Phylogeography of Brachyplatystoma rousseauxii in Amazon 723

Phylogeography of Brachyplatystoma
rousseauxii (Siluriformes - Pimelodidae) in the
Amazon Basin offers preliminary evidence for
the first case of “homing” for an Amazonian
migratory catfish
J.S. Batista1 and J.A. Alves-Gomes2
1
Instituto Nacional de Pesquisas da Amazônia (INPA),
Coordenação de Pesquisas (COPE),
Laboratório Temático de Biologia Molecular, Manaus, AM, Brasil
2
Instituto Nacional de Pesquisas da Amazônia (INPA),
Coordenação de Pesquisas em Biologia Aquática, Manaus, AM, Brasil
Corresponding author: J.A. Alves-Gomes
E-mail: [email protected]

Genet. Mol. Res. 5 (4): 723-740 (2006)

Received March 31, 2006
Accepted October 9, 2006
Published December 1, 2006

ABSTRACT. The large pimelodid, Brachyplatystoma rousseauxii, is

one of the two most important catfish species for the fisheries in the
Amazon. It is captured by commercial and artisanal fishing fleets in at
least five Amazonian countries, at fishing grounds more than 5000 km
apart. Current evidence suggests a complex life cycle that includes the
longest reproductive migration known for a freshwater fish species.
Experimental fisheries have pointed to a decrease in yield in the West-
ern Amazon. However, reliable information about the capture and status
of this fishery resource is still nonexistent, and no study has ever ad-
dressed its genetic diversity. We sequenced the entire D-loop of 45 indi-
viduals of B. rousseauxii, fifteen from each of three different fishing
locations along the main channel of the Solimões-Amazonas System
covering a distance of around 2200 km. Results of phylogenetic analy-

Genetics and Molecular Research

Research 55 (4):
(4): 723-740
723-740 (2006) FUNPEC-RP www.funpecrp.com.br
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 J.S. Batista and J.A. Alves-Gomes 724
ses, molecular diversity estimations, analysis of molecular variance, and
nested clade analysis, together show that there is no genetic segregation
associated with location in the main channel, as one would expect for a
migratory species. However, the significant decrease found in genetic
diversity towards the western part of the Amazon could be explained by
a non-random choice of tributary to spawn. It is possible that the genetic
diversity of the migrating schools decreases towards the west because
portions of the species’ genetic diversity are being “captured” by the
different effluents, as the fish migrates to spawn in the headwaters.
Like the salmon in North America, B. rousseauxii may be returning to
their home tributary to spawn.

Key words: Siluriformes, Brachyplatystoma, Phylogeography,

Amazon, Fish homing, Conservation

INTRODUCTION

The Amazon, with a geographical area of about 6,500,000 km2, holds an incomparable
network of river and water habitats which harbors the richest icthyofauna of the planet. Fish
represent not only a very accessible source of protein, but also an important source of income
for the Amazonians, especially for families in the lower socio-economic range. In fact, fishing
has always been a major economic activity in the entire region, but for most of the commercially
important fish groups we still do not know the basic parameters of their biology or the status of
their stocks. Finding the point where the fishing activity becomes sustainable is a major goal still
to be accomplished, and modern molecular techniques can definitely contribute to such a com-
plex task.
The large majority of the fish commercially captured for food in the Amazon falls within
a group of approximately 30 species, which are divided into two major classes, based on local
cultural standards: the scaled fish (essentially the representatives of orders Characiformes,
Osteoglossiformes, Perciformes, and Clupeiformes) and the scaleless or “leathered” fish (the
large catfish of the order Siluriformes). Considering the latter, 95% of the capture is centered
over around five species of the family Pimelodidae and more than 60% of that is made up by
only two pimelodids: Brachyplatystoma rousseauxii, regionally known as dourada, and B.
vaillantii, known as piramutaba (Barthem and Goulding, 1997). Understanding the life cycle
and population parameters of these two species is crucial to assure their conservation.
B. rousseauxii has been among the two most important and intensively captured cat-
fish for decades along the main channel of the Solimões-Amazonas River System1 (SAS), at
fishing grounds distributed over five countries, more than 5000 km apart (Barthem and Goulding,
1997) (Figure 1). The peak of fishing activity at most of the locations along this extensive
geographical area is seasonal and associated with a complex, and not completely understood,
life cycle. This cycle, which is also found in other large pimelodids, apparently includes the
longest reproductive migration known for a freshwater fish species.

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1
In its entire length, the Amazonas River covers a total of about 5900 km, passing by four countries and receiving seven
different names. Here we refer as Solimões-Amazonas System as the stretch of the main channel and its associated white
water tributaries.
 Phylogeography of Brachyplatystoma rousseauxii in Amazon 725

Figure 1. In the Amazon, the life cycle of Brachyplatystoma rousseauxii is associated with an extensive area that includes
the habitats near the Atlantic, around Belém, up to the headwaters of the Solimões’ River affluents, near the Andes. Along
this entire region, B. rousseauxii is an important item in the local economy as a source of income and fish protein
resource. We sampled 15 individuals at each of the three locations: Belém, Manaus and Tabatinga, covering more than
2200 km of river extension.

In 1998, a study comparing the capture of B. rousseauxii in Belém to previous years

(JICA/MPEG/IBAMA, 1998) pointed to overfishing, based on the reduction of the yield in that
region. More recently, Alonso (2002) confirmed the overfishing of dourada in the SAS, reinforc-
ing the need for studies focused on this species. The same situation appears to be applicable for
other large pimelodidae such as B. vaillantii (Barthem and Petrere, 1995) and B. filamentosum,
the largest pimelodidae in the Amazon (Petrere et al., 2004).

REPRODUCTIVE MIGRATION OF BRACHYPLATYSTOMA ROUSSEAUXII

A model for the life cycle of B. rousseauxii, and other pimelodid catfishes, was pro-
posed by Barthem and Goulding (1997), based on an extensive review of the available evidence
complemented by the analyses of several thousands of samples obtained from 25 rivers of the
Amazon Basin. For instance, the average size of capture at the main fishing locations along the
SAS increases towards the west, with the large mature fish being only found in the Western

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 J.S. Batista and J.A. Alves-Gomes 726
Amazon. Furthermore, not a single mature individual was ever registered east of Manaus, re-
gardless of the tremendous fishing activity over the last couple of decades. According to the
authors, schools of immature adults and pre-adults of “dourada” concentrate in the lower por-
tion of the Amazon River, near the estuary. Between June and November, or just about at the
time the river level starts to rise due to the rainy season in the headwaters, these large schools
start to congregate and move towards the Western Amazon. The fish travel about 5 to 6 months
to spawn exclusively in the headwaters of the white water tributaries of the SAS. These head-
water tributaries are distributed over at least five different countries (Brazil, Peru, Colombia,
Ecuador, and Bolivia), and the total distance covered by these schools from the mouth of the
Amazon may reach up to 5500 km. After spawning in the Western Amazon, the larvae are
passively carried by the water currents back to the main river channel of the SAS. By calculat-
ing the water speed, it is estimated that B. rousseauxii born in the headwaters may take be-
tween 13 and 20 days to reach the estuary again. In the protected and nutrient-rich estuarine
environment, the young grow for approximately three years, during which time they move up to
the lower portion of the main channel of the Amazon River. There, the fish may stay for another
year surviving on larger prey. After this growing period, schools form again and start migrating
west towards the headwaters, to complete the cycle.
The conception of such a migratory model represents a major advance towards a better
knowledge of the natural history of the migrating catfish in the Amazon, but several points still
remain to be elucidated. For instance, for the practical aspects of management and conserva-
tion policies, it is mandatory to know if the species considered is really composed of a single,
genetically homogeneous stock, or if there is some kind of genetic segregation associated with
space (between affluents) and time (between cohorts), or if the entire species performs the
migratory journey at the same time. Depending on the answer obtained, different conservational
units can be defined and appropriate policies adopted. It is also important to know if there are
resident populations along the area considered. Another relevant aspect related to this life cycle
is the importance of the different tributaries for the reproduction of B. rousseauxii. Do the
individuals return to the same tributary where they were born, forming “monophyletic” popula-
tion groups in each sub-basin, or is the tributary chosen for spawning defined by chance or other
fluctuating/momentary, environmental parameters? It is also not known how many times a fish
can perform the reproductive migration during its lifetime. Given the species’ complex life cycle
plus the nature and size of the area involved, traditional methods of fishery biology (meristic and
morphological characters) to define stocks have not proven to be enough. The general migra-
tory scenario proposed (Barthem and Goulding, 1997) has never been tested before from a
genetic perspective, and it has been established that such questions can receive a substantial
help from molecular markers (Avise, 2004), as we associated genetic diversity with geographi-
cal location. Here, we report the first assessment of the intraspecific genetic variability with its
correlation to geography for an Amazonian migratory fish species based on mitochondrial DNA
sequences.
The molecular marker used was the complete sequence of the mitochondrial control
region (also know as D-loop). Our main objective was to obtain preliminary and reliable infor-
mation about the phylogeography of the species and to establish if the genetic variability found
was related to geographical distribution. Such information can help us to compile meaningful
scientific information about the life cycle of this species and serve as a basis to subsidize man-
agement and conservation policies in the near future.

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 Phylogeography of Brachyplatystoma rousseauxii in Amazon 727
MATERIAL AND METHODS

Sampling

Specimens of B. rousseauxii were collected at three points along the SAS: Belém,
Manaus and Tabatinga (Figure 1). At each location, 15 individuals were sampled from the
artisanal fishing fleet during 1998 and 1999. Special attention to sample from the artisanal fish-
eries was because this kind of activity uses small boats with little autonomy, and therefore, all
their fishing activity is within a radius of no more than 20 km from the town selected. In this way,
we were assured that our samples represented the genetic diversity of individuals occurring
around each location, which is crucial if we wanted to test the possibility of differentiating
possible resident populations. Special care was also taken in order to sample the largest variety
of sizes possible and on different days, as a measure to reduce the chance of sampling the same
cohort. Besides the individuals of B. rousseauxii, one specimen of B. vaillantii collected in
Belém was also included in the phylogenetic analysis as the sister group. Samples of muscle of
each individual were excised with sterilized blades and pliers and conditioned in tubes with 80%
ethanol until DNA extraction in the laboratory.

DNA extraction, PCR and sequencing

An SDS-phenol/chloroform/isoamyl alcohol protocol was used to obtain total DNA

(Alves-Gomes et al., 1995). The quality of the extracted DNA was determined on 0.8% aga-
rose gels stained with ethidium bromide and photographed with a digital photo-documentation
apparatus (Eagle Eye - Stratagene). A fragment encompassing the entire mitochondrial control
region of each individual was amplified in a PCR reaction with a total volume of 30 µL with 10-
100 ng of total DNA, 1.5 mM MgCl2, 1X Taq buffer, 200 µM dNTPs, 0.5 µM of each primer,
and 0.5 unit Taq DNA Polymerase. The primers used have the following sequences: F-TTF: 5’
GCC TAA GAG CAT CGG TCT TGT AA 3’ and F-12R: 5’ GTC AGG ACC ATG CCT TTG
TG 3’ (Sivasundar et al., 2001). The amplification was carried out in 30 cycles with the follow-
ing temperature profile: the first five cycles were executed with 1 min at 94°C, 1 min at 53°C
and 1.5 min at 72°C. In the remaining 25 cycles the annealing temperature was dropped to
50°C. The efficiency of the amplification was determined on 0.8% agarose gels, with a molecu-
lar weight marker (λ DNA digested with HaeIII). All the amplified products were purified with
the Wizard kit (Promega) following the protocol of the manufacturer. The purified product
(mtDNA control region) was sequenced with the ABI PRISM BigDye Terminator Cycle Se-
quencing in an ABI 377 automated DNA sequencer (Perkin Elmer), following the protocol
accompanying the cycle sequencing kit. Sequences were checked, edited and aligned using the
program SEQED 1.0.3 (ABI, 1992).

Phylogenetic and statistical analyses

The phylogenetic estimations were performed by three methodological principles: maxi-

mum parsimony (MP), maximum likelihood (ML) and distance, with the program PAUP* ver-
sion 4.0 (Swofford, 1999). For the parsimony analyses, two weight matrices for transitions (TS)
and transversions (TV) were used: TS1TV1, where the same cost of one step was given to the

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 J.S. Batista and J.A. Alves-Gomes 728
two types of mutations, and TS1TV2, where each TV costs twice the number of steps of each
TS. No analyses were performed considering gaps as a fifth character state because no gaps
were present among the B. rousseauxii sequences (all gaps were autapomorphies for B.
vaillantii). All parsimony analyses were carried out including an individual of B. vaillantii as
the outgroup. The remaining options for parsimony in PAUP* were: characters were unor-
dered, multistate characters were considered uncertain, gaps were treated as missing, and un-
informative sites were excluded. Most parsimonious trees were generated by heuristic search
and the taxa were added by random stepwise addition. One hundred replications were per-
formed with TBR as a method for clade permutation. For each tree we obtained the length (L),
measured by the number of steps, the consistency index (C.I.) and the retention index (R.I.).
The consensus trees for both weight matrices were obtained by strict consensus and by the
50% majority rule. The degree of confidence for each branch of the tree was estimated by
bootstrap (Felsenstein, 1985) with 1000 replications.
For ML searches, the evolutionary model was defined with the program MODELTEST
3.0 (Posada and Crandall, 1998). The model adopted was the Hasegawa-Kishino-Yano (HKY
85) (Hasegawa et al., 1985) considering the gamma distribution (Γ) for the number of mutations
per site, and the proportion of invariable sites (I). The empirical frequency of nucleotide bases
and the TS/TV ratio were also defined by the program.
For the distance method, we created an uncorrected distance matrix (p distance) from
which we extracted the number of TSs and TVs for all pairwise comparisons. In order to check
any possible saturation of TS and TV, we plotted the p distance versus both types of mutations
for each pairwise distance. Subsequently, the distances were corrected for multiple hits with the
same model used in ML (HKY + Γ + I). From this corrected matrix, we also estimated a tree by
the neighbor joining (NJ) method.

Population genetics analyses

The number of haplotypes within each of the populations (location) sampled was deter-
mined with the program COLAPSE 1.2 (http://darwin.uvigo.es/software/collapse.html). Sev-
eral other parameters which estimate the DNA polymorphism were determined with the pro-
gram DNASP 4.0 (Rozas and Rozas, 1999). The following parameters were estimated: haplo-
typic diversity - HD, nucleotide diversity - Pi (Nei, 1987), average pairwise distance - K and
respective variances [observed V(k)o, and estimated V(k)e] (Tajima, 1983), total number of muta-
tions - ETA; number of polymorphic sites - S; the average number of substitutions per site - Dxy, net
number of substitutions per site - Da (Nei, 1987), and genetic flux, or number of migrants between
populations per year - Nm (Nei, 1982). The Jukes-Cantor model was also adopted (Jukes and
Cantor, 1969) in order to determine if the molecular variability found among and between the
populations of B. rousseauxii were significant. An analysis of molecular variance (AMOVA)
(Excoffier et al., 1992) was performed with the program ARLEQUIN 2.0 and the level of
significance for the indices and for the total variance was tested by 1000 non-parametric per-
mutations of the haplotypes between the populations (Schneider et al., 2000).

Nested clade analysis

The nested clade analysis (NCA) (Templeton et al., 1987, 1988, 1992, 1995) is a method

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 Phylogeography of Brachyplatystoma rousseauxii in Amazon 729
developed to estimate intra-specific genealogies and to test if the topology obtained for the
haplotypes is associated with some type of geographic structure. This analysis was performed
with the 45 B. rousseauxii sequences. The intra-specific cladogram was produced with the
program TCS 1.06 (Clement et al., 2000) and the final nested clade topology was obtained
manually, according to the procedures described by Crandall and Templeton (1993, 1996) and
Crandall (1996). Subsequently, we used the program GEODIS 2.0 (Posada et al., 2000) to
calculate the statistics related to the clade distances and to test if the several levels of clades
and sub-clades obtained were also associated with a geographic structure. The significance
levels of the analyses were obtained by 10,000 random permutations considering the number of
individuals sampled at each location and the geographic distance between each location where
the haplotypes were collected. The distances between locations were estimated on a map,
following the course of the river: Belém-Manaus ≈ 1000 km, Manaus-Tabatinga ≈ 1200 km and
Belém-Tabatinga ≈ 2200 km. The analyses also include an exact contingency test, where the
null hypothesis of no association between the clades and the geographic locality was tested.
Two types of distances for each sub-clade were calculated: the clade’s internal distance (Dc)
and the distance between clades (Dn). The distance between clades is the average of these
distances. The program also calculates two other distances between interior (I) and tip (T)
clades: I-Tc and I-Tn (Templeton et al., 1995; Templeton, 1998). Once all the distances were
calculated, the results were interpreted according to a biological key conceived by Templeton
(1998).

RESULTS

Nucleotide sequences and base composition

The complete DNA sequence for each individual totaled 1037 bp and comprised 12 bp
of threonine tRNA, the complete 70 bp of proline tRNA, the complete sequence of 911 bp of the
D-loop, and another 44 bp corresponding to the partial sequence of the phenylalanine tRNA.
When the specimen of B. vaillantii (genbank accession number DQ779047) was incorporated
into the matrix as a sister group, the alignment of the 46 sequences generated a data matrix with
1069 bp, with the necessary introduction of 32 indels (insertion/deletion events). In the matrix
without the outgroup, 983 bp were constant, 54 were variable and, among these, 30 were phy-
logenetically informative, whereas with the outgroup included 905 sites were constant, 129
variable and 35 informative.
The average base composition for the 45 mitochondrial DNA control region sequences
of B. rousseauxii was 32.49 ± 0.17% for thymine, 32.18 ± 0.12% for adenine, 21.25 ± 0.17%
for cytosine, and 14.07 ± 0.08% for guanine. In addition, in the 54 polymorphic sites of the 1037-
bp matrix, 57 substitutions were observed, which were 45 TS and 12 TV, giving a transition/
transversion (TS/TV) ratio of 3.9. No saturation was detected for both TS and TV when the
absolute number of the two types of mutations was plotted against the uncorrected “p” distance
(Figure 2).

Phylogenetic analysis

For MP searches, the analyses produced four trees equally parsimonious for each of

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 J.S. Batista and J.A. Alves-Gomes 730

Figure 2. Number of substitutions (transitions = TS and transversions = TV) plotted against the uncorrected genetic
distance (p) for all pairwise comparisons of the D-loop matrix excluding the outgroup.

the weight matrix used (TS1TV1 and TS1TV2). For TS1TV1, the L was 208 steps, the C.I. =
0.822 and R.I. = 0.809 and for TS1TV2, L = 271; C.I. = 0.838 and R.I. = 0.802. The strict
consensus trees of both types of weighting schemes were identical. For searches under the MP
criterion, the HKY + Γ + I was used as an evolutionary model. These parameters and values
were defined as: proportion of invariable sites = 0.633; gamma parameter = 0.6723; TS/TV
ratio = 4.55, and the proportion of bases were A = 0.3194, C = 0.2115, G = 0.1428, T = 0.3263.
With these parameters, we obtained a topology with -Ln = 2508.26406. We also used the model
HKY + Γ + I to calculate the average distance among and between the individuals collected in
the three sampling locations (Table 1) and to generate a topology based upon the NJ algorithm.
We found distances varying between 0.0 and 3.5% among B. rousseauxii, whereas the uncor-
rected “p” genetic distance between B. rousseauxii and the outgroup varied between 12.5 and
13.7%. A 50% majority rule consensus tree was computed among the 10 trees found under all
conditions used (4 resultant trees for MP TS1TV1, 4 trees for MP TS1TV2, 1 tree for ML and
1 tree for NJ) (Figure 3).

Table 1. Average pairwise percentual distance, corrected with HKY evolutionary model, estimated among and
between the Brachyplatystoma rousseauxii individuals collected in three sampling locations.

Belém Manaus Tabatinga

Belém 1.2 ± 0.5

Manaus 1.2 ± 0.7 1.0 ± 0.7
Tabatinga 1.1 ± 0.4 0.9 ± 0.6 0.8 ± 0.4

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 Phylogeography of Brachyplatystoma rousseauxii in Amazon 731

Figure 3. A general 50% majority rule consensus tree computed among the 10 trees found under all phylogenetic
conditions used. Individuals from Belém are identified in blue, Manaus in green and Tabatinga in red. The numbers over the
branches represent the percentage in which those branches were recovered among the resultant trees.

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 J.S. Batista and J.A. Alves-Gomes 732
Haplotype frequency and distribution

A total of 34 haplotypes were identified when each location was computed separately,
consisting of 15 in Belém, 10 in Manaus, and 9 in Tabatinga. As we proceeded to define the
singleton distribution (haplotypes represented by a unique sequence or individual in the popula-
tion) for these 34 haplotypes, Belém had 15 (no repeated haplotype), Manaus had 8 (haplotypes
D17 and D18 repeated four and three times, respectively) and Tabatinga 6 (haplotypes D17 and
D28 twice, and D18 five times). Because some haplotypes were present in Belém and Tabatinga
(D14) and in Manaus and Tabatinga (D17 and D18), 31 haplotypes and 27 singletons were
identified when the three locations (45 individuals) were considered altogether (Table 2).

Table 2. Frequency of the 31 haplotypes of Brachyplatystoma rousseauxii sampled in three Amazonian localities.

Haplotype Belém Manaus Tabatinga Total GenBank

accession number

D1 1 1 DQ779016
D2 1 1 DQ779017
D3 1 1 DQ779018
D4 1 1 DQ779019
D5 1 1 DQ779020
D6 1 1 DQ779021
D7 1 1 DQ779022
D8 1 1 DQ779023
D9 1 1 DQ779024
D10 1 1 DQ779025
D11 1 1 DQ779026
D12 1 1 DQ779027
D13 1 1 DQ779028
D14 1 1 2 DQ779029
D15 1 1 DQ779030
D16 1 1 DQ779031
D17 4 2 6 DQ779032
D18 3 5 8 DQ779033
D19 1 1 DQ779034
D20 1 1 DQ779035
D21 1 1 DQ779036
D22 1 1 DQ779037
D23 1 1 DQ779038
D24 1 1 DQ779039
D25 1 1 DQ779040
D26 1 1 DQ779041
D27 1 1 DQ779042
D28 2 2 DQ779043
D29 1 1 DQ779044
D30 1 1 DQ779045
D31 1 1 DQ779046
Total 15 15 15 45

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 Phylogeography of Brachyplatystoma rousseauxii in Amazon 733
Population diversity analysis

The results obtained for estimation of the intra- and inter-population DNA variability
and the other parameters related to DNA polymorphism are shown in Tables 3 and 4. Figure 4
shows an illustrative summary of the variability found. For the most relevant parameters calcu-
lated (H, S, ETA, and HD), the variability found among the individuals collected in Belém is
larger than in Manaus. In turn, the genetic variability of the individuals collected in Manaus is
higher than the variability found in Tabatinga.

Table 3. Summary of genetic parameters estimated from the mtDNA control region of the Brachyplatystoma
rousseauxii sampled in Belém, Manaus and Tabatinga.

Locality N H S ETA HD Pi K V(k)o V(k)e

Belém 15 15 38 39 1.000 0.0095 9.705 12.17 22.24

(0.024) (0.0007)
Manaus 15 10 34 36 0.914 0.0079 8.143 27.20 16.03
(0.056) (0.0015)
Tabatinga 15 9 22 23 0.886 0.0064 6.543 11.93 10.74
(0.069) (0.0007)
N: number of samples, H: haplotype frequence, S: total number of polymorphic sites, ETA: total number of mutations,
HD: haplotypes diversity, Pi: nucleotide diversity, K: average number of nucleotide differences and respective variances
observed, V(k)o, and estimated, V(k)e. Numbers in parentheses for HD and Pi are the standard deviations.

Table 4. Summary of genetic parameters estimated from the mtDNA control region of the Brachyplatystoma
rousseauxii between sampled from Belém, Manaus and Tabatinga.

N S ETA(t) P1m2 P2m1 DF SM Pi K Dxy Da K (bp) Nm

Belém (1)
x 30 52 55 19 16 0 20 0.00912 9.416 0.0064 0.00093 9.876 5.39
Manaus (2)

Manaus (1)
x 30 38 40 17 4 0 19 0.00702 7.271 0.0070 -0.00014 7.204 19.52
Tabatinga (2)

Belém (1)
x 30 42 43 20 4 0 19 0.00833 8.595 0.0088 0.00089 9.036 5.22
Tabatinga (2)
N - number of samples, S - total number of polymorphic sites, ETA - total number of mutations, P1m2 - polymorphic in
population 1 but monomorphic in population 2, P2m1 - polymorphic in population 2 but monomorphic in population 1,
DF - fixation differences, SM - number of shared mutations, Pi - nucleotide diversity, K - average number of nucleotide
differences, Dxy - average number of substitutions per site, Da - net number of substitutions per site, K (bp) average
number of nucleotide differences between populations, Nm - number of migrants among populations per year.

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 J.S. Batista and J.A. Alves-Gomes 734

Figure 4. Comparison of main genetic diversity parameters among Brachyplatystoma rousseauxii individuals collected at
three localities along the Solimões/Amazonas axis. The parameters related to genetic diversity show a recurrent pattern:
Belém has the highest genetic diversity and Tabatinga the lowest, for the locations sampled, i.e., the genetic diversity
decreases towards the Western Amazon, as one approaches the Andes. H: haplotype frequency; S: number of polymorphic
sites; ETA: number of mutations, K: average number of nucleotide differences.

More than 93% of the total variance was observed within the locations, whereas 6.61%
was distributed among the locations (Fst = 0.0661, P < 0.05). The genetic variability was signifi-
cantly different between the locations at Belém and Manaus (Fst = 0.10; P < 0.01) and between
Belém and Tabatinga (Fst = 0.10; P < 0.05). Thus, the genetic variability found in Belém was
significantly larger than the diversity found in Manaus and Tabatinga, corroborating the data in
Tables 4 and 5. No significant difference was found between the genetic diversity of Manaus
and that of Tabatinga.

Nested clade analysis

Figure 5 depicts the cladogram of the haplotypes (Table 2), for a 95% degree of confi-
dence. In Table 5 we show the steps followed for the biological inference, using the key of
Templeton (1998). The results obtained establish that for step-clades from 1 to 4, there were no
significant distances (Dc or Dn). A significant value was only obtained for step-clade of level 5,
which represents the entire cladogram. The analyses performed with the objective to determine
if there was any genetic structure associated with geographic distribution failed to reveal such
association. Therefore, we could not distinguish isolated (sub) populations of B. rousseauxii in
the SAS main channel.

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 Phylogeography of Brachyplatystoma rousseauxii in Amazon 735

Figure 5. Nested cladogram structure estimated with the haplotypes from the three Amazonian locations: Belém, Manaus
and Tabatinga. The circles represent not sampled or hypothetical haplotypes. Each line between haplotypes represents
one mutation.

Table 5. Key for biological inference (Templeton, 1998) results obtained according to nested clade cladogram
analysis (Figure 5).

Clade Steps followed Biological inference

in the key

All clades of steps 1 to 4 1-No It is not possible to reject Ho (There is no geographic

association between haplotypes)
Step-5 (whole cladogram) 1-2-3-5-6-7-Yes Restricted gene flow and/or dispersion, but with some
long distance dispersion

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 J.S. Batista and J.A. Alves-Gomes 736
DISCUSSION

Phylogenetic analyses

The absence of genetic segregation of B. rousseauxii associated with geographic loca-

tion is well characterized at the 50% majority rule consensus tree for all 10 resultant topologies
(Figure 3) obtained for the fish collected in Eastern, Central and Western Amazon. This result
supports the hypothesis of a single stock of B. rousseauxii migrating between the estuary and the
headwaters to spawn (Barthem and Goulding, 1997). In principle, we have a genetically mixed
stock that uses the main river channel along the entire extension of the SAS main channel.
The genetic distance among the 45 individuals of B. rousseauxii, corrected by the
HKY85 model, varied between 0.0 and 3.5% (average = 0.8 ± 0.3). When the pairwise dis-
tance was computed for each location separately, we obtained values varying between 0.0 and
2.3% (average = 1.2 ± 0.5) for Belém, 0.0 and 3.5% (average = 1.0 ± 0.7) for Manaus and 0.0
and 1.5% (average = 0.8 ± 0.4) for Tabatinga. When we computed distances for pairs of
locations, we found values between 0.2 and 3.5% (average = 1.2 ± 0.7) between Belém and
Manaus, 0.0 and 2.3% (average = 1.1 ± 0.4) between Belém and Tabatinga, and 0.0 and 2.9%
(average = 0.9 ± 0.6) between Manaus and Tabatinga. The largest absolute distance in the
matrix was 3.5%, between two individuals from Manaus (individuals 294MA and 188MA), but
when the average pairwise distance was computed for each locality, Belém had the largest
variability and Tabatinga the smallest (Table 1).
In all the phylogenetic trees, the specimen 294MA, collected in Manaus, was depicted
as the most basal taxon for the group of individuals analyzed. The genetic distances associated
with this specimen were always the largest found in the pairwise calculations, going from 1.1 to
3.5%, pushing the average distance found in Manaus to the levels found in Belém. Additional
sampling may reveal if this haplotype is also found in other locations and if it might, in fact, really
be a representative of an “ancestral” population of B. rousseauxii in the Amazon.
The genetic distances found in the present study are within the range found for other
Amazonian species, for the same DNA marker, such as Prochilodus lineatus (0.3-3.5%)
(Sivasundar et al., 2001) and Mylesinus paraschomburgkii (0.0-6.9%) (Porto, 1999).

Population analyses: haplotype distribution and DNA polymorphism

The decrease in the genetic variability of B. rousseauxii from Belém to Manaus to

Tabatinga (Tables 3 and 4 and Figure 4) can be unambiguously inferred from the several param-
eters calculated such as, HD, S, ETA, Pi, K, and number of non-shared polymorphic sites
between populations. On the other hand, the absence of fixed mutations between the individuals
of the three locations may be considered as a strong indication that the individuals are not
genetically isolated in the SAS main channel.
The haplotype distribution and occurrence suggest a higher similarity between the popu-
lations of Manaus and Tabatinga (Table 2). According to haplotype diversity, Belém contains the
higher diversity, supporting the results also related to nucleotide diversity (Tables 3, 4 and Figure
4). In fact, by all parameters analyzed, the variability tends to decrease towards the west, from
Belém to Tabatinga. Among the possible explanations that can account for that is that there are
resident and genetically more divergent populations in the middle and lower Amazon that do not

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 Phylogeography of Brachyplatystoma rousseauxii in Amazon 737
reach the headwaters. However, this is not likely considering the strong evidence for a migra-
tory cycle in B. rousseauxii based on experimental fisheries, gonadal maturation and informa-
tion from fishermen. A second possible explanation is the very high capture rate in Manaus and
Tabatinga, which causes a decrease in genetic diversity in these places. In other words, parts of
the total gene pool are being captured in the several white water affluents along the way. Only
a fraction of the individuals really reaches the furthest tributaries. The most likely third plausible
phenomenon is that part of the genetic diversity of the species does not reach the western part
of the Amazon because the schools formed near the estuary return to the river where they were
born, and not to a randomly chosen tributary.
According to Kimura and Maruyama (1971), two populations can be differentiated if
the gene flow estimator Nm (where N is the effective population size and m is the rate of
mutation/site/generation) assumes values below 1, whereas a single and panmictic population
unit would be considered for values above 4. In this study, the Nm estimated between the
populations was always larger than 4 (Table 4). The values of Nm point to migration and/or
mixing between populations, and consequently gene flow between the three locations, which
defines a single population of B. rousseauxii in the SAS main channel. The relatively low values
of intra- and inter-population nucleotide diversity (Pi), the average number of substitutions/site
(K) and the net rate of substitutions (Da), reinforce such a concept (Tables 3 and 4). However,
we still need to confirm if the different tributaries hold genetically segregated populations.

Nested clade analysis

Intra-specific phylogenies constitute a different substrate for evolutionary principles

than phylogenetic relationships between species, and therefore their study requires a distinct
approach (Posada and Crandall, 2001). In this regard, the NCA can help us to understand the
evolutionary relationship among the individuals/haplotypes, also considering the geographic in-
formation related to the clade under study.
In the nested cladogram we constructed (Figure 5), we observed the same basic result
of no genetic differentiation associated with collection site or geographical distribution, as we
observed in the phylogenetic analysis. There was no significant value for any level up to the four-step
clades. As we examine the values for Dc, Dn, I-Tc, and I-Tn found under the biological key
(Templeton, 1998), we cannot reject H0, i.e., there is no association between geographic distri-
bution and the genetic diversity of the haplotypes. For the results obtained, three possibilities are
suggested (Templeton, 1998): either an interbreeding population in panmixia, a wide distribution
of a non-interbreeding population or reduced sample size. In this study, we believe the first
alternative seems to be more likely the case. The only nested clade that showed significant
values was the five-step clade (5-1) which encompasses the entire cladogram (Figure 5). By
inference from the key, the explanation invokes restricted gene flow and/or long distance dis-
persion in the populations/individuals considered, i.e., between individuals of Belém and Tabatinga.

An alternative hypothesis for the life cycle of Brachyplatystoma rousseauxii

According to all the results obtained in the different analyses (phylogenetics, DNA
polymorphism, AMOVA, and NCA), with the specimens collected from the three locations
(Belém, Manaus and Tabatinga), we proposed the following scenario: we cannot reject the

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 J.S. Batista and J.A. Alves-Gomes 738
hypothesis of a single, mixed population along the SAS main axis (Barthem and Goulding, 1997).
In Belém, however, we have a significantly higher genetic variability of the species. In our
model, this is because the estuary congregates individuals originating from all the white water
affluents of the SAS. Furthermore, if the choice of affluent was driven by chance only, we
should not detect any decrease in DNA polymorphism in the western part of the Amazon, since
the complete gene pool would represent all locations. Tabatinga, on the other hand, possesses
the lowest genetic variability among the locations sampled. This would be possible because the
whole gene pool of the species, initially concentrated in Belém, is diminished as the migratory
schools move from the estuary to the headwaters of the tributaries, and a large majority of the
migrating fish are able to return to the same tributary where they were born, not choosing their
affluent to spawn by chance or momentary circumstances during the reproductive migration,
i.e., the species is homing, as do the salmon in North America.
There is only one large white water river between Belém and Manaus, the Madeira,
whereas between Manaus and Tabatinga there are two large rivers on the right (Purus and
Juruá) and two on the left (Japurá and Içá) margins. Therefore, if the hypothesis outlined before
is correct, we should expect a larger drop in genetic variability between Manaus and Tabatinga
than between Belém in Manaus. This can be observed for the S and ETA parameters (Figure
4). The white water affluents of the SAS appear to serve as a “sink” of the genetic diversity for
the migrating of B. rousseauxii in the Amazon. This hypothesis is relatively easy to test, as the
samples obtained within the different tributaries should show a very low genetic divergence or
“monophyletic” assemblages. These populations mix in the SAS during a considerable portion
of their life cycle, but segregate for reproduction.
According to Barthem and Goulding (1997), migration of this species is associated with
the white water tributaries of the Amazon basin. There is no information, if or to what extent B.
rousseauxii also uses other non-white water rivers of the SAS for reproduction, such as the
rivers Tocantins, Tapajós, Xingu, Trombetas, Uatuma, Jari, and Negro. We know from local
fishermen that B. rousseauxii may reach up to the middle of the Negro River, but this is not a
common event. We also have information from fishermen that B. rousseauxii may be found in
the upper parts of the Tapajós and Xingu Rivers, but we do not know how far or if there is any
reproductive activity of this fish at these locations. It is well known that the nutrient-poor and
acidic black waters of the Negro River do not provide enough food for the large and voracious
schools of migratory pimelodids. It is not surprising, therefore, that all the commercial fishery
activity in the Amazon is associated with the nutrient-rich and highly productive white water
rivers. The main white water rivers of the Amazon are the Madeira, Purus and Juruá on the
right margin and the Napo, Putumayo/Içá and Caquetá/Japurá on the left margin. We obtained
one sample of a young B. rousseauxii from one of the main affluents of the Negro River (the
Branco River) which may imply an important role of the Negro River Basin in the life cycle of
B. rousseauxii.
There are B. rousseauxii also in the Orinoco River Basin where migrations similar to
that in the Amazon also occur (Ramirez-Gil H, personal communication), but we know even less
about the Orinoco populations than the Amazonian. There is an intermittent contact between the
Orinocan and Amazon basins through the headwaters of both basins, but such contact is unlikely to
allow the transpositions of large fish or schools, such as those of Brachyplatystoma, between
basins. The degree of genetic differentiation between the Amazonian and Orinoco populations still
remains to be studied. Isozyme and RAPD data were used to assess the variability between

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 Phylogeography of Brachyplatystoma rousseauxii in Amazon 739
populations of Pseudoplatystoma fasciatum and P. tigrinum, two apparently non-migratory
pimelodid species with a larger distribution in the Neotropics than Brachyplatystoma from the
Orinoco and the Amazonian Basin. Despite the lack of variation at the enzyme level, the author
could tell the Orinocan and Amazonian population apart based on RAPD for both species (Ramirez
Gil, 2001). Probably the same is true for B. rousseauxii, but no information about the genetic
variability or gene flow between the B. rousseauxii of the two river systems is available.
Considering this scenario, it is important to call attention to the potential biological im-
portance that the different tributaries may represent for the life cycle of B. rousseauxii. Poli-
cies for its conservation and management and that of other migratory species such as B. vaillantii
and possibly B. filamentosum, should carefully consider the hypothesis proposed here. If differ-
ent rivers have different populations, then any action involving a single river may affect a con-
siderable portion of the genetic variability of the species studied. It is also important to consider
the fact that B. rousseauxii uses different areas for growth and for reproduction. This whole
situation gains additional importance if we consider that the species is present and captured at
least in five different countries. The social and economic benefit resulting from the commercial
exploitation of this species depends, intimately, on correct management policies that can guaran-
tee the sustainable use of this resource.

ACKNOWLEDGMENTS

The authors thank Dr. Iracilda Sampaio from Universidade Federal do Pará (UFPA)
for help on getting sequences, Juan Carlos Alonso, SINCHI/Colômbia, for help on sample col-
lection, Guillermo Orti from University of Nebraska for the primers, Izeni Farias from LEGAL/
ICB/UFAM, and Tomas Hrbek from University of Puerto Rico for help in some analyses. We
also thank Fundação de Amparo à Pesquisa do Estado do Amazonas (FAPEAM), CNPq and
MCT-INPA (grant PPI 1-3550) for financial support.

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