DOI: 10.1111/jbi.
13207
ORIGINAL ARTICLE
Bayesian analyses detect a history of both vicariance and
geodispersal in Neotropical freshwater fishes
Carolina Barros Machado1
1
Departamento de Gene
tica e Evolucß~ao,
Universidade Federal de S~ao Carlos, S~ao
Carlos, SP, Brazil
2
Department of Biology, City College of
New York and The Graduate Center of the
City University of New York, New York,
NY, USA
Correspondence
Carolina Barros Machado, Departamento de

tica e Evolucß~
Gene ao, Universidade Federal
de S~ao Carlos, S~
ao Carlos, SP, Brazil.
Email: carolbioms@gmail.com
Funding information
Coordenacß~ao de Aperfeicßoamento de
Pessoal de N
ıvel Superior; National Science
Foundation, Grant/Award Number: DEB
1120487, 1343578; Conselho Nacional de

gico,
Desenvolvimento Cient
ıfico e Tecnolo
Grant/Award Number: CNPq 308385/2014-
4, 563299/2010-0; Fundacß~ao de Amparo a
Pesquisa do Estado de S~ao Paulo, Grant/
Award Number: BIOTA 2013/50297-0,
FAPESP 10/52315-7
Editor: Luiz Rocha
Journal of Biogeography. 2018;1–13.
| Pedro Manoel Galetti Jr1 | Ana Carolina Carnaval2
Abstract
Aim: Among the main biogeographical hypotheses explaining the remarkable diver-
sity of fishes in the Neotropics is the “palaeogeographical hypothesis”, focusing on
vicariance, and the “hydrogeological hypothesis”, focusing on geodispersal. Yet while
reflecting different processes, they may result in similar biogeographical patterns.
We employed a model-based Bayesian approach to test these alternative hypothe-
ses and determine which shaped the phylogeographical patterns observed in a
group of Neotropical fishes.
Location: South America.
Taxon: Salminus.
Methods: We used mitochondrial and nuclear markers to infer phylogenetic rela-
tionships and estimate divergence times among Salminus species, associating the
results with known geological events. We then employed approximate Bayesian
computation (ABC) to explore changes in population size over time, asking whether
vicariance or geodispersal events best explain the phylogeographical signature
observed in the data. Because geodispersal captures a few individuals from a paren-
tal population, which can then expand and lead to a new lineage, we expect to find
genetic signatures of a founder event following population expansion under this
scenario, but not under vicariance.
Results: The analyses suggest that the diversification process in Salminus
began in Upper Miocene, and ABC indicates that it involved both vicariance
and geodispersal events: while a vicariance event better explains the
phylogeographical structure within S. brasiliensis and the genetic patterns of
differentiation between S. sp. Amazon and S. sp. Araguaia, geodispersal
appears to have been the most important event structuring lineages of Salminus
hilarii.
Main Conclusions: Both vicariance and geodispersal signatures were detected in
our biological model, inferring a complex yet realistic demographic history of
Salminus lineages. The correspondence between the ABC results and traditional
phylogeographical interpretations provide further confidence in the models
drawn and tested. This study reinforces the value of applying an ABC
framework in phylogeographical studies, particularly for those interested in testing
alternative and biologically plausible processes underlying similar biogeographical
patterns.
wileyonlinelibrary.com/journal/jbi © 2018 John Wiley & Sons Ltd | 1
2 |
KEYWORDS
ABC analysis, fish diversification, historical biogeography, phylogeography, Salminus, South
America
1 | INTRODUCTION
Multiple hypotheses have been invoked to explain the remarkable
diversity and the complex process of diversification in the Neotropics
(Leite & Rogers, 2013). Yet, most phylogeographical studies to date
have focused on terrestrial organisms (Beheregaray, 2008). Freshwa-
ter species, through their direct associations with hydrographic
basins, offer an unmatched opportunity to study the dynamics of the
Neotropical hydrological landscape and the historical geomorphologi-
cal processes that shaped it (Hubert & Renno, 2006; Hubert et al.,
2007; Montoya-Burgos, 2003; Sivasundar, Bermingham, & Ort
ı,
2001; Tagliacollo, Roxo, Duke-Sylvester, Oliveira, & Albert, 2015).
The major river systems in South America began their develop-
ment with the breakup of Gondwana (c. 90 million years ago—Ma).
However, most of them reached their modern configuration recently,
after being heavily influenced by the middle Miocene-Pleistocene
phase of uplift of the Andes (Hoorn, Guerrero, Sarmiento, & Lorente,
1995; Lundberg et al., 1998). Through compressional tectonics,
Andean orogeny is thought to have promoted both disruption and
coalescence of drainage systems (Lundberg et al., 1998). The fluctu-
ating sea levels and climate changes also largely impacted connec-
tions across rivers (Albert, Petry, & Reis, 2011). This dynamism of
hydrographic systems is the main promoter of diversification events
in freshwater species.
Based on the transformations of the hydrological landscape, two
main hypotheses have been employed to explain the (presumably)
largely allopatric processes of diversification of Neotropical freshwa-
ter species (Hubert & Renno, 2006). On the one hand, the “palaeo-
geographical hypothesis” has focused on the emergence of physical
barriers fragmenting an ancestral aquatic area, particularly resulting
from tectonic readjustment, as major drivers of diversification; stud-
ies of palaeoarches of the Amazon basin are one such example
(Albert, Lovejoy, & Crampton, 2006). This hypothesis invokes vicari-
ance scenarios related to the formation of impermeable barriers and
consequent interruption of gene flow, leading to speciation (Ho
et al., 2015). According to Hubert et al. (2007), it also predicts that
in the taxon-area relationships, the lineages or species occurring in
hydrographic basins separated by geological barriers constitute recip-
rocally monophyletic clades. On the other hand, the “hydrogeological
hypothesis” has emphasized headwater capture events between
basins as an important trigger of colonization of new areas, and sub-
sequent differentiation (Hubert & Renno, 2006; Montoya-Burgos,
2003). This scenario implies in the temporary removal of barriers
between adjacent basins, enabling mixing of local assemblages, gene
flow and range expansion—a process often referred to as geodisper-
sal (Ho et al., 2015; Lieberman & Eldredge, 1996). Under this scenar-
io, lineages or species newly formed through geodispersal are
MACHADO ET AL.
expected to be nested within a clade of fishes that occupy adjacent
basins (Hubert et al., 2007).
Although they reflect different processes, identifying whether
allopatric sister species diverged through vicariance or geodispersal
has been challenging, particularly when new barriers emerge after
headwater capture, or in the face of local extinctions (Burridge,
Craw, & Waters, 2006; Kodandaramaiah, 2010). Model-based analy-
sis such as approximate Bayesian computation (ABC; Beaumont,
Zhang, & Balding, 2002) has allowed the distinction between models
involving vicariance and dispersal processes in terrestrial organisms,
resulting in evidences of founder events to distinguish them (Perez,
Bonatelli, Moraes, & Carstens, 2016; Prates, Rivera, Rodrigues, &
Carnaval, 2016). ABC is a flexible class of Monte Carlo algorithms
that bypass the calculation of exact likelihoods using summary statis-
tics and simulation. Its use has increased in population genetics and
phylogeographical studies, allowing a quantitative evaluation of the
demographic and evolutionary history by strictly contrasting realistic
models defined a priori and estimating relevant parameters (Inoue,
Lang, & Berg, 2015; Vanhaecke et al., 2015; Willis et al., 2015).
Building on recent advances in how to test alternative demo-
graphic hypotheses, and on the availability of a user-friendly of ABC
plataform (Cornuet et al., 2014), we explored the use of this analyti-
cal tool to distinguish between phylogeographical signatures of
vicariance (palaeogeographical hypothesis) versus geodispersal (hy-
drogeological hypothesis) in the evolutionary history of South Ameri-
can fishes. For that, we focused on the genus Salminus. The
monophyly of this group (Machado, Ishizuka, Freitas, Valiati, &
Galetti, 2017), the large migratory capacity of its species (Lima &
Britski, 2007), and their allopatric ranges along South America’s
major hydrographic basins render it a biological model to clarify the
role of vicariance and geodispersal processes on the diversification
of South American fishes, and to ask how local landscape changes
may have impacted the riverine biota.
To study how former landscape changes impacted the distribution
patterns of lineage diversity in Salminus species, we used mitochon-
drial and nuclear markers to reconstruct the phylogenetic relationships
within the genus, including samples from all molecular operational tax-
onomic unit defined previously by Machado et al. (2017). Following
other phylogeographical studies of South American freshwater sys-
tems (Ramirez, Birindelli, & Galetti, 2017; Tagliacollo et al., 2015; Wil-
lis et al., 2015), we used our trees to estimate divergence times among
lineages, and tentatively associated them with geological events
known to have changed the hydrologic landscape (thus potentially
flagging relevant processes in the group diversification). Going beyond
that, however, we employed an ABC statistical framework to specifi-
cally identify which biogeographical scenario (palaeogeographical or
hydrogeological hypotheses) better explains the genetic structure
MACHADO ET AL. | 3

observed within our focus group. Given the loci available, we were
able to investigate which process(es) was(were) influential in structur-
ing phylogeographical diversity within Salminus brasiliensis and Salmi-
nus hilarii, as well as those underscoring the divergence between
Salminus sp. Amazon and Salminus sp. Araguaia.
Throughout this analysis, we expected that diversification events
triggered by geodispersal (that is, facilitated by headwater capture
events that characterize the hydrogeological hypothesis) would leave
a signature of genetic founder events following population expansions
in colonized areas. This reasoning is justified by the fact that headwa-
ter capture is expected to isolate a small number of individuals from
their parental population (Yeung et al., 2011). This is particularly evi-
dent when widely distributed large and medium sized species that
occupy first- and second-order rivers are used as biological model.
Although many authors propose that the headwater captures can
involve both geodispersal and vicariance events (Albert et al., 2011),
we here adopt headwater captures as a geodispersal scenario because
the genetic signature of founder event leaves strong footprint in the
genome. We expected that the severe population reduction associated
with geodispersal would lead to a reduction in genetic variation that
can be detected by the ABC analysis (Perez et al., 2016; Prates et al.,
2016). On the other hand, diversification caused by vicariance, as per
the palaeogeographical hypothesis, is not expected to be tied to foun-
der bottlenecks (Figure 1). To verify which signature is prevalent
within species of Salminus, we explicitly simulated alternative scenar-
ios of temporal shifts in population size concomitant with lineage
divergence, and compared them to our observed (empirical) data.
2 | MATERIALS AND METHODS
2.1 | Sampling of molecular data
We sampled 94 specimens of Salminus across distinct South America
basins, including representatives of four named species (Salminus affi-
nis [Steindachner, 1880], Salminus brasiliensis [Cuvier, 1816], Salminus
franciscanus (Lima & Britski, 2007), and Salminus hilarii [Valenciennes,
1850]) and two undescribed species (Salminus sp. Amazon and Salmi-
nus sp. Araguaia) identified by Machado et al. (2017) (Figure 2, see
Appendix S1 in Supporting information for more sample details).
Partial sequences of three mitochondrial regions (cytochrome C
oxidase I [COI], cytochrome b [CytB], and D-loop) and two nuclear
markers (the recombination activating gene 2 [RAG2] and intron I of
the S7 ribosomal protein gene [S7]), were amplified with primers
described in Appendix S2. Polymerase chain reaction (PCR) condi-
tions, PCR products purification, and sequencing were carried out as
described in Machado et al. (2017). All mitochondrial and nuclear
sequences are available through GenBank (see Appendix S1 for
accession numbers). We also combined available COI and RAG2
sequences (Machado et al., 2017) with our dataset.
Sequences were aligned and edited in GENEIOUS R7 6.1.6 (http://
www.geneious.com, Kearse et al., 2012). Because insertion/deletion
heterozygote individuals were found in S7 gene sequences from S.
brasiliensis, S. hilarii and S. sp. Amazon, hence leading to overlapping
peaks, we used CODONCODE ALIGNER 4.2.7 (CodonCode Corporation,
Dedham, MA, USA) to find and process the heterozygous indels. For
all nuclear sequences, we used GENEIOUS’ plugin Find heterozygotes
with a 0.80 threshold to identify heterozygous positions and assign
ambiguity codes. Then, we reconstructed the haplotypic phase of
the heterozygotes with PHASE 2.1 (Stephens & Donnelly, 2003), using
SEQPHASE to format the input files (Flot, 2010).
2.2 | Phylogenetic inference, divergence time
estimation and ancestral area estimation
Before conducting a phylogenetic analysis, we checked for possible
recombination in the nuclear markers using RDP 3.44 (Martin &
Rybicki, 2000) and setting the highest acceptable p value to infer
recombination to 0.01. We also assessed substitution saturation for
all markers through an Index of substitution saturation based on
entropy in DAMBE (Xia, Xie, Salemi, Chen, & Wang, 2003). Models of
nucleotide evolution were detected with JMODELTEST 2.1.4 (Posada,
2008) using the Bayesian information criterion.

F I G U R E 1 Evolutionary scenarios drawn to test palaeogeographic and hydrogeological hypotheses. (1) Palaeogeographical hypothesis
shows a vicariance signature. In this scenario, we assumed one ancestral population not sampled, with effective size Na, that at time t2
suffered split and two new populations were formed with effective size lower than Na (N1 and N2). (2) The hydrogeological hypothesis has a
founder event signature following by population expansion in the receiver basin. We assumed a small number of founders (N1f or N2f, where
“f” means founder), originated from source-basin, colonized the adjacent basin after a geodispersal event, caused by headwater capture, at t2,
followed by population expansion at t1
4 |
For phylogenetic inference, we used the species tree method
implemented by the *BEAST tool in BEAST 1.8.1 package (Drummond,
Suchard, Xie, & Rambaut, 2012). Brycon specimens were used to root
the tree, following Oliveira et al. (2011). Because the D-loop data
showed much higher levels of saturation relative to the other mark-
ers, precluding our dating analyses from convergence, we opted to
exclude it from the phylogenetic study and the divergence time
reconstruction, yet kept it in the ABC analysis. The species tree was
thus inferred with three independent loci: the two mitochondrial
genes combined (COI and CytB), and two nuclear loci (S7 and RAG2).
Because species tree method requires a priori assignment of indi-
viduals to species, we considered as putative species both the mito-
chondrial lineages defined by DNA barcoding (Machado et al., 2017)
and the basin of occurrence. Thus, we included samples from the
following lineages: S. affinis, S. franciscanus, S. sp. Amazon, S. sp. Ara-
guaia, S. brasiliensis from the Upper Paran
a basin, S. brasiliensis from
the Paraguay and Uruguay basins and the Lagoa dos Patos drainage
(hereafter named PUL lineage), and three S. hilarii lineages (two lin-
eages from S~ao Francisco basin [hereafter named SF1 and SF2] and
one from Upper Paran
a basin).
Divergence times were estimated under an uncorrelated lognor-
mal relaxed-clock model in *BEAST. We calibrated the phylogeny
using a biogeographical event, the uplift of the Eastern Andes Cordil-
lera (Hoorn et al., 1995), which separated the Magdalena and Ama-
zon palaeobasins and is consistent with the divergence of S. affinis
from all other Salminus. The following features were implemented:
normal prior distribution of the time of the most recent common
MACHADO ET AL.
F I G U R E 2 Sampling sites of Salminus in
South America. Salminus affinis (♦),
Salminus brasiliensis (■), Salminus
franciscanus (★), Salminus hilarii (▲),
Salminus sp. Amazon ( ), and Salminus sp.
Araguaia ( ). Colors represent the
hydrographic basins where samples were
collected
ancestor (MRCA), with offset of 11.8 Ma and standard deviation
2 Ma (95% of the highest posterior density [HPD] = 7.88 and
15.72), uniform prior distribution (interval = 0–1) of the mean rate of
the molecular clock (ucld.mean parameter), Yule model as species
tree prior, with an unlinked clock model across markers, and default
setting of substitution rates and nucleotide frequency parameters.
Three independent Markov chains were started from random
trees, ran independently for 200 million generations, and sampled at
every 10,000 steps. We then combined all runs in LOGCOMBINER 1.8.1
with 25% burn-in, and summarized a maximum clade credibility tree
in TREEANNOTATOR 1.8.1 (BEAST 1.8.1 package). Proper mixing was veri-
fied with TRACER 1.5 (Rambaut & Drummond, 2007), and an effective
sample size (ESS) of 200 or higher was required for all parameters.
To facilitate our understanding about what historical processes
are important, mainly in the early evolutionary history of Salminus,
we reconstructed ancestral area through the maximum likelihood
approach using the “BioGeoBEARS” package (Matzke, 2013) for R
(http://www.R-project.org; detailed analysis in Appendix S3). After
theses procedures, we compared the divergence times of the species
and lineages with know geoclimatic events.
2.3 | Testing biogeographical scenarios by ABC
We used all mitochondrial (COI, CytB and D-loop) and nuclear
(RAG2 and S7) markers in an ABC statistical framework to test
which biogeographical scenario best matched the current phylogeo-
graphical structure of the following lineages: S. brasiliensis from the
MACHADO ET AL.
Upper Paran

a basin, S. brasiliensis from the Paraguay, Uruguay, and
Lagoa dos Patos hydrographic systems (PUL lineage), S. hilarii from
the Upper Paran

a basin, two monophyletic S. hilarii lineages from the
S~ao Francisco basin, and the clade composed of S. sp. Amazon and
S. sp. Araguaia. Because we used a small number of loci, we did not
expect to find clear, strong signatures of old demographic history
among ancestral populations in deep nodes. Instead, we focused our
efforts on testing alternative scenarios that may explain recent diver-
gence processes. The chosen lineages are ideal to an ABC-based test
of alternative biogeographical scenarios because both the vicariance
and the geodispersal hypotheses appear to be plausible explanations
for the distribution patterns observed within Salminus, as well as the
divergence times associated with Salminus lineages.
For the ABC analysis we used the DIYABC 2.0.4 (Do It Yourself
ABC—Cornuet et al., 2014). DIYABC considers four categories of
events: population divergence, discrete change in effective popula-
tion size, admixture and sampling (to allow considering samples
taken at different times). In this study, we evaluated if population
divergence was followed by discrete change in effective population
size to differentiate geodispersal (which implies a founder effect)
from vicariance (no founder effect). Thus, we tested two classes of
biogeographical scenarios independently for each lineage described
above (Figure 1): a geodispersal event with founder effect (simulat-
ing a hydrogeological hypothesis) and a vicariance scenario (simulat-
ing a palaeogeographical hypothesis). For each scenario, present-day
lineages were set at time t0, and divergence was set occur at t2. In
the case of the two S. brasiliensis lineages, and of the clade com-
posed of S. sp. Amazon and S. sp. Araguaia, the scenarios were
drawn as follows: the vicariance scenario assumed one (unsampled)
ancestral population with an effective population size of Na, which
split into two populations of effective size lower than Na (N1 and
N2) at t2. These daughter populations were then simulated to evolve
independently, and at t0 represent different lineages within a named
species (in the case of S. brasiliensis), or different species (in the case
of S. sp. Amazon and S. sp. Araguaia). In turn, the geodispersal sce-
nario assumed that a small number of founders (N1f or N2f, where
“f” means founder) originated from a sampled source-basin, colo-
nized the adjacent basin after a headwater capture event at t2, and
underwent population expansion at t1. There are two scenarios
within this class because we allowed the source-basin identity to
vary. As with the vicariance scenario class, the populations were sim-
ulated to evolve independently after the founder event, until t0 (Fig-
ure 3a, b). The major differences between the vicariance and
geodispersal scenarios are: (1) an (unsampled) ancestral population is
incorporated in the vicariance scenario, while it does not exist in the
geodispersal scenario given that one daughter population came from
the source-basin; (2) changes in the effective size of the daughter
populations at t2: while the vicariance scenario does not include sig-
nificant differences between the size of daughter populations, the
geodispersal model simulates a severe reduction in the effective
population size of the lineage that is putatively colonizing a new area
due to the headwater capture event; (3) demographic expansion is
simulated after colonization in geodispersal scenario, but not under
| 5
the vicariance scenario. This founder event is expected in Salminus
species because they occur in first and second-order rivers (Lima &
Britski, 2007); a headwater capture event is thus able to move just a
few individuals.
In the case of S. hilarii, in which three well-structured mitochon-
drial lineages are observed, we added more parameters to the model
and modified the scenarios to be tested. Because there are two S~ao
Francisco lineages, and given that the identity of which one emerged
first is important, the choice of the best scenario was made in two
steps. Initially, each biogeographical scenario was tested separately
(geodispersion with the S~ao Francisco as the source basin, geodis-
persion with the Upper Paran
a as the source basin, and a vicariance
scenario class—Figure S4.2 and results are in Table S4.4,
Appendix S4). The second round compared the models with highest
posterior probabilities resulting from each biogeographical scenario
class calculated in the first round (Figure 3c). Details of the prior dis-
tribution of parameters, summary statistics, model comparison,
parameters estimation and precision, and model checking are
described in Appendix S4.
3 | RESULTS
3.1 | Phylogenetic relationship among Salminus
species
The phylogeny recovered S. affinis, the only trans-Andean species, as
an early divergent lineage that is sister to two main clades: one com-
posed of S. sp. Amazon and S. sp. Araguaia (hereafter called the
Northwest clade), and other comprised of S. brasiliensis, S. francis-
canus and S. hilarii (hereafter called the Southeast clade; Figure 4). In
the Northwest clade, two groups of S. sp. Amazon were identified,
each from unique localities (Upper Amazon and Madeira basins), yet
with low support (<70%). For this reason, we pooled data from both
areas and consider S. sp. Amazon as a single lineage in our biogeo-
graphical tests. In the Southeast clade, S. franciscanus was placed as
the sister group of S. brasiliensis and S. hilarii. The relationship
between S. brasiliensis and S. hilarii was weakly supported (node 6),
and the nuclear and mitochondrial markers supported different
topologies regarding this pair of taxa: S. brasiliensis was paraphyletic
in the mitochondrial tree, while the nuclear intron S7 recovered it as
a monophyletic species (data not shown).
3.2 | Divergence times within Salminus and
ancestral area estimation
Dating analyses suggested that the diversification process in Salminus
began in the Late Miocene (Figure 4). Our biogeographical calibration
places the Salminus MRCA in the proto-Amazon region at about
11.05 Ma (95% HPD = 7.02–14.98 Ma). The next divergence event
suggested by the analysis, between the Northwest clade (Amazon and
Araguaia basins) and the Southeast clade (La Plata basin—Paraguay,
Uruguay, Paran
a basins—Lagoa dos Patos drainage, and S~ao Francisco
basin), places the split at 7.76 Ma (95% HPD = 4.01—11.64 Ma). All
6 | MACHADO ET AL.

F I G U R E 3 Evolutionary scenarios tested using DIYABC in (a) Salminus brasiliensis, (b) Salminus sp. Amazon and S. sp. Araguaia, and (c)
Salminus hilarii from South America. Colors represent the hydrographic basins where samples were collected. N: effective population size; Nf:
founder effective population size; t: time. Time and effective population size are not strictly to scale

remaining diversification processes among species were placed in the
Early Pliocene (Figure 4). We also estimated the diversification time of
the S. brasiliensis lineages (Upper Paran
a and PUL lineages diverged at
0.36 Ma, 95% HPD = 0.11–0.72 Ma), and S. hilarii lineages (Upper
Paran
a and S~
ao Francisco lineages split at 1.36 Ma, 95% HPD = 0.51–
2.53 Ma, and the sympatric lineages in the S~ao Francisco basin are esti-
mated to have diverged at 0.68 Ma, 95% HPD = 0.17–1.44 Ma; Fig-
ure 4).
Of the six biogeographical models that we implemented in “Bio-
GeoBEARS”, DIVALIKEj (Disperal-vicariance plus founder-event spe-
ciation) provided the best statistical fit to our data (AIC weights
(>0.90) and see Table S3.3, Appendix S3). The most probable distri-
bution area of MRCA of all Salminus was in the proto-Amazon
region. The model also recovered that the MRCA of the Northwest
and Southeast clades was present in the palaeo-Amazon (Amazon
and Tocantins-Araguaia basins). The MRCA of the Northwest clade
continued in the palaeo-Amazon where it diverged to form S. sp.
Araguaia and S. sp. Amazonas, while the common ancestor of the
Southeast clade was in the S~ao Francisco basin and later reached
the other basins of the Brazilian shield (Paran
a, Paraguay, Uruguay,
and Lagoa dos Patos—Figure S3.1, Appendix S3).
3.3 | Testing biogeographical hypotheses with ABC
The demographic reconstruction resulting from the ABC analysis
identified vicariance (palaeogeographical hypothesis) as the most
highly supported event to explain the current phylogeographical
structure of S. brasiliensis, and the divergence process between S. sp.
Amazon and S. sp. Araguaia. In contrast, geodispersal (hydrogeologi-
cal hypothesis) was the event more highly supported to explain
diversity within S. hilarii (Table 1).
All estimated parameters of the winning scenarios and their rela-
tive median of the absolute error (RMAE) can be found in Table 2.
The RMAE values were moderately low, indicating reliable estimation
of all parameters, except NUPf (0.835) and NSF2 (0.337) estimated
from the S. hilarii winning scenario. Because both parameters were
MACHADO ET AL. | 7

F I G U R E 4 Bayesian phylogenetic analysis and divergence time estimative in BEAST 1.8.1 using two mitochondrial (CytB and COI) and two
nuclear (RAG2 and S7) markers. We used one biogeography event calibration (red circle). Numbers below nodes represent divergence time
estimates, while numbers above nodes represent the posterior probability. Blue bars represent 95% highest posterior probability of the time
estimated

not used here, this result did not affect our evaluation about drawn
and tested scenarios. Winning scenarios for each species showed
high type I error rates, yet type II error rates were low (Table S4.6—
Appendix S4). This provides greater confidence in the best scenario
proposed by the ABC analysis.
The winning scenario for S. brasiliensis inferred the median
effective population sizes for the Upper Paran
a and PUL lin-
eages as NUP = 85,300 and NPUL = 136,000, respectively. Both lin-
eages were inferred to have diverged from an ancestral population
of median effective population size of 685,000, at t2 = 0.892 Ma.
For the clade comprising S. sp. Amazon and S. sp. Araguaia, the
scenario with highest posterior probability recovered an ancestral
population (Na = 582,000) splitting at t2 = 2.36 Ma. Median
effective population sizes at present were inferred as
NAM = 155,000 for S. sp. Amazon and NToA = 30100 for S. sp. Ara-
guaia.
The winning scenario for S. hilarii modeled the Upper Paran
a
basin as having captured part of the S~ao Francisco basin (source-
basin). This headwater capture was inferred to have happened at
t2 = 1.952 Ma. A small number of founders (NUPf = 5,040), origi-
nated from SF1 lineage, was recovered as having colonized the
Upper Paran
a basin and then expanded at 1.592 Ma, t1. Divergence
time between the two sympatric lineages in the S~ao Francisco basin
was inferred as ts = 0.606 Ma. The median effective population
sizes of these lineages at present were estimated at NSF1 = 446,000,
NSF2 = 441,000, and NUP = 460,000.
8 |
T A B L E 1 Bayesian posterior probabilities for each tested scenario estimated using DIYABC for Salminus brasiliensis, Salminus sp. Amazon and
S. sp. Araguaia, and Salminus hilarii. The highest posterior probability is in bold. Colors in the winner scenario column represent the
hydrographic basins where samples were collected.
Salminus brasiliensis
Scenario 1- Vicariance event
Scenario 2- Paraguay and Uruguay basins, and Lagoa dos
Patos drainage captures part of Upper Paran
a basin
(geodispersal event)
Scenario 3- Upper Paran
a basin captures part of Paraguay
and Uruguay basins, and Lagoa dos Patos drainage
(geodispersal event)
Salminus sp. Amazon and Salminus sp. Araguaia
Scenario 1- Vicariance event
Scenario 2- Tocantins-Araguaia basin captures part of
Amazon basin (geodispersal event)
Scenario 3- Amazon basin captures part of Tocantins-Araguaia
basin (geodispersal event)
Salminus hilarii (Round 2)
Scenario 1 - Upper Paran
a and S~ao Francisco_2 lineages
diverge from ancestral population (Vicariance event)
Scenario 2 - Upper Paran
a basin captures part of S~ao Francisco
(SF1) (geodispersal event)
Scenario 3 - S~ao Francisco (SF2) basin captures part of Upper
Paran
a basin (geodispersal event)
4 | DISCUSSION
4.1 | Salminus phylogeny
Our multiloci phylogenetic reconstruction of Salminus detected the
intraspecific structure and solved most interspecific relationships
within the group. All nodes were strongly supported, except for
the relationship between S. brasiliensis and S. hilarii (node 6,
Figure 4). Poor resolution in that portion of the tree may result
from incongruence among the mitochondrial and nuclear markers,
which is commonly observed in other organisms and expected
under the coalescent theory (D
avalos, Cirranello, Geisler, & Sim-
mons, 2012).
The inferred tree (Figure 4) conflicts with two other phylogenies
previously proposed for the group, which were based on morpholog-
ical (F.C.T. Lima, personal communication) and molecular data (Abe,
Mariguela, Avelino, Foresti, & Oliveira, 2014). Our analysis strongly
supports S. affinis as a sister species to all remaining taxa. In con-
trast, both previous phylogenies recovered S. affinis as sister to S. sp.
(a species composed of S. sp. Amazon and S. sp. Araguaia). More-
over, Abe et al. (2014) recovered S. hilarii as the early divergent spe-
cies and sister of all remaining Salminus, a scenario completely
different from ours results.
To understand the reasons why Abe et al.’s (2014) molecular
data led to such distinct conclusions, we re-analysed the gene trees
MACHADO ET AL.
Posterior probability (95% CI) Winner scenario
0.4255 (0.4184-0.4325)
0.3566 (0.3497-0.3634)
0.2180 (0.2117-0.2242)
0.5044 (0.4929-0.5158)
0.2063 (0.1975-0.2151)
0.2893 (0.2788-0.2998)
0.2695 (0.2376-0.3013)
0.6596 (0.6212-0.6981)
0.0709 (0.0000-0.1431)
from that study, built from sequences of 16SrRNA, Cytochrome B,
recombination activating gene 1 and 2, and the myosin heavy chain
6 cardiac muscle. Among those molecular markers, only RAG2
showed a distinct topology, placing S. hilarii as the early divergent
species. Gene trees built with all other molecular markers utilized in
the Abe et al. (2014) analysis, as well as with those used in the pre-
sent study, recovered S. hilarii as phylogenetically closer to S. francis-
canus and S. brasiliensis. Given that inconsistency, we ran a new
Bayesian Inference analysis with RAG2 sequences of all Bryconidae
species available in GenBank (Abe et al., 2014; Oliveira et al., 2011),
also including our own RAG2 dataset (data not shown). All Salminus
sequences except by one—the S. hilarii specimen used by Abe et al.
(2014)—formed a monophyletic group with strong support. That S.
hilarii sequence, in turn, clustered with Brycon specimens, suggesting
that it had been mislabeled at some point of the laboratory work
that led to the original Abe et al. (2014) study. This finding indicates
that mistake led to a misinterpretation of the real evolutionary rela-
tionship among Salminus species; the molecular phylogeny obtained
here is coherent.
4.2 | Possible reconstructions of the early
evolutionary history of Salminus
Our dating analysis placed the MRCA of all species of Salminus in the
late Miocene (11.05 Ma, 95% HPD = 7.02–14.98 Ma; node 1—
MACHADO ET AL. | 9

T A B L E 2 Posterior parameter values for scenarios with high probability (winners) in Salminus brasiliensis, Salminus sp. Amazon and S. sp.
Araguaia, and Salminus hilarii estimated using DIYABC. The unit of time, counted in generation, was converted into 106 years before present
(Ma), based in an average generation time of 2 years (de Godoy, 1975)a
Posterior parameter estimation

Parameters Median 95% CI RMAE

Salminus brasiliensis
PUL lineage effective population size NPUL 136,000 61,400–332,000 0.265
Upper Paran
a lineage effective population size NUP 85,300 37,900–214,000 0.247
Ancestral effective population size Na 685,000 269,000–970,000 0.119
Time of divergence between PUL and Upper Paran
a lineages t2 0.892 Ma 0.416–1.17 Ma 0.269
Salminus sp. Amazon and Salminus sp. Araguaia
Salminus sp. Araguaia effective population size NToA 30,100 12,300–115,000 0.288
Salminus sp. Amazon effective population size NAm 155,000 57,800–442,000 0.279
Ancestral effective population size Na 582,000 204,000–956,000 0.136
Time of divergence between Salminus sp. Araguaia and Salminus sp. Amazon t2 2.36 Ma 1.178–4.54 Ma 0.190
Salminus hilarii
S~ao Francisco_1 lineage effective population size NSF1 446,000 156,000–882,000 0.267
S~ao Francisco_2 lineage effective population size NSF2 441,000 135,000–907,000 0.337
Upper Paran
a lineage effective population size NUP 460,000 179,000–832,000 0.254
Founder effective population size into Upper Paran
a basin NUPf 5,040 358–9,740 0.835
Time of divergence between S~ao Francisco_1 and S~ao Francisco_2 lineages ts 0.606 Ma 0.216–1.086 Ma 0.272
Time of expansion for Upper Paran
a lineage t1 1.592 Ma 0.77–2.58 Ma 0.255
Time of founder event between S~ao Francisco and Upper Paran
a lineages t2 1.952 Ma 1.046–3.24 Ma 0.135

N: effective population size; t: time in years; Nf: founder effective population size; RMAE: relative median of absolute error.
a
See Appendix S4.

Figure 4), hence prior to the uplift of the Eastern Andean Cordillera.
At that time, the modern hydrographic basins northwest of South
America were not individualized, and the region was covered by a
lagoon system known as the Pebas lake (Lundberg et al., 1998). It is
likely that the Salminus MRCA was then distributed across all proto-
Amazon, and that the appearance of the Eastern Cordillera divided
ancestral populations into the Magdalena and Amazon/Tocantins-Ara-
guaia palaeobasins—with the population from Magdalena palaeobasin
leading to the modern S. affinis, while that in the Amazon palaeobasin
becoming the MRCA of the remaining species. We were unable to use
ABC to test whether this vicariance scenario was more strongly sup-
ported than the geodispersal event. However, vicariant reconstruc-
tions as the one discussed above have been widely employed to
explain cis- and trans-Andean ichthyofauna divergence (Albert et al.,
2006; Montoya-Burgos, 2003; Ramirez et al., 2017; Sivasundar et al.,
2001).
The data suggests that the next cladogenetic event within Salmi-
nus resulted in the isolation of a Southeastern clade species from
the Northwestern clade (node 2—Figure 4; Figure S3.1, Appendix 3).
The MRCA of this Southeast+Northwest clade was estimated at
approximately 7.76 Ma (95% HPD = 4.01–11.64 Ma), and likely pre-
sent in the Amazon/Tocantins-Araguaia. Species distribution pat-
terns, tied to the inferred order of events, suggests that an ancestral
group of individuals colonized the Brazilian Shield basins, probably
through a headwater capture event to the S~ao Francisco basin, and
gave rise to Southeast clade.
Two possible palaeo-routes of geodispersal have been proposed
between the Amazon/Tocantins-Araguaia and the S~ao Francisco
palaeobasins. One of them involves the Serra Geral do Tocantins
(SGT), a tectonic structure that dates to the Cretaceous and which
stands between the two basins, acting as physical barrier to their
headwaters (Lima & Caires, 2011). It has been proposed that former
erosion processes in the SGT resulted in a contact area where both
basins share their headwaters, creating a potential for faunal inter-
change (Lima & Caires, 2011). Another possible geodispersal palaeo-
route between these two hydrographic systems may have been
established through a Northern-Northeastearn Brazil coastal corridor.
According to Hubert et al. (2007), two geodispersal events involving
these basins occurred in the Serrasalmus genus, and one of them
coincides with our estimated date. Hubert et al. (2007) proposed
that Miocene marine regressions promoted coalescence at the
mouths of several rivers, creating transient connections between the
palaeobasins of the Amazon/Tocantins-Araguaia and the Brazilian
Northeast. Although speculative, this event may have allowed the
MRCA of the Southeast clade of Salminus to reach the Brazilian
Shield. Hubert and Renno (2006) also implied this route in historical
reconstructions of other Characiformes.
Within the Southeast clade, the cladogenetic events involving
the Upper Paran
a and S~ao Francisco basins were not consistent with
speciation by vicariance but by successive geodispersal, because the
hydrographic basins do not form monophyletic clades (Hubert et al.,
2007). In fact, the geological structure of the Brazilian Shield is
10 | MACHADO
conducive to headwater captures because it has several geological
faults that are continually activated, leading to rearrangements
among adjacent basins and consequent faunistic interchange
(Buckup, 2011; Ribeiro, 2006; Ribeiro & Menezes, 2015). The
appearance of both hydrographic systems is very old and often asso-
ciated with the Gondwana break-up (Potter, 1997; Ribeiro, 2006).
Between their adjacent headwaters there is a geological fault system
denominated Upper S~ao Francisco River Crustal Discontinuity
(DCARSF), which has been in tectonic activity since the Pre-Cam-
brian (Buckup, 2011). Due to continuous DCARSF reactivations, it
thus appears that headwater captures became important diversifica-
tion events for Salminus, Hyspostomus (Montoya-Burgos, 2003) and
some Characiformes (Hubert & Renno, 2006). Our analyses placed
the MRCA of the Southeast clade in S~ao Francisco basin nearly at
4.28 Ma (95% HPD = 1.89–7.24 Ma), when part of the lineage gave
rise to S. franciscanus and part of it geodispersed to the Upper
Paran
a basin. The latter appears as the probable ancestral area for
the MRCA of S. brasiliensis and S. hilarii (node 5, Figure 4; Fig-
ure S3.1).
Although we were unable to use the ABC approach to infer the
demographic history of the early Salminus divergences, the estimated
divergence times are in close agreement with previous studies
(Albert et al., 2006; Hubert & Renno, 2006; Montoya-Burgos, 2003;
Ramirez et al., 2017; Sivasundar et al., 2001).
4.3 | ABC, vicariance vs. geodispersion, and the
recent diversification history of Salminus
Phylogeographical studies of the Neotropical ichthyofauna usually
provide estimates of divergence times and correlate them with
known historical events. However, due to the complexity of histori-
cal events, similar biogeographical patterns often can emerge from
distinct diversification processes (Moritz, Patton, Schneider, & Smith,
2000)—attesting the importance of incorporating statistical infer-
ences into hypothesis testing. Among the few studies using this
approach in South America freshwater fishes are the reconstruction
of potential historic colonization routes for Aplochiton zebra species
(Vanhaecke et al., 2015), an investigation of the performance of dis-
tinct divergence pattern resulted by nuclear and mitochondrial mark-
ers in Cichla temensis lineages (Willis et al., 2015), and the
identification of headwater captures events that promoted the diver-
sification in species of the Pimelodidae family in Amazon West
(Tagliacollo et al., 2015). Our study is the first to implement a
model-based Bayesian framework to test alternative biogeographical
scenarios potentially responsible for the diversification process in
Neotropical freshwater fishes.
Within the Northwestern clade, our phylogenetic analysis sug-
gested a cladogenetic event to have happened at 2.7 Ma (95%
HPD = 0.98–5.17 Ma), splitting S. sp. Amazon and S. sp. Araguaia
(node 3, Figure 4). The ABC analysis supported a vicariance scenario as
the most probable event responsible for the divergence between these
species that happened at 2.36 Ma (95% HPD = 1.178–4.54 Ma). This
ET AL.
event represented the break-up of a connection between the Amazon
and Tocantins-Araguaia palaeobasins. Geological and geomorphological
data point out that the intense tectonic reactivation of the geological
faults located in the Lower Amazon basin changed the course of the
Tocantins palaeoriver during the Plio-Pleistocene: the ancient Tocan-
tins river discharged on the opposite course of the modern drainage,
into the Amazon palaeoriver (Rossetti & Valeriano, 2007). The interrup-
tion of direct discharge of the Tocantins palaeoriver into the Amazon
palaeoriver was also suggested as the vicariance event responsible to
lead speciation between Inia araguaiaensis and Inia geofrensis at
2.08 Ma (Hrbek et al., 2014).
Within the Southeastern clade, the ABC-based test supported
vicariance as the main driver of diversification across lineages of S.
brasiliensis, while geodispersal was responsible for diversification
across lineages of S. hilarii. S. brasiliensis showed two well-defined
allopatric lineages (Upper Paran
a and PUL), and their diversification
process was likely driven by the split of the hydrographic basins
where both occurred. Other co-distributed species also have
marked genetic structure between the Upper Paran
a basin and the

ıcan,
remaining hydrographic systems of the La Plata basin (Pi
alek, R

n, & Zrzavy
Casciotta, Almiro
, 2012; Sivasundar et al., 2001). In fact,
the Upper Paran
a basin is considered a distinct ecoregion relative
to the other areas here considered (Abell et al., 2008). Its biogeo-
graphical history is complex, and it is associated with successive
reactivation of geological faults during the Quaternary (Bartorelli,
2004). These neotectonic activities promoted drainage rearrange-
ments (Ribeiro & Menezes, 2015) and the formation of geographical

, Iguacßu, Mocon
a and Sete Quedas falls, for example;
barriers (Apipe
Bartorelli, 2004).
The best supported scenario in the DIYABC analysis supports an
ancestral S. brasiliensis population that was widely distributed across
all the La Plata basin, followed by the appearance of an effective bar-
rier that promoted allopatric divergence between lineages approxi-
mately 0.89 Ma (95% HPD = 0.416–1.17 Ma). The divergence time
between the lineages of S. brasiliensis seems consistent with the Sete
Quedas falls appearance. Prior to its inundation, caused by the con-
struction of the Itaipu Reservoir construction, the Sete Quedas falls
was an important impermeable barrier between the Upper Paran
a
basin and the remaining hydrographic basins that composed the La
Plata. The absolute age of the falls is unknown. According to Bar-
torelli (2004), tectonic reactivation events happening during the Med-
ium Pleistocene and until the Maximum Last Glacial (1.5–0.1 Ma)
were responsible for the genesis of several falls in the Upper Paran
a
basin, including Sete Quedas. This vicariant event is also used to
explain diversification process in Prochilodus lineatus (Sivasundar
et al., 2001) and Crenicichla lacustris group (Pi
alek et al., 2012).
Allopatric divergence processes involving species in the S~ao
Francisco and the Upper Paran
a basins have been previously associ-
ated with geodispersal events promoted by successive headwater
captures between basins (Hubert et al., 2007). Our ABC analysis
supports this interpretation: there is strong support for a geodisper-
sal event being responsible for the diversification of the lineages of
MACHADO ET AL.
S. hilarii in the Upper Paran
a and S~ao Francisco basins. The most
probable scenario recovered the S~ao Francisco hydrographic system
as the source-basin in the first headwater capture event (not the
Upper Paran
a basin, as observed in species tree-node 8, Figure 4;
Figure S3.1). The chosen scenario recovered that part of S~ao Fran-
cisco lineage (SF1) was captured by the Upper Paran
a basin approxi-
mately at 1.95 Ma. Although it was possible to estimate the
divergence time between sympatric lineages (0.606 Ma, 95% HPD =
0.216–1.086 Ma, as per the ABC analysis, 0.67 Ma, 95% HPD =
0.17–1.44 Ma, as per *BEAST), their underlying diversification pro-
cess remains unclear.
5 | CONCLUSION
Through an ABC analysis, we were able to build and distinguish phy-
logeographical signatures of two main events that promote diversifi-
cation in freshwater fishes—namely, vicariance and geodispersal. The
hydrological hypothesis, detected through signatures of founder
events in colonized areas following by expansion population size,
represents a geodispersal event. In contrast, vicariance events failed
to leave signatures of founder events, source-basin identity, and
expansion event. Detection of these sequence of events in the
geodispersal was only possible because the biological model (large
and medium-sized migratory fishes) used in this study is appropriate.
Had we studied fishes restricted to headwaters, in which most of
the population may be moved in geographical space through head-
water captures, this unique signature left by a founder event would
likely be absent. This investigation is thus unique in Neotropical
freshwater species diversification studies: for the first time, at the
genetic level, vicariance and geodispersal events were distinguished
by phylogeographical signatures.
As with any approach to data analysis, phylogeographical model
choice using ABC has limitations, and decisions about which models to
include in the comparison set can be challenging. Overall, ABC loses

& Car-
power to differentiate among large numbers of models (Thome
stens, 2016), similar candidate models increased the failure to recover
the true model (Cabrera & Palsbøll, 2017), and the choice insufficient
summary statistics led to a loss of information that can bias the calcu-
lation of the relative posterior probability (Pelletier & Carstens, 2014).
None of these affected our simulations. Results obtained through tra-
ditional phylogeographical analyses (divergence time estimations, fol-
lowed by associations with known historical events) were consistent
with the model-based Bayesian approach. This is reassuring, and
speaks of the reliability and robustness of the biogeographical scenar-
ios here inferred and reinforces the value of an ABC framework in
phylogeographical studies, particularly those interested in testing
alternative processes underlying similar biogeographical patterns.
ACKNOWLEDGEMENTS
We are thankful to Dr. Jorge Ramirez (Universidade Federal de S~ao
Carlos) and Dr. Ivan Prates (Smithsonian’s National Museum of
| 11
Natural History) for their insights and discussions of the methods
and results of this paper. This research was funded by Coordenacßa~o
de Aperfeicßoamento de Pessoal de N
ıvel Superior (CAPES), Fundacß~ao
de Amparo a Pesquisa do Estado de S~ao Paulo (FAPESP 10/52315-
7) and Conselho Nacional de Desenvolvimento Cient
ıfico e Tec-

gico (CNPq 563299/2010-0; 308385/2014-4). A. Carnaval
nolo
acknowledges support by the National Science Foundation (DEB
1120487, 1343578), FAPESP (BIOTA 2013/50297-0) and NASA.
This study was developed in accordance with the Brazilian laws,
approved by the ethic committee on animal use at Universidade
Federal de S~ao Carlos (CEUA number 5765010416), and carried out
under a temporally scientific collection license number 32217-4 pro-
vided by ICMBio-SISBIO. We also thank the anonymous reviewers
and Editor for all comments and suggestions, which were very help-
ful in improving the final manuscript.
ORCID
Carolina Barros Machado http://orcid.org/0000-0001-5195-5577
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081. https://doi.org/10.11646/zootaxa.3949.1.2 Carolina Machado is developing her Postdoc project at Universi-
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Characiformes) in major South American Rivers. Molecular Ecology, His research is focused on Genetic Conservation, involving aqua-
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tic and terrestrial animals. Studies related to forensic genetic,
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for haplotype reconstruction. The American Journal of Human Genet- systematic, taxonomy phylogeny and phylogeography are also
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Tagliacollo, V. A., Roxo, F. F., Duke-Sylvester, S. M., Oliveira, C., & Albert, Ana Carnaval is assistant professor at City University of New York
J. S. (2015). Biogeographical signature of river capture events in
and researcher associate at The American Museum of Natural His-
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Thome
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tion leads to insight into the evolutionary history of four-eyed frogs. improving diversity prediction and conservation in tropical regions.
Proceedings of the National Academy of Sciences, 113, 8010–8017.
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the ideas; C. B. M. collected and analysed the data; C. B. M. led
G., & Consuegra, S. (2015). Genetic signatures of historical dispersal
of fish threatened by biological invasions: The case of galaxiids in the writing with assistance from P. M. G. J. and A. C. C.
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