,Carlana, and Astyanacinus, sup-
ported by five morphological synapomorphies (Fig. 2). One of these
synapomorphies is the absence or reduction of the fourth infraor-
bital canal bone (ch. 66: 0 > 1) that is derived independently in a
clade comprised of Aphyocharax and Prionobrama (Fig. 3). The
reduction of the infraorbital series in different characiform groups,
especially the fourth infraorbital bone, is widely thought to be
homoplastic (Vari, 1995; Zanata and Vari, 2005; Toledo-Piza,
2007; Mirande, 2010). Other synapomorphies for the clade com-
prised of Inpaichthys and its close relatives include aspects of the
third infraorbital canal bone (ch. 64: 1 > 0), premaxilla (ch. 104:
0 > 1), maxillary teeth (ch. 136: 0 > 1), and gill rakers (ch. 196:
0 > 1). Although these characters are homoplastic, most are not
optimized as basal within Aphyocharacinae.
3.2. Monophyly of Aphyocharax, Prionobrama, Paragoniates,
Phenagoniates, Leptagoniates, and Xenagoniates
The clade comprised of Aphyocharax,Prionobrama,Paragoniates,
Phenagoniates,Leptagoniates steindachneri, and Xenagoniates, but
excluding Inpaichthys,Rachoviscus, and Leptagoniates pi is a statis-
tically well supported group in both the model-based and parsi-
mony-based analyses (Figs. 1 and 2); additionally, this clade is
supported by eleven morphological synapomorphies (Fig. 3),
including seven identical to those proposed by Mirande (2010).
In relation to internal relationships, phylogenies recovered in this
study are congruent with molecular hypotheses of Oliveira et al.
(2011), however slightly different from morphological hypotheses
of Mirande (2009, 2010) in relation to the sister group of
Aphyocharax.
3.3. Aphyocharax as the sister to Prionobrama
The morphological phylogenies of Mirande (2009, 2010) pro-
posed that the three species of Aphyocharax studied form a natural
group that is the sister group of a clade comprised of Prionobrama,
Paragoniates,Phenagoniates, and Xenagoniates. Including also Lep-
tagoniates steindachneri, which shares a general resemblance to
Xenagoniates, this group is similar to that proposed by Géry
(1977). Herein, phylogenetic analyses recovered identical relation-
ships to that published by Oliveira et al. (2011), and a very similar
topology to those proposed by Mirande (2009, 2010), except for
the relationship between Aphyocharax and Prionobrama (Figs. 1
and 2). While morphological phylogenies indicated that Aphyocha-
rax is the sister group of all remaining aphyocharacins (Mirande,
2009, 2010), molecular phylogenies have placed Aphyocharax as
the sister to Prionobrama (Oliveira et al., 2011;Figs. 1 and 2).
Mirande’s (2010) hypothesis of relationship between Prionobra-
ma and all remaining aphyocharacins is supported by seven non-
exclusive synapomorphies. The TE parsimony-based phylogeny,
alternatively, indicates that Aphyocharax and Prionobrama form a
monophyletic group based on three morphological synapomorphies
(ch. 66: 0 > 1; ch. 138: 1 > 0; ch. 366: 0 > 1). One of these
synapomorphies is remarkable and unique; the lateral line is inter-
rupted with a single perforated scale on the posterior region of cau-
dal peduncle (ch. 366: 0 > 1; Fig. 4). This atypical state has not been
reported to any other characid species (Malabarba, 1998; Zanata and
Vari, 2005; Mirande, 2009, 2010), and therefore represents strong
Fig. 2 (continued)
V.A. Tagliacollo et al. / Molecular Phylogenetics and Evolution 64 (2012) 297–307 303
Author's personal copy
evidence for a close relationship between Aphyocharax and Prionob-
rama. Another synapomorphy is the absence or reduction of the
fourth infraorbital canal bone (ch. 66: 0 > 1; Fig. 5). This character
state is one of the synapomorphies proposed by Mirande (2010)
for the clade he called Aphyocharacinae; however it is only shared
by Inpaichthys,Aphyocharax, and Prionobrama, with a reversion to
the condition of having well developed fourth infraorbital in other
aphyocharacin species (Mirande, 2010). If Inpaichthys is excluded
from consideration as argued above, the absence or reduction of this
infraorbital bone may be viewed as shared only by Aphyocharax and
Prionobrama. A third synapomorphy recovered is the presence of a
single large cusp on anterior maxillary teeth (ch. 138: 1 > 0).
Although a single cusp is also present in Aphyodite grammica,Exodon
paradoxus, and Acestrocephalus sardine, the tooth morphology of Par-
agoniates,Phenagoniates,Leptagoniates and Xenagoniates is distinct
due to the presence of three or more small cuspids.
Fig. 3. Cladogram of the subfamily Aphyocharacinae (as defined in the present study) showing morphological synapomorphies along its branches. Numbers correspond to
characters reported by Mirande (2010), except 366 (0 > 1), 369 (0 > 1), 370 (0 > 1), and 371 (0 > 1) which are proposed here. See legend for symbols and RI values onbranches.
Fig. 4. Character 366 (0 > 1; CI: 1.0/RC: 1.0): lateral line interrupted with only the last scale of the series perforated (USNM 361472, Aphyocharax pusillus).
304 V.A. Tagliacollo et al. / Molecular Phylogenetics and Evolution 64 (2012) 297–307
Author's personal copy
3.4. Monophyly of Paragoniates, Phenagoniates, Leptagoniates, and
Xenagoniates
The clade comprised of Paragoniates,Phenagoniates,Leptagoni-
ates steindachneri, and Xenagoniates is statistically well supported
based on both mode-based and parsimony-based analyses (Figs.
1 and 2). Five morphological synapomorphies reinforce its mono-
phyly (Fig. 3), of which four were reported by Mirande (2010). Fur-
thermore, internal relationships recovered support the hypotheses
of Mirande (2009, 2010) and Oliveira et al. (2011).
The monophyly of the clade comprised of Phenagoniates,Leptag-
oniates steindachneri and Xenagoniates is statistically well supported
(Figs. 1 and 2); additionally seven synapomorphies reinforce its
monophyly (Fig. 3). Among these synapomorphies, six are identical
to those recovered by Mirande (2010). One additional synapomor-
phy is the higher number of dorsal-fin pterygiophores (ch. 276:
0 > 1), which evolved independently in Prionobrama, and also in a
clade comprised of some species of Stevardiinae (Fig. 2).
The monophyly of the clade comprised of Leptagoniates steind-
achneri plus Xenagoniates is also statistically well supported (Figs.
1 and 2). Although morphological synapomorphies are not yet
known for this clade, in part because Leptagoniates steindacheneri
was not examined by Mirande (2010), both species share at least
three unusual traits which could be evidence of their relationship.
These traits include a high number (30–40 vs. 15–17) of caudal
vertebrae, absence of the third postcheithrum, and a complete lat-
eral line. The former two characteristics are currently known only
from species that are distantly related to Aphyocharacinae (see
Mirande, 2009, 2010). Also, a complete lateral line has been con-
cluded to represent the plesiomorphic state in many characid taxa
(Malabarba, 1998; Zanata and Vari, 2005; Toledo-Piza, 2007;
Mirande, 2010). However, a complete lateral line may be a synapo-
morphy in the case of Leptagoniates steindachneri plus Xenagoni-
ates, since other aphyocharacins possess an incomplete lateral
line. Future studies may test this hypothesis, which if confirmed,
would represent the first case where a complete lateral line was re-
gained within the Characidae.
3.5. Monophyly of Aphyocharax and Prionobrama and polyphyly of
Leptagoniates
As previously recognized by Mirande (2010) Aphyocharacinae
was thought to include four monotypic genera and four genera
with multiple species: Prionobrama (2 spp.), Rachovischus (2
spp.), Leptagoniates (2 spp.), and Aphyocharax (11 spp.). Excluding
Rachoviscus due to its close relation to Hollandichthys, results of this
study indicate that Prionobrama and, most probably, Aphyocharax
are both monophyletic, while Leptagoniates is polyphyletic (Figs.
1 and 2).
The monophyly of Prionobrama is statistically well supported
(Fig. 1 and 2); additionally three morphological synapomorphies
reinforce this hypothesis (Fig. 3). According to the TE parsimony-
based phylogeny P. paraguayensis and P. filigera share characteris-
tics of the pelvic girdle and suspensorium including: coracoid as
long as deep (ch. 369: 0 > 1), mesocoracoid oriented obliquely with
respect to the posterior margin of the cleithrum (ch. 370: 0 > 1)
and bifurcated anterior border of the metapterygoid (ch. 371:
0>1;Fig. 6).
The monophyly of Aphyocharax is also well supported statisti-
cally (Figs. 1 and 2); additionally three morphological synapomor-
phies reinforce this hypothesis (Fig. 3). Although four described
species (A.erythrurus,A.yekwanae,A.gracilis, and A.agassizii) were
not included in this study, these species share the features of red
fin coloration, modest body elongation, single series of tricuspid
teeth on the premaxilla and mandible, and maxilla with teeth on
up to two thirds of its ventral margin that characterize the genus
Aphyocharax. The synapomorphies recovered herein are identical
to those proposed by Mirande (2010), which include: trigemino-
facialis foramen narrow, as a cleft with sphenotic almost excluded
from its margin (ch. 42: 0 > 1), dorsal projection of maxilla overlaps
the second infraorbital (ch. 102: 0 > 1), and dorsal development of
third postcleithrum not projecting dorsally to posterior region of
scapula (ch. 251: 0 > 1).
As noted above, the monophyly of Leptagoniates was not con-
firmed since L. steindachneri belongs to Aphyocharacinae while L.
Fig. 5. Character 66 (0 > 1; CI: 0.40/RC: 0.31): Fourth infra-orbital absent (LBP 4046, Aphyocharax pusillus) or reduced (LBP not cataloged, Prionobrama filigera).
Fig. 6. Character 371 (0 > 1; CI: 1.0/RC: 1.0): Anterior border of metapterygoid
bifurcated (LBP 3230, Prionobrama paraguayensis).
V.A. Tagliacollo et al. / Molecular Phylogenetics and Evolution 64 (2012) 297–307 305
Author's personal copy
pi is more closely related to species of Cheirodontinae (Figs. 1 and
2). The subfamily Cheirodontinae is currently diagnosed by four
synapomorphies including the lack of a humeral spot, presence
of a pseudotympanum, pedunculate teeth, and a single row of reg-
ular teeth on the maxilla (Malabarba, 1998). Both L.pi and L.steind-
achneri possess all these characteristics, and, additionally, share a
high number of anal-fin rays (usually more than 40). Nevertheless,
L.steindachneri has an elongate and slender body, like that of Xen-
agoniates, whereas the body shape of L. pi is much deeper. Also, un-
like L.steindachneri, the fin rays in L.pi rarely transcend a vertical
line through the last dorsal-fin ray. Furthermore, these species can
be distinguished by the number of vertebrae: 13 vs. 10 pre-caudal
vertebrae in L. pi, and 27 vs. 38 caudal vertebrae in L.steindachneri.
3.6. Phylogenetic position of Aphyocharacidium
The phylogenetic results of this study indicate that Aphyocha-
racidium bolivianum is the sister group to Aphyocharacinae (sensu
Mirande, 2010 – excluding Rachoviscus and Inpaichthys)(Figs. 1
and 2). This hypothesis is the same as that of Oliveira et al.
(2011). According to Mirande (2010),Aphyocharacidium is a mem-
ber of the Aphyoditeinae, which is itself the sister to Cheirodonti-
nae, and these subfamilies together represent the sister group of
Aphyocharacinae. However, the molecular results of Oliveira
et al. (2011) and those presented herein suggest that Aphyoditei-
nae is not monophyletic.
Although Mirande (2010) recognized eight genera in
Aphyoditeinae, the position of three of these genera (Leptobrycon,
Oxybrycon,Tyttobrycon) was provisional due to a lack of material
available for examination. The species sampling of Aphyoditeinae
used in this study included four of the five genera and species
examined by Mirande (2010):Aphyodite grammica,Microschemo-
brycon casiquiare,Parecbasis cyclolepis, and Aphyocharacidium
bolivianum. The results are similar to those of Oliveira et al.
(2011), since they suggest that Aphyodite grammica is closely
related to Hemigrammus ulrey,Microschemobrycon casiquiare is
closely related to Characinae (sensu Lucena and Menezes, 2003),
and Parecbasis cyclolepis is the sister group of a clade comprised of
Thayeria,Bario,Hollandichthys, and Rachoviscus (Figs. 1 and 2). The
molecular phylogenies additionally suggest that Aphyocharacidium
bolivianum is more closely related to Aphyocharacinae than
Cheirodontinae (Figs. 1 and 2).
Under a TE parsimony analysis, the relationship between
Aphyocharacidium bolivianum and all remaining aphyocharacins
(as recognized herein) is supported by a single morphological syn-
apomorphy: two dorsal-fin rays articulating with the first dorsal
pterygiophore (ch. 266: 1 > 0). Although this state is polymorphic
in some characid species (see also Mirande, 2010) it is invariable
in A. bolivianum and other aphyocharacin species examined
(n= 45 specimens – see Supplementary material B). Consequently,
character 266 is taken as evidence for a close relationship between
A. bolivianum and other aphyocharacins.
4. Conclusion
The relationships recovered in this study are similar to many of
those proposed by Mirande (2009, 2010), with some notable
exceptions. The results largely concur with the inclusion of Aphyo-
charax,Prionobrama,Paragoniates,Phenagoniates,Leptagoniates,
and Xenagoniates. However, the subfamily Aphyocharacinae as pro-
posed herein differs from that of Mirande (2010) in that it ex-
cludes: (1) Inpaichthys, which is closer to the distantly related
characid taxa Nematobrycon and Carlana;(2) Rachoviscus, which
is the sister group to the distantly related characid taxon Holland-
ichthys; and (3) Leptagoniates pi, which is more closely related to
species of Cheirodontinae than to the type of the genus L. steind-
achneri. The results also differ from Mirande (2010) in recognizing
a sister-group relationship between Aphyocharax and Prionobrama.
In conclusion, this study is the first to propose the inclusion of
Aphyocharacidium within the Aphyocharacinae. Given the phyloge-
netic results recovered herein, we propose to include Aphyocharac-
idium within the subfamily Aphyocharacinae, rather than place it
in Aphyoditeinae. Under this proposal the Aphyocharacinae in-
cludes seven genera (Aphyocharacidium,Aphyocharax,Prionobrama,
Paragoniates,Phenagoniates,Leptagoniates, and Xenagoniates) diag-
nosed by a single morphological synapomorphy: two dorsal-fin
rays articulating with the first dorsal pterygiophore.
Acknowledgments
We are grateful to all the individuals and institutions that as-
sisted us in the collection and identification of the specimens that
served as the basis for this study, with special thanks to the Aca-
demic of Natural Science of Philadelphia (ANSP). For loan of spec-
imens, hospitality during visits and other assistances we thank
Paulo A. Buckup, Marcelo Britto, Osvaldo T. Oyakawa, Mauro Nir-
chio, Mark H. Sabaj, John G. Lundberg, Carolina Doria, Hernán Ort-
ega, Ricardo M Corrêa e Castro, Janice Cunha, Gisela Farinelli,
Riviane Garcez, and Gislene Torrente-Vilara. We are also thankful
to Cristiano Moreira, Fábio F. Roxo, Flávio C.T. Lima, James S. Albert,
Juan M. Mirande, Mark Sabaj, Ricardo Britzke, and an anonymous
reviewer from their comments and suggestions. This paper is part
of the Thematic Project ‘‘Phylogenetic relationships in the Characi-
dae (Ostariophysi: Characiformes)’’ which was funded by Fundação
de Apoio à Pesquisa do Estado de São Paulo (FAPESP – proc. 04/
09219-6). VAT was financially supported by Conselho Nacional
de Desenvolvimento Científico e Tecnológico (CNPq) – proc.
132938/2009-0. R.C.B. was supported by Fundação do Instituto
de Biociências de Botucatu (FundBio). C.O. is CNPq researcher
(proc. 309632/2007-2).
Appendix A. Supplementary material
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.ympev.
2012.04.007.
References
Albert, J.S., Knouft, J.H., Johnson, D.M., 2009. Fossils provide better estimates of
ancestral body size than do extant taxa in fishes. Acta Zool. 90, 308–335.
Eigenmann, C.H., 1909. The fresh-water fishes of Patagonia and an examination of
the Archiplata – Archelenis Theory. In: Scott, W.B. (Ed.), Reports of the Princeton
University Expeditions to Patagonia, 1896–1899, Princeton, NJ, III(1), 8 pl.,
1905/1911, pp. 225–374.
Eigenmann, C.H., 1910. IV. Catalogue of the fresh-water fishes of tropical and south
temperate America. In: Scott, W.B. (Ed.), Reports of the Princeton University
Expeditions to Patagonia, 1896–1899, Princeton, NJ, III (2), pp. 375–511.
Eigenmann, C.H., 1912. The freshwater fishes of British Guiana, including a study of
the ecological grouping of species and the relation of the fauna to the plateau to
that of the lowlands. Mem. Carnegie Mus. 5, 1–578.
Eigenmann, C.H., 1915. The Cheirodontinae, a subfamily of minute characid fishes of
South America. Mem. Carnegie Mus. 7 (1), 1–99.
Eschmeyer, W., 2010. Catalog of Fishes. <http://www.calacademy.org/research/
ichthyology/catalog> (accessed 05.01.12).
Géry, J., 1960. Contributions to the study of characoid fishes, n°6. New
Cheirodontidae from French Guiana. Senck. Biol. 41, 15–39.
Géry, J., 1977. Characoids of the World. TFH publications, Neptune, 672pp.
Géry, J., Boutiére, H., 1964. Petitella Georgiae g. sp. nov. ov. (Pisces, Cypriniformes,
Characoidei). Vie & Millieu 17 (Suppl.), 473–484.
Géry, J., Junk, W.J., 1977. Inpaichthys kerri n. g. n. sp., um novo peixe caracídeo do
alto rio Aripuanã, Mato Grosso, Brasil. Acta Amazon 7 (3), 417–422.
Goloboff, P.A., Farris, J.S., Nixon, K.C., 2008. TNT, a free program for phylogenetic
analysis. Cladistics 24, 774–786.
Gregory, W.K., Conrad, G.M., 1938. The phylogeny of the Characin Fishes. Am. Mus.
Nat. Hist. 23, 319–360.
306 V.A. Tagliacollo et al. / Molecular Phylogenetics and Evolution 64 (2012) 297–307
Author's personal copy
Günther, A., 1864. Catalogue of the Physostomi, containing the families Siluridae,
Characinidae, Haplochitonidae, Sternoptychidae, Scopelidae, Stomiatidae. In:
Catalogue of the Fishes in the British Museum. British Museum of Natural
History, London, v. 5, xxii + 455p.
Günther, A., 1868. Diagnoses of some new fresh-water fishes from Surinam and
Brazil in the collection of the British Museum. Ann. Mag. Nat. Hist. Lond. 1 (4),
475–481.
Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor
and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41,
95–98.
Hillis, D.M., Bull, J.J., 1993. An empirical test of bootstrapping as a method for
assessing confidence in phylogenetic analysis. Syst. Biol. 42, 182–192.
Huelsenbeck, J.P., Ronquist, F., 2001. MrBayes: Bayesian inference of phylogenetic
trees. Bioinformatics 17, 754–755.
Javonillo, R., Malabarba, L.R., Weitzman, S.H., Burns, J.R., 2010. Relationships among
major lineages of characid fishes (Teleostei: Ostariophysi: Characiformes),
based on molecular sequence data. Mol. Phylogenet. Evol. 54, 498–511.
Katoh, K., Misawa, K., Kuma, K., Miyata, T., 2002. MAFFT: a novel method for rapid
multiple sequence alignment based on fast Fourier transform. Nucleic Acids
Res. 30, 3059–3066.
Katoh, K., Kuma, K., Toh, H., Miyata, T., 2005. MAFFT version 5: improvement in
accuracy of multiple sequence alignment. Nucleic Acids Res. 33 (2), 511–518.
Lucena, C.A.S., Menezes, N.A., 2003. Subfamily Characinae. In: Reis, R.E., Kullander,
S.O., Ferraris, C.J., Jr. (Eds.), Check List of the Freshwater Fishes of South and
Central America (CLOFFSCA). Porto Alegre, EDIPUCRS, pp. 200–208.
Malabarba, L.R., 1998. Monophyly of Cheirodontinae, caracters and major clades
(Ostariophysi: Characida). In: Malabarba, L.R., Reis, R.E., Vari, R.P., Lucena, Z.M.,
Lucena, C.A.S. (Eds.), Phylogeny and Classification of Neotropical Fishes.
EDIPUCRS, Porto Alegre, pp. 193–233.
Malabarba, L.R., Weitzman, S.H., 2003. Description of a new genus with six species
from Southern Brazil, Uruguay and Argentina with discussion of a putative
characid clade (Teleostei: Characiformes: Characidae). Comun. Mus. Ciênc.
Tecnol. PUCRS Ser. Zool. Porto Alegre 16, 67–151.
Mirande, J.M., 2009. Weighted parsimony phylogeny of the family Characidae
(Teleostei: Characiformes). Cladistics 25, 574–613.
Mirande, J.M., 2010. Phylogeny of the family Characidae (Teleostei: Characiformes):
from characters to taxonomy. Neotrop. Ichthyol. 8, 385–568.
Nylander, J.A.A., 2004. MrModeltest v2. Program Distributed by the Author.
Evolutionary Biology Centre, Uppsala University, Sweden.
Oliveira, C., Avelino, G.S., Abe, K.T., Mariguela, T.C., Benine, R.C., Ortí, G., Vari, R.P.,
Castro, R.M.C., 2011. Phylogenetic relationships within the speciose family
Characidae (Teleostei: Ostariophysi: Characiformes) based on multilocus
analysis and extensive ingroup sampling. BMC Evol. Biol. 11, 275. http://
dx.doi.org/10.1186/1471-2148-11-275.
Posada, D., 2008. JModelTest: phylogenetic model averaging. Mol. Biol. Evol. 25,
1253–1256.
Posada, D., Buckley, T.R., 2004. Model selection and model averaging in
phylogenetics: advantages of Akaike information criterion and Bayesian
approaches over likelihood ratio tests. Syst. Biol. 53, 793–808.
Rambaut, A., Drummond, A.J., 2004. Tracer v1.4. <http://beast.bio.ed.ac.uk/Tracer>.
Reis, R.E., Kullander, S.O., Ferraris, C.J., Jr., 2003. Check List of the Freshwater Fishes
of South and Central America (CLOFFSCA). Porto Alegre, EDIPUCRS, p. 729.
Ronquist, F., Huelsenbeck, J.P., 2003. MrBayes 3: Bayesian phylogenetic inference
under mixed models. Bioinformatics 19, 1572–1574.
Stamatakis, A., 2006. RAxML-VI-HPC: maximum likelihood-based phylogenetic
analyses with thousands of taxa and mixed models. Bioinformatics 22, 2688–
2690.
Stamatakis, A., Hoover, P., Rougemont, J., 2008. A rapid bootstrap algorithm for the
RAxML web servers. Syst. Biol. 57, 758–771.
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5:
molecular evolutionary genetics analysis using maximum likelihood,
evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28
(10), 2731–2739.
Taylor, W.R., Van Dyke, G.C., 1985. Revised procedures for staining and clearing
small fishes and other vertebrates for bones and cartilage. Cybium 9, 107–119.
Thomaz, A.T., Malabarba, L.R., Bonatto, S.L., 2010. The phylogenetic placement of
Hollandichthys Eigenmann 1909 (Teleostei: Characidae) and related genera.
Mol. Phylogenet. Evol. 57, 1347–1352.
Toledo–Piza, M., 2007. Phylogenetic relationships among Acestrorhynchus species
(Ostariophysi: Characiformes: Acestrorhynchidae). Zool. J. Linnean Soc. 151,
691–757.
Vari, R.P., 1995. The Neotropical fish family Ctenoluciidae (Teleostei: Ostariophysi:
Characiformes): supra and infrafamilial phylogenetic relationships, with a
revisionary study. Smithson Contrib. Zool. 564, 1–97.
Xia, X., Lemey, P., 2009. Assessing substitution saturation with DAMBE. In: Lemey,
P., Salemi, M., Vandamme, A.M. (Eds.), The Phylogenetic Handbook: A Practical
Approach to DNA and Protein Phylogeny, second ed. University Press,
Cambridge, pp. 615–630.
Xia, X., Xie, Z., 2001. DAMBE: data analysis in molecular biology and evolution. J.
Heredity 92, 371–373.
Xia, X., Xie, Z., Salemi, M., Chen, L., Wang, Y., 2003. An index of substitution
saturation and its application. Mol. Phylogenet. Evol. 26, 1–7.
Zanata, A.M., Vari, R.P., 2005. The family Alestidae (Ostariophysi: Characiformes): a
phylogenetic analysis of a Trans-Atlantic Clade. Zool. J. Linnean Soc. 145, 1–144.
V.A. Tagliacollo et al. / Molecular Phylogenetics and Evolution 64 (2012) 297–307 307
