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Syst. Biol. XX(XX):XX–XX, 2023

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https://doi.org/10.1093/sysbio/syad034
Advance Access Publication June 1, 2023

Speciation in Coastal Basins Driven by Staggered Headwater Captures: Dispersal of

a Species Complex, Leporinus bahiensis, as Revealed by Genome-wide SNP Data
Jorge L. Ramirez1,2,*, , Carolina B. Machado3, Paulo Roberto Antunes de Mello Affonso4 and

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Pedro M. Galetti Jr3
Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos, Lima, Peru
1

Museo de Historia Natural de la Universidad Nacional Mayor de San Marcos, Lima, Peru
2

3
Departamento de Genética e Evolução, Universidade Federal de São Carlos, São Carlos, SP, Brazil and
4
Departamento de Ciências Biológicas, Universidade Estadual do Sudoeste da Bahia, Jequié, BA, Brazil
*Correspondence to be sent to: Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos, Calle Germán Amézaga N° 375,
15081, Lima, Perú; E-mail: jramirezma@unmsm.edu.pe

Received 3 March 2022; reviews returned 22 May 2023; accepted 30 May 2023
Associate editor: James Albert

Abstract.—Past sea level changes and geological instability along watershed boundaries have largely influenced fish
distribution across coastal basins, either by dispersal via palaeodrainages now submerged or by headwater captures,
respectively. Accordingly, the South American Atlantic coast encompasses several small and isolated drainages that
share a similar species composition, representing a suitable model to infer historical processes. Leporinus bahiensis is
a freshwater fish species widespread along adjacent coastal basins over narrow continental shelf with no evidence
of palaeodrainage connections at low sea level periods. Therefore, this study aimed to reconstruct its evolutionary
history to infer the role of headwater captures in the dispersal process. To accomplish this, we employed molecular-
level phylogenetic and population structure analyses based on Sanger sequences (5 genes) and genome-wide SNP
data. Phylogenetic trees based on Sanger data were inconclusive, but SNPs data did support the monophyletic status
of L. bahiensis. Both COI and SNP data revealed structured populations according to each hydrographic basin. Species
delimitation analyses revealed from 3 (COI) to 5 (multilocus approach) MOTUs, corresponding to the sampled
basins. An intricate biogeographic scenario was inferred and supported by Approximate Bayesian Computation
(ABC) analysis. Specifically, a staggered pattern was revealed and characterized by sequential headwater captures
from basins adjacent to upland drainages into small coastal basins at different periods. These headwater captures
resulted in dispersal throughout contiguous coastal basins, followed by deep genetic divergence among lineages. To
decipher such recent divergences, as herein represented by L. bahiensis populations, we used genome-wide SNPs data.
Indeed, the combined use of genome-wide SNPs data and ABC method allowed us to reconstruct the evolutionary
history and speciation of L. bahiensis. This framework might be useful in disentangling the diversification process in
other neotropical fishes subject to a reticulate geological history. [ABC, freshwater fishes, genotype by sequencing,
neotropics, phylogeography.]

Freshwater fish are restricted to aquatic habitats, and
their dispersal modes depend on temporal connectiv-
ity between hydrographic systems. As such, their evo-
lutionary history is largely correlated to the geological
history of river basins (Hubert et al. 2007; Reis et al.
2016). Therefore, 2 major processes can affect fish dis-
tribution along coastal basins: 1) past sea level changes
(Weitzman et al. 1988; De Bruyn et al. 2013; Dias et al.
2014; Thomaz et al. 2017) and 2) geological instability
of the watershed that divides adjacent basins (Ihering
1898; Buckup 2011). In the former, periods of sea level
retreats, particularly during the Pleistocene (i.e., Last
Glacial Maximum), would create putative migration
routes via palaeodrainages across the exposed con-
tinental shelf, allowing colonization of new areas.
However, after sea level transgression, populations
would become isolated, again resulting in interpopula-
tion differentiation and, eventually, speciation (Buckup
2011; Dias et al. 2014). The role of palaeodrainages in
dispersal and divergence of aquatic organisms has been
amply reported (Abreu and Calliari 2005; Dias et al.
2014; Thomaz et al. 2015; Thomaz and Knowles 2018).
In contrast, some authors have argued that sea level
fluctuations could not account for the presence of sister
groups in both coastal and inland basins (Ribeiro 2006;
Menezes et al. 2008; Buckup 2011). This distribution pat-
tern could be explained by geological instability along
watershed boundaries. In this case, headwater cap-
tures would play a key role in the distribution of sister
taxa by introducing lineages from upland basins into
coastal basins (Ribeiro 2006). This process could take
place repeatedly at different times, thus increasing the
diversity of coastal basins via merging (geodispersal)
and further separation of geographic areas (vicariance)
(Albert and Carvalho 2011). Therefore, geodispersal
would promote biotic dispersal while vicariance would
result in posterior biotic isolation. Once introduced into
coastal basins, the variation in sea levels could then act
as a secondary driver of dispersal along adjacent coastal
basins (Buckup 2011; Thomaz et al. 2017; Thomaz and
Knowles 2020).
Molecular studies have thrown some cold water on
the role of palaeodrainages in dispersal and divergence
of aquatic organisms. In particular, these studies have

1
2 SYSTEMATIC BIOLOGY
reported a significant divergence among isolated pop-
ulations of fishes from coastal basins. Such divergence
suggests longer periods of isolation than those expected
under a scenario of dispersal across palaeodrainages
during sea level retreats in the Pleistocene (Menezes et
al. 2008; Lima et al. 2017; Ramirez et al. 2017b; Barreto
et al. 2022). Accordingly, headwater captures have been
consistently reported as the main process involved in
dispersal and differentiation of lineages among coastal
basins, particularly across the northeastern region of
South America (Menezes et al. 2008; Lima et al. 2017,
2021; Souza et al. 2020; de Sousa et al. 2021). However,
these studies are still reduced in number when com-
pared to other Neotropical regions.
The South American Atlantic coast comprises sev-
eral small and currently isolated drainages, and they
are characterized by high levels of endemism and dis-
tinct biogeographic ecoregions (Buckup 2011; Lujan
and Armbruster 2011). Most of these basins share a
similar species composition, including representatives
also found in inland basins, reflecting their historical
interrelationships (Buckup 2011; Lujan and Armbruster
2011; Thomaz and Knowles 2018). Leporinus bahiensis
Steindachner (1875) is one such fish species described
for Atlantic coastal basins. It is widespread in several
hydrographic systems of Bahia (northeastern Brazil), an
area characterized by a narrow continental shelf with
no evidence of submersed palaeodrainage connectivity
between adjacent basins (Thomaz and Knowles 2018).
Because of these features, L. bahiensis stands out as a
good model to evaluate the role of headwater capture in
dispersal and speciation processes along coastal basins
without the influence of palaeodrainage connections at
low sea level periods.
Therefore, in this study, we herein reconstructed
the evolutionary history of L. bahiensis along its range
based on molecular phylogenetics, population struc-
ture analyses and Approximate Bayesian Computation
(ABC), using Sanger sequencing of 2 mitochondrial
and 3 nuclear genes, as well as genome-wide SNP data
obtained by high-throughput Genotype by sequencing
(GBS). Taking into account the distribution of L. bahien-
sis across distinct and isolated basins, we raised 3 main
questions: (i) is L. bahiensis a monophyletic group?;
(ii) does the genetic structure among populations cor-
respond to speciation events?; (iii) could sequential
headwater captures explain the current distribution of
lineages/populations along coastal basins?.”
Material and Methods
Sample Collection
Specimens of L. bahiensis were sampled from 5 coastal
basins, including Paraguaçu (n = 6), Jequiriçá (n = 5),
Almas (n = 4), Contas (n = 9), and Almada (n = 9), total-
ing 33 samples (Supplementary Table S1 and Fig. 1).
Another 2 closely related Leporinus species (Ramirez
2015) were included for comparative analyses: Leporinus
taeniatus Lütken 1875 from São Francisco, Itapicuru,
and Jaguaribe basins and Leporinus octofasciatus
Steindachner 1915 from Paraná basin (Supplementary
Table S1). Leporinus bahiensis could be separated from
all congeners, except L. paranensis from Upper Paraná,
by having a dental formular of 3/4, 35 scales in the lat-
eral line, 16 circumpeduncular scales, and 3 round dark
blotches on lateral line supplemented by several brown
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bars across the dorsum (Steindachner 1875; Garavello
and Britski 1987). Collection and transportation of
specimens were carried out under permission (license
#32215) provided by the Institute Chico Mendes de
Conservação da Biodiversidade (ICMBio). Vouchers
were deposited in recognized ichthyological collections
(Supplementary Table S1).
All procedures were performed under ethical and
legal recommendations as proposed by the Federal
Council of Biologists, Resolution 301 (8 December 2012)
and approved by the Ethics Committee of Utilization of
Animals from the Universidade Estadual do Sudoeste
da Bahia (CEUA/UESB, number 32/2013). Genetic her-
itage access was registered under number AAA03B9 at
SisGen.
DNA Extraction and Sanger Sequencing
Total DNA was isolated using DNeasy blood and
tissue kit (Qiagen) following the manufacturer’s
instructions. We sequenced the partial mitochondrial
Cytochrome C Oxidase Subunit I (COI; 698 bp) and
Cytochrome b (Cytb; 1110 bp) genes using AnosCOIF-
AnosCOIR and AnosCytBF-AnosCytBR primers,
respectively (Ramirez and Galetti 2015). The nuclear
Myosin heavy chain 6 cardiac muscle alpha gene
(Myh6, ~750 bp), recombination activating gene 1
(RAG1, ~1500 bp), and recombination activating gene
2 (RAG2, ~1100 bp) were sequenced using primers and
protocols described elsewhere (Li and Ortí 2007; Li et al.
2007; Oliveira et al. 2011). All sequences were deposited
in GenBank (Supplementary Table S1).
Genotype By Sequencing Library and Sequencing
GBS libraries were constructed using commercial
services (Ecomol Consultoria, Brazil). Total DNA (10
ng) was digested with PstI restriction enzyme and
ligated with barcode adaptors. The libraries were puri-
fied, quantified, pooled and size-selected, following
sequencing in the Illumina HiSeq 2500 using 1 × 100
(single read). Raw data were quality filtered, demul-
tiplexed, and the output files were generated using
the ipyrad v. 0.9.5 pipeline (Eaton and Overcast 2020).
GBS data summary is shown in Supplementary Table
S2 and available on Dryad via the link included in the
Supplementary Material.
Phylogenetic Analyses
Two data sets were used for phylogenetic recon-
struction: 1) 2 mitochondrial and 3 nuclear genes and
2) SNPs from GBS. The first data set included genes
2023 RAMIREZ ET AL. - SPECIATION IN COASTAL BASINS DRIVEN BY STAGGERED HEADWATER CAPTURES
Figure 1.  Map of Eastern Brazil showing hydrographic basins and collection sites of L. bahiensis and L. taeniatus.
successfully used to infer phylogenetic relation-
ships within Anostomidae (Ramirez and Galetti 2015;
Ramirez et al. 2016, 2017b, 2020). The final Sanger data
set included 4 individuals of L. bahiensis and 3 speci-
mens of L. taeniatus and 1 specimen of L. octofasciatus.
These 3 species, together with Leporinus paranensis
Garavello and Britski 1987, share a dental formula 3/4
and are closely related (Ramirez 2015). Leporinus lacus-
tris Campos, 1945 was included as an outgroup since it is
related to the L. taeniatus clade (Silva-Santos et al. 2018).
Each gene was independently aligned using Clustal
X2 (Larkin et al. 2007), and no gaps were observed. A
maximum parsimony analysis for the concatenated
sequences was performed in PAUP* 4.0b10 (Swofford
2003) with 1000 bootstrap replicates. A maximum like-
lihood (ML) tree was reconstructed using the concate-
nated alignments in RAxML for XSEDE (Stamatakis et
al. 2008) on the CIPRES Science Gateway web server
(Miller et al. 2010), using a mixed partition model, as
determined by PartitionFinder (Lanfear et al. 2012), with
a GTR+G substitution model and 1000 bootstrap repli-
cates. A multilocus Bayesian species tree was estimated
on *BEAST v2.6.3 (Heled and Drummond 2010), using
K80+I, HKY+I, K80, K80, and JC substitution models for
3
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COI, Cytb, Myh6, RAG1, and RAG2, respectively, based
on the Bayesian Information Criterion, using jModelT-
est 2 (Darriba et al. 2012). The mitochondrial genes were
linked to share a single tree and were set to have an
effective population size one-quarter that of the nuclear
genes. We used an uncorrelated log-normal relaxed
clock in all partitions and birth and death model as tree
prior. The tree was calibrated using the clock rate from
previous Anostomidae studies (Ramirez et al. 2020) as
7.8 × 10−9, 8.9 × 10−9, 5.3 × 10−10, 8 × 10−10, 8.3 × 10−10 muta-
tions/site/year COI, Cytb, Myh6, RAG1, and RAG2,
respectively. Markov Chain Monte Carlo (MCMC) was
implemented using 100 million generations, sampling
every 5000 runs and a 10% burn-in. Convergence of
analyses and effective sample size (>200) were evalu-
ated in Tracer v. 1.7.1 (Rambaut et al. 2018).
Phylogenetic analyses using the SNP data set encom-
passed 31 individuals (27 L. bahiensis, 3 L. taeniatus, and 1
L. octofasciatus). All trees were rooted using L. octofascia-
tus as the outgroup. A ML tree using RAxML for XSEDE
(Stamatakis et al. 2008) was generated by assembling
the loci present in at least 22 individuals, thus total-
ing 19,540 loci. A quartet-based coalescent phylogeny
was inferred using Tetrad available in ipyrad v. 0.9.5
4 SYSTEMATIC BIOLOGY
(Eaton and Overcast 2020) based on the SVDquartets
algorithm (Chifman and Kubatko 2014) and optimized
for SNPs data. In this case, only those loci present in
all individuals (5728 bp) and a randomly sampled sin-
gle SNP from each locus were used since the algorithm
relies on multilocus unlinked SNPs. All possible quar-
tets (subset of 4 samples) were sampled, and the sup-
port values were assessed using 1000 non-parametric
bootstrap replicates.
Genetic Structure Analyses
A COI haplotype network was calculated using
Median-Joining (Bandelt et al. 1999) with POPART soft-
ware (Leigh and Bryant 2015). To estimate the number
of genetic clusters, a hierarchical Bayesian clustering of
individuals was performed using the SNPs data set (27
individuals, 10,438 bp) in STRUCTURE (Pritchard et
al. 2000), as implemented in ipyrad v. 0.9.5 (Eaton and
Overcast 2020). We tested k = 2 to 6 populations and
ran 10 replicates for each K with 100,000 MCMC
and a burn-in of 5000. Replicates were aligned using
CLUMPP (Jakobsson and Rosenberg 2007), and the
best K was selected based on ΔK statistics (Evanno et al.
2005). Hierarchical analyses were performed on genetic
clusters comprising more than 1 watershed to assess
fine-scale patterns of population structure.
Species Delimitation Analyses
Species delimitation analyses were carried out based
on 2 data sets. The first corresponded to COI fragments,
widely used to evaluate single-locus species delim-
itation (Roxo et al. 2015; Ramirez et al. 2017a, 2020;
Hofmann et al. 2019), comprising 42 individuals that
included 24 samples of L. bahiensis. For this marker, 3
methods of species delimitation were used: the gen-
eral mixed Yule coalescent model with a single thresh-
old (Pons et al. 2006), the Poisson tree processes (PTP)
(Zhang et al. 2013), and the Bayesian implementation of
the PTP model (bPTP) (Zhang et al. 2013). Because these
methods require a phylogenetic tree as input, we gener-
ated an ultrametric tree using BEAST v. 2.6.3 (Bouckaert
et al. 2014) with an HKY+I substitution model chosen
by jModelTest2 (Darriba et al. 2012), a birth and death
tree model, and an uncorrelated log-normal relaxed
clock. Finally, all delimitation results were compared,
and consensus Molecular Operational Taxonomic Units
(MOTUs) were generated using the SPdel pipeline
(https://github.com/jolobito/SPdel) (Ramirez et al.
2020). Additionally, COI intra- and inter-group genetic
distances were estimated using the K2P model in the
same SPdel pipeline.
In addition, the SNPs data set was selected for multilo-
cus species delimitation using Bayes Factor Delimitation
(BFD) (Leaché et al. 2014) and BPP v. 4.38 (Yang and
Rannala 2010). For both algorithms, a maximum of 4
individuals for each basin was included, with no missing
data, owing to computational constraints. Therefore, the
data set included 17 samples of L. bahiensis and one each
of L. taeniatus and L. octofasciatus. For BFD, we estimated
a coalescent-based species tree using SNAPP (Bryant et al.
2012) to test 5 alternative species delimitation models, sep-
arating the populations of L. bahiensis according to basin
(Table S3). We assigned a Gamma distribution to Lambda
prior (Alpha = 2.5, Beta = 2.0, and Mean = 5) and set Theta
= 0.01 (Alpha = 1.0, Beta = 100, and Lambda = 5). SNAPP
analyses were performed in BEAST v. 2.6.3 (Bouckaert
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et al. 2014) with a path sampling of 24 steps and 200,000
MCMC generations, sampling every 1000 generations.
Marginal likelihood (ML) estimates for each model were
ranked using BFD, and BFD was calculated based on
twice the difference in marginal likelihood estimates for
2 competing methods (Leaché et al. 2014). For BPP, we
conducted species delimitation based on a guide tree
(Yang and Rannala 2010; Rannala and Yang 2013), using
options speciesdelimitation = 1 and speciestree = 0. The
fixed tree was based on the Tetrad result, and the putative
species were designated by basin. The analyses were per-
formed with 500 loci randomly sampled and 2 replicate
runs under different seeds. Prior θ (genetic diversity) and
τ (root age) were calculated using the Minimalist BPP app
found at https://brannala.github.io/bpps/. We set the
inverse-gamma prior θ to the average pairwise difference
among sequences within each population, IG (3, 0.12),
and tested 2 alternative τ corresponding to the maximum
pairwise differences: 1) all L. bahiensis populations refer to
distinct species, IG (3, 0.0014) or 2) a single species, IG (3,
0.0018). A range of fine-tuning algorithm parameters was
used: algorithm 0 with ε = 1, 2, and 5 and algorithm 1 with
α = 1, 1.5, and 2 and m = 0.5, 1, and 2. For all runs, 200,000
MCMC generations were sampled every 2 generations
with a burn-in of 20,000.
Dispersal Routes Assessment Using ABC approach
To understand the role of geodispersal events in
the diversification of L. bahiensis, we tested alterna-
tive demographic scenarios using the ABC approach
with DIYABC v2.1.0 software (Cornuet et al. 2014).
Evolutionary scenarios were based on phylogenetic
reconstruction analyses, placing Paraguaçu or Contas
basin as the source population (Fig. 2) since both basins
share boundaries with the São Francisco basin where
the sister species (L. taeniatus) is distributed (Fig. 1).
Scenarios 1 and 2 considered a unique event of diver-
gence among all sampled basins. This could be expected
when dispersal is driven by a single palaeodrainage
connection during a period of sea level retreats (Fig. 2).
Scenarios 4 and 6 considered an initial dispersal driven
by headwater capture from Paraguaçu to Contas or
vice versa, followed by a subsequent and simultane-
ous dispersal event among daughter populations by a
palaeodrainage connection during a period of sea level
retreats (Fig. 2). Scenarios 3 and 5 correspond to a stag-
gered pattern in which the initial source population var-
ies (Fig. 2). In this case, dispersal occurred from basins
adjacent to upland drainages to small coastal basins by
consecutive headwater captures at different times.
2023 RAMIREZ ET AL. - SPECIATION IN COASTAL BASINS DRIVEN BY STAGGERED HEADWATER CAPTURES 5

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Figure 2.  Models explaining the staggered dispersal processes. A) Alternative phylogenetic scenarios to be tested through an approximate
Bayesian computation approach (ABC). N: effective population size; t: time given in generations, db: time during the bottleneck event. Width
on the tree branch indicates changes in effective population size. Vertical bar indicates time backwards. B) Geographic scenario showing the
staggered dispersal by sequential headwater captures as hypothesized in scenario 3 (scenario chosen by the ABC analysis). River networks
are illustrative. T4–T0, time steps, from past to present. Shaded regions are occupied basin. The captured river in each time is in red. In blue,
coastline during the low sea level period. Dashed rivers are palaeodrainages now submerged, showing no connection between them.
6 SYSTEMATIC BIOLOGY
In all the above-described scenarios, an increase in
effective population size (Ne) of daughter populations
after initial geodispersal was considered (Machado et
al. 2018). Details about the tested demographic and
time priors are provided in the Supplementary Table S4.
We simulated 1 million data sets for each scenario.
To check the likelihood that both scenarios and prior
distributions of parameters could produce a simulated
data set close to the observed data set, we performed
a pre-evaluation using Principal Components Analysis.
The posterior probabilities of alternative scenarios were
estimated by polychotomous logistic regression analy-
sis, considering 1% of the total simulated data set clos-
est to the observed data (Cornuet et al. 2008). The most
probable scenario was then determined based on the
highest and non-overlapped values of posterior proba-
bility within a 95% confidence interval (CI).
Estimates of Divergence Times
DIYABC software does not require mutation model
parameterization in SNPs data sets. However, this would
result in the absence of any calibration brought by priors
on mutation parameters. Therefore, we used a compos-
ite-likelihood method based on the site frequency spec-
trum (SFS) implemented in FASTSIMCOAL2 (Excoffier
et al. 2013, 2021) to estimate divergence times between
lineages according to the scenario obtained by ABC.
The FASTSIMCOAL2 approach allowed us to simu-
late a scenario whereby 2 populations diverged and
then estimate the time divergence by using a mutation
rate and the effective population size of 1 population
as reference. The SFS was constructed from the variant
call format file without missing data and calculated
by each pair of basins using easySFS (https://github.
com/isaacovercast/easySFS). FASTIMCOAL2 analysis
was performed using “removeZeroSFS” by assuming
no migration between populations after splitting, as
expected in a scenario of headwater capture. Then Ne of
1 of the 2 populations was calculated from nucleotide
diversity, π, of fixed and variable sites, and a mutation
rate (μ) of 2.24 × 10−8 was chosen (Thomaz et al. 2017).
For each pair of basins, 40 replicates were run with
100,000–250,000 simulations, a stopping criterion of
0.001, and 10–40 expectation conditional maximization
cycles for the calculation of composite likelihood. The
95% CIs of parameter estimates, including divergence
time, were calculated from 100 parametric bootstrap
replicates. Each replicate used a simulated SFS with the
same number of individuals, loci and parameters from
the maximum composite likelihood estimates to re-esti-
mate parameters.
Results
Phylogenetic Analysis
The aligned sequences of COI, CytB, Myh6, RAG1
and RAG2 included 595, 1005, 733, 1160, and 1023 bp,
and 30, 33, 3, 4, and 3 parsimony-informative sites,
respectively. No gaps were observed in the alignments.
All phylogenetic trees recovered the same topology,
supporting a well-defined clade per basin, but the
monophyletic status of L. bahiensis was not supported.
Instead, the phylogenetic trees based on mitochondrial
and nuclear genes recovered L. bahiensis and L. taeniatus
as paraphyletic (Fig. 3, left, Supplementary Fig. S1).
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On the other hand, the phylogenetic trees based on
SNPs recovered L. bahiensis as a monophyletic group
divided into highly supported subclades from each
coastal basin (Fig. 3, right), placing L. taeniatus as a sis-
ter group of L. bahiensis. Two reciprocally monophyletic
subclades were observed within L. bahiensis, compris-
ing the samples from Paraguaçu, Jequiriçá, and Almas
basins and another cluster that included populations
from Contas and Almada basins.
Genetic Structure of Leporinus bahiensis
Eight COI haplotypes described in L. bahiensis (Fig.
4a) were distributed according to each basin, except for
samples from Jequiriçá and Almas which shared the
same haplotype.
Structure analysis revealed K = 4 with the highest ΔK,
splitting L. bahiensis into 3 distinct clusters, Almada,
Contas and a third one joining the samples from
Paraguaçu, Jequiriçá and Almas, along with the signal
of an admixture from a fourth and unsampled popula-
tion in Paraguaçu and Almas (Fig. 4b, Supplementary
Table S5). A hierarchical analysis within the latter
cluster found K = 3 with the highest ΔK. Accordingly,
Paraguaçu was separated from Jequiriçá and Almas, and
it remained as a single group (Fig. 4b, Supplementary
Table S5). The admixture signal from the unsampled
population was once again suggested for the popula-
tions from Paraguaçu and Almas.
Species Delimitation
The final aligned COI sequences resulted in 619
characters with 51 parsimony-informative sites. All
single-gene species delimitation analyses invariably
recovered 8 molecular units, regarded as consensus
MOTUs, out of which 3 were related to L. bahiensis (Fig.
4c). These MOTUs are correlated with basins since most
of them encompassed a unique MOTU, except for the
populations from Paraguaçu/Jequiriçá/Almas that
were clustered in a single MOTU. Genetic distances for
both nominal species and consensus MOTUs are shown
in Supplementary Table S6. The maximum intraspecific
distance of L. bahiensis was 2.65%, while the maximum
intra-MOTU distance was 0.49%, and the minimum
inter-MOTU was 0.65%.
For the SNPs data set, we tested 5 species delimi-
tation scenarios using BFD coalescent delimitation.
The best-supported model comprised 7 lineages, 5
of which belonged to L. bahiensis, according to BFD
(Supplementary Table S3). This model decisively sup-
ported the division of L. bahiensis according to basin.
2023 RAMIREZ ET AL. - SPECIATION IN COASTAL BASINS DRIVEN BY STAGGERED HEADWATER CAPTURES 7

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Figure 3.  Phylogenetic trees of L. bahiensis and related species. Left: Bayesian species tree generated using 5 genes (approx. 4900 bp). Red
diamonds indicate nodes with support values above 0.9 of posterior probability in Bayesian inference and bootstrap higher than 75% in MP and
ML analyses. Yellow diamonds indicate high support values in 2 out of the 3 methods. Right: ML tree generated using SNPs data from GBS.
Red diamonds indicate nodes with bootstrap values for ML and quartet analyses higher than 75%. Numbers in node indicate divergence time
estimated using FASTSIMCOAL, in millions of years. Scale bar indicates nucleotide substitution per site.

BPP delimitation also inferred 7 potential species with
the lowest τ values for all tested priors (posterior proba-
bilities > 0.9). This model also divided L. bahiensis into 5
distinct units. However, considering the highest τ prior,
5 of the 12 tests suggested the presence of 6 species
(posterior probabilities lower than 0.9) joining Jequiriçá
and Almas populations, but with low support values.
The remaining tests supported the model that included
7 species (posterior probabilities > 0.9) (Supplementary
Table S7).
Dispersal Routes Assessment Using ABC
Demographic scenario 3 (Fig. 2) had the highest poste-
rior probability using raw summary statistics (posterior
probability = 1.0; 95% CI: 1.0–1.0), suggesting Paraguaçu
basin as a source for 2 independent dispersal events
towards Contas and Jequiriçá basins. Afterwards, the
specimens from Contas would have colonized Almada
basin, while another dispersal event was inferred from
Jequiriçá to Almas basin. The confidence estimated for
this scenario based on the logistic approach revealed a
low posterior error rate (0.074), confirming the reliabil-
ity of this analysis. As shown in Supplementary Table
S8, most tested parameters presented low RMAE values
(<0.2), except for 5 [NContas (N1), NAlmada (N2), Nb_Almada
(N2b), Nb_das Almas (N5b), and Tbot (db)], indicating that
the estimates are trustworthy (Supplementary Table S8;
Cornuet et al. 2008). These results squarely place sce-
nario 3 as the most appropriate model to explain the
current genetic subdivision in L. bahiensis.
Estimates of Divergence Times
FASTSIMCOAL2 divergence time estimates using
the best model obtained by ABC analysis dated the first
8 SYSTEMATIC BIOLOGY

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Figure 4.  Genetic structure of L. bahiensis. A) Median-joining (MJ) haplotype network using COI data. Small dark dots indicate missing
or unsampled haplotypes. B) STRUCTURE analysis based on SNPs data. The first layer included all individuals (K = 4), while the second
layer encompassed only the specimens from Almas/Jequiriçá/Paraguaçu (K = 3). Vertical bars indicate individual membership coefficient. C)
Bayesian tree showing the clustering of MOTUs obtained by species delimitation analyses based on COI. Blue line represents nominal taxonomy,
and red line represents consensus MOTUs. Red diamonds indicate nodes with support values higher than 0.9 of posterior probability. Scale bar
indicates nucleotide substitutions per site.
2023 RAMIREZ ET AL. - SPECIATION IN COASTAL BASINS DRIVEN BY STAGGERED HEADWATER CAPTURES
dispersal event from Paraguaçu to Contas around 1.95
Ma (95% CI: 1.94–2.1) (Fig. 3 right). The second disper-
sal event (Contas to Almada) took place about 1.36 Ma
(95% CI: 1.36–1.47), while the dispersal from Paraguaçu
to Jequiriçá occurred about 0.4 Ma (95% C.I. 0.39–0.43),
followed by a dispersal event from the latter to Almas
basin near 0.33 Ma (95% CI: 0.34–0.37).
Discussion
The specimens morphologically recognized as
L. bahiensis have been sampled in distinct coastal
basins along the state of Bahia, northeastern Brazil.
Surprisingly, the phylogenetic trees inferred from
nuclear and mitochondrial markers, successfully used
in studies of Anostomidae (Ramirez and Galetti 2015;
Ramirez et al. 2016, 2017b, 2020), failed to recover the
monophyly of L. bahiensis (Fig. 3). In fact, populations of
L. taeniatus were placed as a subgroup within the cluster
of L. bahiensis in spite of their differences in morphol-
ogy and range. L. taeniatus, distributed in São Francisco
and other rivers in northeastern Brazil (Garavello and
Santos 2009), is characterized by a continuous narrow
dark-brown stripe over a lateral line (Santos and Jégu
1996), while L. bahiensis is restricted to a few coastal
basins and presents 3 large-rounded blotches along the
midlateral line.
On the other hand, the SNPs data set resolved the
phylogenetic relationships between both closely related
species, supporting their reciprocal monophyletic sta-
tus (Fig. 3), probably because genomic approaches, such
as GBS, provided a larger amount of high-throughput
sequencing data. Actually, the monophyletic signal,
which is based on multiple loci (e.g., Sanger sequenc-
ing), requires longer periods of divergence (Rosenberg
2003; Knowles and Carstens 2007) and is, therefore,
limited to the inference of evolutionary relationships in
groups of rapid initial divergence. Different processes
like incomplete lineage sorting and ancestral polymor-
phism can hinder phylogenetic reconstruction based on
sequencing of a few loci, especially in cases of recent
speciation (Knowles and Carstens 2007).
On the other hand, the genetic structure of L. bahiensis
based on either COI or SNPs data usually supported
the discrimination of populations according to each
river basin (Fig. 4), except for samples from Jequiriçá
and Almas basins that shared the same COI haplotype.
Furthermore, the number of mutation steps between
Jequiriçá/Almas and Paraguaçu samples was lower
than that observed between other populations of L.
bahiensis. Since these coastal basins have no evidence
of past connections via palaeodrainages during low sea
level periods (Thomaz and Knowles 2018), the genetic
structure herein reported likely refers to allopatric
processes.
When the structured lineages observed in L. bahiensis
were tested for the putative presence of MOTUs using
COI data, a total of 3 consensus MOTUs was obtained,
corresponding, in part, to the sampled basins. Again,
9
the only exception was observed in the samples from
Jequiriçá/Almas/Paraguaçu, recovered as a single
MOTU. It should be pointed out that the lowest genetic
divergence in MOTUS of L. bahiensis based on COI was
relatively high (1.71–1.89%) in comparison to previous
reports in Anostomidae (Ramirez and Galetti 2015;
Ramirez et al. 2017a, 2020).
In contrast, multilocus (SNPs) species delimita-
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tion analyses invariably indicated 5 MOTUS within
L. bahiensis, each one corresponding to a single basin.
These results reinforce the idea that multilocus anal-
yses usually show higher resolution power than the
single-locus species delimitation approach (Liu et al.
2017), especially when considering very recent events,
such as that inferred among samples from Paraguaçu,
Jequiriçá and Almas (<400 Kya). Nonetheless, follow-
ing a conservative perspective, we suggest at least 3
MOTUs within L. bahiensis, as represented by the lin-
eages from Almada, Contas, and Paraguaçu/Jequiriçá/
Almas basins. This calls for a thorough and integrative
taxonomic reevaluation to determine species status of
these MOTUs. Moreover, the description of L. bahiensis
should be reevaluated with emphasis on the definition
of type locality since syntypes have been lost (Garavello
and Britski 2003).
Results based on the ABC approach revealed that the
diversification processes among populations of L. bahien-
sis could not be explained by simultaneous dispersal
events via submersed palaeodrainages in Pleistocene,
thus differing from other biogeographic reports in
Neotropical freshwater fish (e.g., Hirschmann et al.
2015; Thomaz et al. 2015; Abreu et al. 2020; Thomaz and
Knowles 2020). Indeed, the northeastern coast of Brazil,
site of the present study, presents a relatively narrow
continental shelf that would restrain the dispersal of
populations by the lack of submersed palaeodrainages
connecting coastal basins (Thomaz and Knowles 2018).
Thus, the best model to explain the present results
would comprise a staggered pattern characterized by
sequential events of headwater capture at different
periods among basins. This inference is supported by
previous reports, including distinct fish taxa from the
sampled region, revealing the key role of headwater
captures in the dispersal and genetic divergence among
currently isolated populations, even before sea level
changes in late Pleistocene (Menezes et al. 2008; Lima
et al. 2017; Ramirez et al. 2017b; de Sousa et al. 2021;
Barreto et al. 2022). In several cases, the remarkable
genetic differentiation among populations of a single
nominal taxon could only be explained by a complex
network of headwater captures followed by isolation
of drainages (Souza et al. 2020; de Sousa et al. 2021).
Accordingly, this study reinforces the importance of
headwater captures to the dispersal and further differ-
entiation of freshwater fish populations along the South
American Atlantic coast (Lima et al. 2017), as particu-
larly validated through the ABC approach combined
with a genome-wide SNPs data set (Fig. 2).
Finally, we can conclude that different approaches
are required to overcome the difficulties in studying
10 SYSTEMATIC BIOLOGY
the evolutionary history of species with recent diver-
gence, such as that observed in L. bahiensis. In partic-
ular, genome-wide SNPs data might be decisive in
resolving cases where other markers (e.g., partial gene
sequences) fail, as clearly demonstrated in the present
phylogenetic reconstruction and species delimitation
analyses. Leporinus bahiensis is recognized as a species
complex composed of at least 3 MOTUs. This calls for
a reexamination of its taxonomic status. Furthermore,
combining the ABC approach with genome-wide SNPs
data proved to be effective in reconstructing complex
biogeographic scenarios. Going forward, this will help
us understand speciation in other highly diverse groups
like Neotropical fishes.
Supplementary Material
Data available from the Dryad Digital Repository:
http://dx.doi.org/10.5061/dryad.0zpc8670j.
Acknowledgments
We are grateful to D. Carvalho, E. Feldberg, L.
Carvalho-Costa, P. Venere and W. Troy for their assis-
tance with tissue samples, ICMBIO/MMA for permis-
sion to conduct fish sampling (32215- 1), and SISGEN
for granting access to genetic material (AAA03B9).
Funding
This work was supported by the Conselho Nacional
de Desenvolvimento Científico e Tecnológico, CNPq
(405309/2016-3 and 303524/2019-7 to PMGJ). CBM
thanks PNPD/CAPES (Finance code 001).
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