High genetic variability and lack of population structure in Prochilodus lineatus

in the upper Pilcomayo and Bermejo River systems in South-east Bolivia

Ruud van den Heuvel

_________________________________________________________________________________________________________

Abstract: Prochilodus lineatus is a highly migratory fish species inhabiting the Paraná-Paraguay basin. In the Pilcomayo River system decline of

P. lineatus due to anthropogenic activities and isolation of the Pilcomayo River from Paraguay River have caused interest in creating efficient

management and conservation strategies. Currently, information to create these strategies is lacking. In particular, migratory patterns for P.

lineatus in the upper Paraná are poorly known and only few population genetic studies are available. To asses genetic population structure we

genotyped 201 individuals from four locations in the upper Pilcomayo and Bermejo Rivers (Villa Montes, El Pibe, Rio Pilaya and Bermejo). All

individuals were genotyped for 5 microsatellite loci found to be informative to assess populations of P. lineatus. Our results show high genetic

variability and lack of population structure within the Pilcomayo River system supporting our hypothesis that during the 1990’s a ‘reserve’ fish

stock might had been present in the lower floodplains of the Grand Chaco area able to revitalize fish decline. Continued isolation of the Pilcomayo

River with the Paraguay basin could pose a threat for this fish stock due to limited possibilities for new genetic diversity to be generated via gene

flow.

_________________________________________________________________________________________________________

Introduction

In recent years, phylogeography and phylogeny have

become powerful tools in understanding evolution of

migratory fish populations, species and communities in

distinct neotropic environments (Piorski, Sanches et al.

2008). By giving insight in how fish populations are

influenced by genetic factors such as the impact of mutation,

genetic drift and selection as well as historical factors

(Piorski, Sanches et al. 2008) these tools provide a basis for

assessment and development of efficient management and

conservation strategies. Assessment and preservation of

biodiversity of migratory fish populations is crucially

important to minimize the loss of genetic variation,

especially when these species are under threat of decline

due to anthropogenic stressors. Decline in species genetic

variation may reduce the ability of a species to adapt to

environmental changes, ultimately decreasing long term

survival (Frankham, Briscoe et al. 2002). So far, little is known

about the genetic variation and population structure in

migratory freshwater fish populations inhabiting large

complex Neotropical River systems. Understanding how

their seasonal migratory patterns influence their genetic

makeup could have implications for fisheries, aquaculture,

management and conservation.

Prochilodus lineatus,

(Characiformes, Prochilodontidae), commonly known as

“sábalo” (in Argentina, Bolivia, Paraguay, Uruguay) or

“curimbatá” (in Brazil) is a native freshwater fish with a

geographical distribution in the Paraná-Uruguay and the

Paraíba do Sul River basins located in South America (Reis,

Kullander et al. 2003). Within these basins, P. lineatus is

considered the most abundant fish species in terms of

absolute biomass (Sverlij, Ros et al. 1993). The fish species is

illiophagous, feeding on algae, detritus and associated

microorganisms (Bayol and de Yuan Cordiviola 1996, Fugi,

Hahn et al. 1996). Therefore Prochilodus is considered a key

species for the role it plays in the cycle of organic matter and

its position in the river food-chain (Pimm , Bowen 1983,

Flecker 1996, Taylor, Flecker et al. 2006). Furthermore,

sábalo is of great economic importance to commercial and

artisanal fisheries in the Paraná River basin (Sivasundar,

Bermingham et al. 2001, Smolders, Guerrero Hiza et al. 2002,

Baigun, Minotti et al. 2013).

Within the Paraná basin the

Pilcomayo and Bermejo Rivers are considered important

migration routes for sábalo as they serve as “fish highways”

towards the spawning grounds located upstream in the

foothills of the Andean mountains. The Pilcomayo and

Bermejo Rivers are tributaries to the large La Plata system

and arise in the Bolivian Andes (Cordillera Oriental, at c 5000

m and 2000 meter above sea level respectively) after which

they cut down through the Andes through an inaccessible

terrain with rapids and narrow canyons ultimately meeting

with the Rio Paraguay in the lower Chaco plains (Figure 1).

Each year during the late dry season, when water levels and

water flow velocity are lowest and water temperature is cool

(<25°C) large shoals of adult sábalo start undertaking

upstream migrations towards the upper parts of the

Bermejo and Pilcomayo Rivers (Smolders, Guerrero Hiza et

al. 2002, Lucas and Baras 2008). During this migration

Prochilodus disperses from feeding grounds located in the

lower parts of the Gran Chaco area and the Paraguay-

Uruguay River system, often covering large distances. After

a migration covering a timespan of several months large

shoals of migrants reach the upstream spawning grounds

located in the Andean foothills. They gather in the open

water of the main channel or tributaries and spawn when

river discharges start increasing at the onset of the rainy

season (Bayley 1973). The peak of reproduction falls in

November-January (Mochek and Pavlov 1998). An

advantage of this reproduction strategy is that Individuals

who survive the migration will move, together with the

spawned eggs and larvae, downstream where they are

distributed over the newly inundated feeding grounds by the

increase in river discharge (Bayley 1973, de Yuan Cordiviola

1992, Araujo‐Lima and Oliveira 1998, Welcomme, Bene et al.

2006, Stassen, van de Ven et al. 2010).

In the Pilcomayo river a

considerable decline of sábalo catches was an important

concern during the 1990’s (Smolders, Guerrero Hiza et al.

2002). Since then, various explanations have been put

forward to explain the decline of P. lineatus in the 1990’s.

Mining operations located near the city of Potosi have

caused heavy metal pollution in the upper parts as well as in

the lower reaches of the river (Hudson-Edwards, Macklin et

al. 2001, Smolders, Lock et al. 2003, Strosnider, Lopez et al.

2014). Sábalo, being a detritivorous species which filters

Figure 1. Map of the Pilcomayo and Bermejo River systems part of the Erosive Andean sub-basin. The dashed lines are (ancient) river beds that remain dry for large parts of the year. The shaded area marks the actual floodplain of the river in 2002. Sample locations are highlighted as red dots on the map. (Edited from Smolders et al. 2002)

large amounts of sediment, could be expected to be very

sensitive to contamination of the sediment. This is

supported by studies showing the effect of heavy metal

contamination through the consumption of sábalo on

communities living along the upper and lower banks of the

river (Miller, Hudson-Edwards et al. 2004, Stassen, Preeker

et al. 2012). Intensive fishing, whose magnitude in the

Pilcomayo River is unknown, and yearly fish kills due to

suspended sediment (Swinkels, Van de Ven et al. 2014) are

also considered to be factors contributing to the decline of

sábalo. It is assumed that the main reason of the observed

sábalo decline during the 1990’s is the loss of the surface of

breeding and feeding areas (marshes in the lower basin) and

a decrease of interconnectivity between these areas and the

main river channel due to river discharge and sedimentation

processes (Smolders, Guerrero Hiza et al. 2002, Stassen, van

de Ven et al. 2010). The Pilcomayo River is characterized by

strong inter-annual variations in river discharge and

sediment load. A trend of declining rainfall in the upper basin

during the 1990’s related to the large-scale climatic “El Niño”

Southern Oscillation ( ENSO) system probably was the main

reason for sábalo decline (Smolders et al., 2000).

Since the 1990’s, ongoing

fluctuations in river discharge and sedimentation processes

have caused the Pilcomayo River to frequently change its

course. The mean sediment load of the Pilcomayo River is

among the highest in the world (Guyot, Calle et al. 1990,

Iriondo 1993, Smolders, Guerrero Hiza et al. 2002) with the

mean annual amount of sediment transported by the river

sufficient to cover as much as 5000 ha of land with one meter

of sediment. Huge amounts of sediment are transported

towards the lower reaches of the river in the Chaco plain

where its deposition provokes silting up as well as diversion

of the river bed, creating an alluvial fan with a total surface

area of 21,000 km2 (Iriondo 1993). During the last decades

the river has deposited its sediment into a tectonic

depression called Estero Patiño (Cordini 1947), located 250

km upstream from the Paraguay River. After the depression

filled up with sediment in the 1940’s (Pool, Usai et al. 1993)

the river started to fill up its own river channel, resulting in a

gradual retreat away from the Paraguay River (figure 2). The

gradual retreat of the Pilcomayo River in the lower Chaco

could have important consequences for sábalo and it is

hypothesized that this retreat has caused the sábalo

population of the Pilcomayo to become more or less isolated

from the populations in the La Plata basin (Stassen, van de

Ven et al. 2010). A recent study has shown the presence of a

single panmictic group with high levels of gene flow within

the La Plata basin (Sivasundar, Bermingham et al. 2001), with

specimens captured more than 1000 km away from the

tagging locality. This is in accordance with the assumption

made by (Castro 1990) that each river basin is thought to

contain a single panmictic population. However,

differentiation of a single panmictic sábalo population into

two or more (sub)populations due to siltation and retraction

of the Pilcomayo River could in the long run be detrimental

for the sábalo population(s) living there, as it results in a loss

of access to the La Plata basins gene pool while at the same

time being negatively affected by anthropogenic and

environmental stressors (Smolders, Guerrero Hiza et al.

2002, Smolders, Lock et al. 2003, Stassen, van de Ven et al.

2010, Swinkels, Van de Ven et al. 2014).

Migration patterns for P.

lineatus have been described for most of the Paraná and Rio

de La Plata basin on the basis of micro-data recording tags,

mark-recapture experiments, biotelemetry and elemental

analyses of hard tissues (Bayley 1973, Godinho and Kynard

2006, Lucas and Baras 2008, Peixer and Petrere Júnior 2009).

However, an evaluation of migration patterns and

population structure has not been described for P. lineatus

for the upper Pilcomayo and Bermejo River basins. To

Figure 1. Retreat of the Pilcomayo river and dynamic creation of new flood plains due to self-blockage (silting up) of the river channel. (from Stassen, van de Ven et al. 2010)

contribute to this lack of knowledge the aim of this study is

to provide insight in the population structure of P. lineatus

in the upper Pilcomayo basin and whether populations

inhabiting the Pilcomayo are affected by the retraction of

the Pilcomayo River from the Paraguay main River channel.

To conduct these measurements powerful molecular

markers and statistical approaches are required (Rueda,

Carriquiriborde et al. 2013). Molecular markers play an

important role as indispensable tools for determining the

genetic variation and biodiversity with high levels of

accuracy and reproducibility (Arif, Khan et al. 2011). Among

these molecular markers microsatellites are potentially the

most informative to questions concerning degree of

relatedness of individuals or groups due to their high

heterozygosity and high mutation rate and the advantage of

an relatively easy and low-cost detection by PCR (Hoshino,

Bravo et al. 2012). Combining microsatellite markers with

the Bayesian clustering approach (Pritchard, Stephens et al.

2000, Falush, Stephens et al. 2003) will allow for an

estimation of distinct populations and allocation of

individuals to discrete migratory stocks, potentially

providing insight into the population structure of P. lineatus.

Despite its ecological value to the ecosystem and its

economic importance to the communities living along the

banks of the Pilcomayo and Bermejo River, so far there has

been insufficient information about the genetic variability

and population structure in Prochilodus lineatus in the upper

Pilcomayo and Bermejo River systems. Such information

might help to formulate appropriate guidelines for proper

management and conservation. In particular, there is

uncertainty about the scope of migratory movements in

these basins and whether the isolation of the Pilcomayo

basin from the greater Parana river system has an effect on

genetic variability and population structure. By comparing

specimens from the Pilcomayo River, a system disconnected

from the La Plata basin, with specimens from the still

connected Bermejo River system we hope to contribute to a

better understanding of the genetic variability and

population structure of P. lineatus in the upper Pilcomayo

and Bermejo basins.

Material & Methods

Sampling and molecular methods

A total of 201 specimens were sampled from the Pilcomayo

and Bermejo River systems in the Tarija Department in

South-eastern Bolivia. The sample locations in the Pilcomayo

River were located near the city of Villa Montes ( 1 km

downstream of Villa Montes, 21°16'50.66"S; 63°27'6.44"W

and 5 km upstream of Villa Montes at a location locally

named ‘El Pibe’, 21°15'30.02"S; 63°32'43.44"W). Other

sample locations were located at a tributary 13 km from the

main channel of the Rio Pilaya (20°56'27.42"S;

Microsatellite Primer sequence (5'-3') Size range (bp) Ta (⁰C) Temp Time Cycle

PL-3 F: 5'-TCTGAGCTGTGAGGAATGGA-3' 185-203 50 95 ⁰C 3 min 1

R: 5'-AGAGCGCTCAAGCACAAGAT-3' 95 ⁰C 30 sec

PL-14 F: 5'-TGCCCAACACTGAAACTGAG-3' 104–134 61 variable 30 sec 29

R: 5'-CTCATCAACCTGCCTGGAAT-3' 72 ⁰C 35 sec

PL-23 F: 5'-TTGGCTACTTCCCCAAACAC-3' 244-252 59 95 ⁰C 30 sec

R: 5'-GGGGAACTAGTTTGACGATGC -3' 53 ⁰C 30 sec 8

PL-119 F: 5'-GAAAAAGGCTAGGGGACTGG-3' 161-207 58 72 ⁰C 35 sec

R: 5'-GAGGAAAAT TGCCTT TTGTAGG-3' 72 ⁰C 2 min 1

PL-139 F: 5'-CAGTGGCATGATGAT TAGATGG-3' 169–235 53

R: 5'-CACCTTTTGTTTGGCTTTTAGG-3'

PL-i61 F: 5'-ACCAAGGTGGGATTCCTTGGTC-3' 210-230 69

R: 5'-TTCTCCATCTCTGGCTGCGCA-3'

Table 1. Primer sequence, size range and amplification conditions (Ta, annealing temperature) of microsatellite loci from P. lineatus. The PCR program used is displayed on the right side of the table. M13 protocol recommends that the last eight PCR cycles should be run with an annealing temperature of 53 ⁰C.

64°15'32.11"W) and near the city of Bermejo

(22°43'31.37"S; 64°21'32.60"W). See figure 1 for sampling

locations. Samples were obtained in four different months:

April of 2013 (El Pibe1, N=51 individuals), June of 2013 (El

Pibe2, N=30 individuals; Rio Pilaya, N=43 individuals), July of

2013 (Villa Montes, N=50 individuals; Bermejo, N=13

individuals), and August of 2013 (Bermejo, N=14 individuals).

The specimens were caught using cast nets. Specimens were

euthanized and muscle tissue samples and dorsal fin samples

where obtained and kept in 96% alcohol for further analysis.

Genomic DNA was extracted using an QIAGEN DNeasy Blood

& Tissue Kit. Each individual specimen was genotyped for 6

microsatellite loci and amplified by polymerase chain

reaction using the primers PL-3, PL-14, PL-23, PL-119, PL-139

(Rueda, Sommer et al. 2011) and PL-i61(Yazbeck and

Kalapothakis 2007) (table 1). The M13 (-21) tailed-primer

genotyping protocol (Schuelke 2000) was used to facilitate

fast and inexpensive analysis. PCR reactions were conducted

individually with each primer pair in 25 µl reaction volumes,

as follows: 17.5 µl H2O, 0.5 µl 50 mM MgCl2, 2.5 µl 10x NH4

reaction buffer, 0.5 µl 5 U BIOTAQ DNA polymerase, 0.5 µl

5mM dNTPs and 0.5 µl each primer (¾ M13 primer, ¼

forward primer). The PCR program and microsatellite

specific annealing temperatures are displayed in table 1. Five

of the six loci were shown to be selectively neutral and

unlinked for Prochilodus populations (Rueda, Sommer et al.

2011). After PCR microsatellite fragments were scored for

length with an Beckman Coulter CEQ 8800 analyzer

(GenomeLab™). Fragment lengths were assigned to allelic

classes with Genemarker V2.6.2®(SoftGenetics LLC).

Statistical analysis

To test for the occurrence of genotyping errors resulting

from low DNA quality ( large allele dropouts) or PCR artifacts

(null alleles) and for Hardy-Weinberg equilibrium (HWE) we

used MICRO-CHECKER (Van Oosterhout, Hutchinson et al.

2004). Next, basic descriptive parameters such as allele

number, gene diversity, inbreeding coefficient (Fis) and

observed and expected heterozygosity were calculated using

Arelequin v. 3.5.1.2 (Excoffier and Lischer 2010) and GenAlEx

v. 6.5 (Peakall and Smouse 2012). To measure genetic

differentiation between sample locations we used pairwise

Fst with routines implemented in Arlequin v. 3.5.1.2 and

finally, to assess population structure we used the Bayesian

clustering approach implemented in STRUCTURE (Pritchard,

Stephens et al. 2000, Falush, Stephens et al. 2003). Given the

number of clusters (K) and assuming Hardy-Weinberg and

linkage equilibrium within clusters, STRUCTURE estimates

posterior probabilities (allele frequencies in each cluster and

population memberships for every individual) for these K

clusters. Runs were performed using a burn-in of 100000

iterations followed by a Markov chain MonteCarlo of 150000

steps. POPFLAG in structure was disabled and the

“admixture model” and correlated allele frequencies

between groups were chosen following recommendations

by Falush et al. (2007). Analyses were run with a K ranging

from 1 to 10 recommended by Evanno et al. (2005). Analysis

of STRUCTURE results was performed with STRUCTURE

HARVESTER Web v.0.6.94 (Earl and Vonholdt 2012). K=3 was

chosen based on STRUCTURE HARVESTER results and

CLUMPP v.1.1.2b (Jakobsson and Rosenberg 2007) was used

to permute and match cluster output. Distruct v.1.1

(Rosenberg 2004) was used to graphically display results.

Finally, Bottleneck v.1.2.02 was run under three mutation

models (IAM, SSM & TPM), with proportion of SMM in the

TPM = 0.000 and variance of the geometric distribution for

TPM = 0.36, to detect recent effective population size

reduction (Piry, Luikart et al. 1999).

Table 2. Summary statistics of allelic variation of 6 microsatellite loci for P. lineatus

Locus N Allele number Gene diversity

PL-3 194 13 0.67

PL-14 196 20 0.94

PL-23 199 1 0

PL-119 192 64 0.97

PL-139 169 32 0.94

PL-i61 188 8 0.72

Results

All 201 individuals were genotyped for 6 microsatellite loci.

12 samples for which less than 5 loci could be determined

were excluded from analysis. During PCR 41 samples failed

to amplify and were excluded for further analysis. For all loci

and individuals combined, microchecker results suggest

that this population is possibly in Hardy Weinberg

equilibrium with loci PL139, PL-i61, PL-3, showing signs of a

null allele. The presence of null alleles indicates an excess

of homozygotes compared to the HWE. Null alleles are likely

to be encountered in populations with a large effective size

and their presence can result in the overestimation of Fst

values and genetic drift when these populations differ

significantly from one another (Chapuis and Estoup 2007).

No evidence of genotyping errors due to stuttering or allele

dropout were found. PL-23 was shown to be monomorphic

and also excluded from further analysis.

Table 3. Null allele frequency’s calculated with MICROCHECKER

using the methods described by Oosterhout et al. (2004),

Chakraborty et al. (1992) and Brookfield (1996)

Locus Oosterhout Chakraborty Brookfield 1 Brookfield 2

PL14 -0.04 -0.03 -0.03 0.00

PL139 0.30 0.44 0.29 0.42

PL-119 -0.01 -0.01 -0.01 0.00

PL-i61 0.26 0.38 0.23 0.23

PL-3 0.21 0.28 0.18 0.18

Genetic diversity at each locus ranged between 0.666-0.968,

number of alleles observed per locus between 8-64 (Table

2). Average observed heterozygosity values over all loci Ho=

0.61 (SD 0.24) and expected heterozygosity He= 0.8 (SD

0.16). Null allele frequencies are shown in table 3. These

results confirm that the microsatellite data is informative for

population structure analysis.

Population structure

Comparison of samples taken at different locations suggest

that the genetic signature varies greatly within the

Pilcomayo and Bermejo River basins and between different

sample dates in the Pilcomayo River. Diversity values (mean

of allele number, observed and expected heterozygosity

over all loci for the five sample locations are shown in table

4. Pairwise Fst values are shown in table 5. Inbreeding

coefficient (Fis) is shown in table 6.

Table 5. Fst and Fst P values for the five sample locations Villa

Montes, El Pibe1, El Pibe2, Rio Pilaya and Bermejo

Fst values Villa Montes El Pibe1 El Pibe2 Rio Pilaya

Villa Montes x

El Pibe1 0.00409 x

El Pibe2 0.07182 0.03262 x

Rio Pilaya 0.00180 -0.00181 0.04736 x

Bermejo 0.02596 0.02709 0.09674 0.01400

Fst P-values Villa Montes El Pibe1 El Pibe2 Rio Pilaya

Villa Montes x

El Pibe1 0.162 x

El Pibe2 0.000 0.000 x

Rio Pilaya 0.450 0.748 0.000 x

Bermejo 0.000 0.009 0.000 0.083

Analysis of Pairwise Fst values suggests that there is no

genetic differentiation between Villa Montes - El Pibe1, Villa

Villa Montes (N =49 ) El Pibe1 (N = 51) El Pibe2 (N = 30) Rio Pilaya (N = 36) Bermejo (N = 23)

AN Total = 86 Total = 80 Total = 73 Total = 74 Total = 67

Range = 4-35 Range = 5-34 Range = 1-25 Range = 1-26 Range = 3-20

Avg = 17.2 (±13.7) Avg = 16.0 (±11.7) Avg = 14.6 (±10.8) Avg = 14.8 (±10.0) Avg = 13.4 (±8.5)

He 0.82 (±0.12) 0.81 (±0.13) 0.75 (±0.28) 0,84 (±0,12) 0,79 (±0,20)

Ho 0.57 (±0.30) 0.59 (±0.23) 0.65 (±0.23) 0,66 (±0,20) 0,60 (±0,23)

Table 4. Genetic diversity for each sample location. (AN= allele number, Avg= average, He= expected heterozygosity, Ho= observed heterozygosity over all loci. Standard deviation are given in parentheses

Figure 3. L-shaped mode shift graph showing the absence of a bottleneck in P. lineatus

Montes - Rio Pilaya, El Pibe1 - Rio Pilaya and Rio Pilaya -

Bermejo (however low). Bottleneck results are shown in

figure 3 showing an L-shaped allele frequency distribution

indicating no bottleneck was present. Structure analysis

indicated that there was no discernible pattern of dominant

cluster memberships in any individuals for any K (Figure

4a,b). Allocation of individual samples to cluster number K =

3 is shown in figure 5. Combined, these analyses suggest that

there is high genetic variation between and within

populations sampled but a lack of distinct genetic stocks.

Discussion

Only little is known about population structure and

phylogeography of highly migratory fishes living in large

Figure 4. Results of genetic analysis of microsatellite data for Prochilodus lineatus from the Pilcomayo and Bermejo River systems using STRUCTURE. Individuals are grouped by sampling location (Villa Montes, El Pibe1, El Pibe2, Rio Pilaya, Bermejo). A plot of mean of est. Ln prob of data(a) and a plot of Delta K(b) obtained using Structure Harvester for 10 independent runs varying K= 1 t/m 10

Neotropical River systems. A recent study has shown that

population structure for P. lineatus is present in the Lower

Uruguay River (Rueda, Carriquiriborde et al. 2013) revealing

0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

-0,1 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9

Alle

le F

req

uen

cy

Class interval

Locus Average Fis Villa Montes El Pibe1 El Pibe2 Rio Pilaya Bermejo

PL-14 0.027 -0,057 -0.005 -0.045 0.085 0.265

PL-139 0.443 0.615 0.514 0.258 0.343 0.359

PL-119 0.037 -0.011 0.063 0.106 0.024 0.011

PL-i61 0.390 0.555 0.306 0.301 0.341 0.371

PL--3 0.374 0.448 0.501 -0.144 0.332 0.245

Locus Average Fis Villa Montes El Pibe1 El Pibe2 Rio Pilaya Bermejo

PL-14 0.027 -312.786 -119.152 -268.768 214.879 884.626

PL-139 0.443 38.668 15.847 -41.857 -22.720 -19.011

PL-119 0.037 -128.380 70.222 184.641 -35.214 -69.707

PL-i61 0.390 42.530 -21.462 -22.851 -12.473 -4.687

PL--3 0.374 19.762 33.870 -138.481 -11.222 -34.446

Table 6. Population specific Fis indices per locus (absolute values) and Population specific Fis indices per locus (relative values (%)

compared to average Fis)

seasonal variation between groups migrating upstream

towards the upper Paraná basin. This supports the idea by

Sanches et al. (2012) that different genetic populations of

migratory fishes can co-exist in a single hydrographic system

by segregating during the spawning season. In contrast,

studies done within the upper Paraná basin support the

presence of a single panmictic sábalo population

(Revaldaves, Renesto et al. 1997, Sivasundar, Bermingham et

al. 2001) These studies show high genetic variability and

gene flow but no genetic structure. Our results support these

findings. Within the Pilcomayo and between the Pilcomayo

and Bermejo River basins high levels of genetic variability

and gene flow can be found while population structure does

not seem to be present. The contrast between these studies

shows the that the existence of a single panmictic population

versus different genetic populations can vary greatly for P.

lineatus when sampling locations differ. Furthermore,

temporal effects have shown to be of significance when

sampling during spawning or non-reproductive season

(Sanches 2012). Within our results genetic differentiation is

observed between the El Pibe2 group and the other groups

sampled in the Pilcomayo River. No differentiation was

found between the Villa Montes, El Pibe1 and Rio Pilaya

group suggesting that the specimens collected at these sites

are more closely related than specimens from the El Pibe2

sample site. Specimens were collected in April (El Pibe1),

June (Rio Pilaya, El Pibe2) and July (Villa Montes). These

findings support findings by Sanches et al. (2012) and

highlight the importance of taking into account temporal

effects when doing population genetic research on highly

migratory species.

Observed heterozygosity

in our results is much lower than expected heterozygosity.

This could be ascribed to the presence of null alleles arising

from variation in the nucleotide sequences of flanking

regions that prevent the primer annealing to template DNA

during PCR amplification of the microsatellite locus [Dakin

and Avise, 2004, Pompanon et al., 2005]. The presence of

null alleles results in an excess of homozygous genotypes

within a population as compared to the expected proportion

under Hardy Weinberg Equilibrium (HWE) and Linkage

Equilibrium (LE) [Callen et al., 1993, Paetkau et al., 1995] and

an overestimation of Fst values and genetic drift (Chapuis and

Estoup 2007). Microchecker analysis suggests that loci

PL139, PL-i61, PL-3 show signs of a null allele. This results in

populations sampled having less genetic differentiation than

our analyses suggest. Nevertheless the following conclusions

can be drawn based on our analysis.

Results (table 4) suggest

that genetic variability is present in the Pilcomayo river

system despite the river being isolated from the La Plata

basin since the 1940’s and the massive decline of sábalo

numbers during the 1990’s. Since the last decade sábalo

population size seems to have been increasing. However, it

is well known that genetic variation does not rebound from

a decrease as quickly as population size does (Nei,

Maruyama et al. 1975). The presence of genetic variability

therefore suggests that despite the loss of an important

migration route and a decline in fish numbers the original

gene pool has more or less remained intact. This is

reinforced by Bottleneck results which show that no

bottleneck has occurred based on our allelic data. Our

hypothesis is that this genetic variability has been preserved

in the lower reaches of the Pilcomayo among the floodplains

of the Chaco plain. Each year different sábalo populations

from different regions in the lower floodplains start their

migration upstream. The populations able to start the

migration are chosen depending on the amount of river

Figure 5. Assignment probabilities for each individual (vertical bars) into one of three clusters. Each color represents the amount to which an individual belongs to a certain cluster.

discharge that year and the resulting connectivity of the

main river channel with the floodplains. This interannual and

seasonal variation of interconnectivity between the

floodplains selects the regions from which sábalo is able to

migrate and spawn upstream keeping genetic variation

intact despite anthropogenic and environmental stressors.

For nearly all Pilcomayo

groups differentiation with the Bermejo group can be found

indicating that sábalo inhabiting the Bermejo River are

genetically more distant from the groups in the Pilcomayo

than they are among themselves. These findings support the

idea made by Stassen et al. 2010, that the populations living

in the Pilcomayo River system are more or less isolated from

the bigger sábalo population inhabiting the La Plata basin.

While genetic variation within the Pilcomayo seems to be

currently present a lasting isolation of the river with the

Paraguay channel might have a negative effect on the

sábalo population in the long run. Due to limited possibilities

for new genetic diversity to be generated via gene flow, it

might be expected that sábalo populations living in the

Pilcomayo River will become more vulnerable to extinction

following a population crash. While a ‘reserve’ fish stock

exists in the floodplains able to revitalize sábalo through

interannual and seasonal fluctuations in river discharge,

continued environmental and anthropogenic effects and

isolation of the Pilcomayo River might deplete this reserve

fish stock resulting in a loss of genetic variation for

Prochilodus populations in the Pilcomayo River basin.

Results gained from this

study could be useful for future management and

conservation strategies regarding P. lineatus within the

Pilcomayo River system. Such as the introduction of

Prochilodus from other parts of the Parana River system to

increase genetic diversity or monitoring genetic diversity

within the area of Villa Montes for the detection of possible

future trends.

References

Araujo‐Lima, C. and E. Oliveira (1998). "Transport of larval fish in the Amazon." Journal of fish Biology 53(sA): 297-306.

Arif, I. A., H. A. Khan, A. H. Bahkali, A. A. Al Homaidan, A. H. Al Farhan, M. Al Sadoon and M. Shobrak (2011). "DNA marker technology for wildlife conservation." Saudi Journal of Biological Sciences 18(3): 219-225.

Baigun, C., P. Minotti and N. Oldani (2013). "Assessment of sábalo (Prochilodus lineatus) fisheries in the lower Paraná River basin (Argentina) based on hydrological, biological, and fishery indicators." Neotropical Ichthyology 11: 199-210.

Bayley, P. B. (1973). "Studies on the migratory characin, Prochilodus platensis Holmberg 1889, (Pisces, Characoidei) in the River Pilcomayo, South America." Journal of Fish Biology 5(1): 25-40.

Bayol, V. and E. de Yuan Cordiviola (1996). "Food assimilation of a neotropical riverine detritivorous fish, Prochilodus lineatus, studied by fatty acid composition (Pisces, Curimatidae)." Hydrobiologia 330(2): 81-88.

Bowen, S. (1983). "Detritivory in neotropical fish communities." Environmental Biology of Fishes 9(2): 137-144.

Brookfield, J. (1996). "A simple new method for estimating null allele frequency from heterozygote deficiency." Molecular Ecology 5(3): 453-455.

Castro, R. (1990). "Revisão taxonômica da família Prochilodontidae (Ostriophysi, Characiformes)." Unpublished Ph. D. dissertation. São Paulo: Universidade de São Paulo. 293pp.

Chakraborty, R., M. d. Andrade, S. Daiger and B. Budowle (1992). "Apparent heterozygote deficiencies observed in DNA typing data and their implications in forensic applications." Annals of human genetics 56(1): 45-57.

Chapuis, M.-P. and A. Estoup (2007). "Microsatellite Null Alleles and Estimation of Population Differentiation." Molecular Biology and Evolution 24(3): 621-631.

Cordini, I. R. (1947). Los ríos Pilcomayo en la región del Patiño. Buenos Aires.

de Yuan Cordiviola, E. (1992). "Fish populations of lentic environments of the Paraná River." Hydrobiologia 237(3): 159-173.

Earl, D. A. and B. M. Vonholdt (2012). "STRUCTURE HARVESTER: a website and program for visualizing STRUCTURE output and implementing the Evanno method." Conservation Genetics Resources 4(2): 359-361.

Evanno, G., S. Regnaut and J. Goudet (2005). "Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study." Molecular Ecology 14(8): 2611-2620.

Excoffier, L. and H. E. L. Lischer (2010). "Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows." Molecular Ecology Resources 10(3): 564-567.

Falush, D., M. Stephens and J. K. Pritchard (2003). "Inference of population structure using multilocus genotype data: linked loci and correlated allele frequencies." Genetics 164(4): 1567-1587.

Falush, D., M. Stephens and J. K. Pritchard (2007). "Inference of population structure using multilocus genotype data: dominant markers and null alleles." Molecular ecology notes 7(4): 574-578.

Flecker, A. S. (1996). "Ecosystem Engineering by a Dominant Detritivore in a Diverse Tropical Stream." Ecology 77(6): 1845-1854.

Frankham, R., D. A. Briscoe and J. D. Ballou (2002). Introduction to conservation genetics, Cambridge University Press.

Fugi, R., N. Hahn and A. Agostinho (1996). "Feeding styles of five species of bottom-feeding fishes of the high Paraná river." Environmental Biology of Fishes 46(3): 297-307.

Godinho, A. L. and B. Kynard (2006). "Migration and spawning of radio-tagged zulega Prochilodus argenteus in a dammed Brazilian River." Transactions of the American Fisheries Society 135(3): 811-824.

Guyot, J. L., H. Calle, J. Cortes and M. Pereira (1990). "Transport of Suspended Sediment and Dissolved Material from the Andes to the Rio-De-La-Plata by the Bolivian Tributaries of the Rio Paraguay (Rios Pilcomayo and Bermejo)." Hydrological Sciences Journal-Journal Des Sciences Hydrologiques 35(6): 653-665.

Hoshino, A. A., J. P. Bravo, P. M. Nobile and K. A. Morelli (2012). "Microsatellites as tools for genetic diversity analysis." Genetic Diversity in Microorganisms: 149-170.

Hudson-Edwards, K. A., M. G. Macklin, J. R. Miller and P. J. Lechler (2001). "Sources, distribution and storage of heavy metals in the Rio Pilcomayo, Bolivia." Journal of Geochemical Exploration 72(3): 229-250.

Iriondo, M. (1993). "Geomorphology and Late Quaternary of the Chaco (South-America)." Geomorphology 7(4): 289-303.

Jakobsson, M. and N. A. Rosenberg (2007). "CLUMPP: a cluster matching and permutation program for dealing with label switching and multimodality in analysis of population structure." Bioinformatics 23(14): 1801-1806.

Lucas, M. and E. Baras (2008). Migration of Freshwater Fishes, Wiley.

Miller, J. R., K. A. Hudson-Edwards, P. J. Lechler, D. Preston and M. G. Macklin (2004). "Heavy metal contamination of water, soil and produce within riverine communities of the Rio Pilcomayo basin, Bolivia." Science of the Total Environment 320(2-3): 189-209.

Mochek, A. and D. Pavlov (1998). "The ecology of Sábalo Prochilodus lineatus (Curimatidae, Characoidei) of the Pilcomayo River (South America)." Journal of ichthyology 38(1): 28-36.

Nei, M., T. Maruyama and R. Chakraborty (1975). "The bottleneck effect and genetic variability in populations." Evolution: 1-10.

Peakall, R. and P. E. Smouse (2012). "GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research-an update." Bioinformatics 28(19): 2537-2539.

Peixer, J. and M. Petrere Júnior (2009). "Sport fishing in Cachoeira de Emas in Mogi-Guaçu River, State of São Paulo, Brazil." Brazilian Journal of Biology 69(4): 1081-1090.

Pimm, S. L. "Food webs too food webs: integration of patterns and dynamics." Trends in Ecology & Evolution 11(8): 349.

Piorski, N. M., A. Sanches, L. F. Carvalho-Costa, T. Hatanaka, M. Carrillo-Avila, P. D. Freitas and P. M. Galetti (2008). "Contribution of conservation genetics in assessing neotropical freshwater fish biodiversity." Brazilian Journal of Biology 68(4): 1039-1050.

Piry, S., G. Luikart and J.-M. Cornuet (1999). "BOTTLENECK: a program for detecting recent effective population size reductions from allele data frequencies." Montpellier, France.

Pool, M., E. Usai and J. Baas (1993). "Estudio del Rio Pilcomayo." Brussels, EURAGRI Temporary Association.[Links].

Pritchard, J. K., M. Stephens and P. Donnelly (2000). "Inference of population structure using multilocus genotype data." Genetics 155(2): 945-959.

Reis, R. E., S. O. Kullander, C. J. Ferraris and P. U. C. d. R. G. d. S. M. d. C. e. Tecnologia (2003). Check List of the Freshwater Fishes of South and Central America, EDIPUCRS.

Revaldaves, E., E. Renesto and M. F. Machado (1997). "Genetic variability of Prochilodus lineatus (Characiformes, Prochilodontidae) in the upper Paraná river." Brazilian Journal of Genetics 20(3).

Rosenberg, N. A. (2004). "DISTRUCT: a program for the graphical display of population structure." Molecular Ecology Notes 4(1): 137-138.

Rueda, E. C., P. Carriquiriborde, A. M. Monzon, G. M. Somoza and G. Orti (2013). "Seasonal variation in genetic population structure of sabalo (Prochilodus lineatus) in the Lower Uruguay River." Genetica 141(7-9): 401-407.

Rueda, E. C., J. Sommer, P. Scarabotti, R. Markariani and G. Orti (2011). "Isolation and characterization of polymorphic microsatellite loci in the migratory freshwater fish Prochilodus lineatus (Characiformes:

Prochilodontidae)." Conservation Genetics Resources 3(4): 681-684.

Sanches, A. and P. M. Galetti (2012). "Population genetic structure revealed by a school of the freshwater migratory fish, Brycon hilarii." Latin American Journal of Aquatic Research 40(2): 408-417.

Sanches, A. G. J., Pedro M Galzerani,Felipe Derazo,Janeth Cutilak-Bianchi,Beatriz Hatanaka,Terumi (2012). "Genetic population structure of two migratory freshwater fish species (Brycon orthotaenia and Prochilodus argenteus) from the São Francisco River in Brazil and its significance for conservation." Latin american journal of aquatic research.

Schuelke, M. (2000). "An economic method for the fluorescent labeling of PCR fragments." Nature Biotechnology 18(2): 233-234.

Sivasundar, A., E. Bermingham and G. Ortí (2001). "Population structure and biogeography of migratory freshwater fishes (Prochilodus: Characiformes) in major South American rivers." Molecular Ecology 10(2): 407-417.

Smolders, A. J. P., M. A. Guerrero Hiza, G. van der Velde and J. G. M. Roelofs (2002). "Dynamics of discharge, sediment transport, heavy metal pollution and Sábalo (Prochilodus lineatus) catches in the lower Pilcomayo river (Bolivia)." River Research and Applications 18(5): 415-427.

Smolders, A. J. P., R. A. C. Lock, G. Van der Velde, R. I. M. Hoyos and J. G. M. Roelofs (2003). "Effects of mining activities on heavy metal concentrations in water, sediment, and macroinvertebrates in different reaches of the Pilcomayo River, South America." Archives of Environmental Contamination and Toxicology 44(3): 314-323.

Stassen, M. J., M. W. van de Ven, T. van der Heide, M. A. G. Hiza, G. van der Velde and A. J. Smolders (2010). "Population dynamics of the migratory fish Prochilodus lineatus in a neotropical river: the relationships with river discharge, flood pulse, El Niño and fluvial megafan behaviour." Neotropical Ichthyology 8(1): 113-122.

Stassen, M. J. M., N. L. Preeker, A. M. J. Ragas, M. W. P. M. van de Ven, A. J. P. Smolders and N. Roeleveld (2012). "Metal exposure and reproductive disorders in

indigenous communities living along the Pilcomayo River, Bolivia." Science of the Total Environment 427: 26-34.

Strosnider, W. H. J., F. S. L. Lopez, J. A. LaBar, K. J. Palmer and R. W. Nairn (2014). "Unabated acid mine drainage from Cerro Rico de Potosi, Bolivia: uncommon constituents of concern impact the Rio Pilcomayo headwaters." Environmental Earth Sciences 71(7): 3223-3234.

Sverlij, S. B., A. E. Ros and G. Orti (1993). Sinopsis de los datos biológicos y pesqueros del sábalo, Prochilodus lineatus (Valenciennes, 1847), Food & Agriculture Org.

Swinkels, L. H., M. W. P. M. Van de Ven, M. J. M. Stassen, G. Van der Velde, H. J. R. Lenders and A. J. P. Smolders (2014). "Suspended sediment causes annual acute fish mortality in the Pilcomayo River (Bolivia)." Hydrological Processes 28(1): 8-15.

Taylor, B. W., A. S. Flecker and R. O. Hall (2006). "Loss of a Harvested Fish Species Disrupts Carbon Flow in a Diverse Tropical River." Science 313(5788): 833-836.

Van Oosterhout, C., W. F. Hutchinson, D. P. M. Wills and P. Shipley (2004). "MICRO-CHECKER: software for identifying and correcting genotyping errors in microsatellite data." Molecular Ecology Notes 4(3): 535-538.

Welcomme, R. L., C. Bene, C. A. Brown, A. Arthington, P. Dugan, J. M. King and V. Sugunan (2006). Predicting the water requirements of river fisheries. Wetlands and Natural Resource Management, Springer: 123-154.

Yazbeck, G. M. and E. Kalapothakis (2007). "Isolation and characterization of microsatellite DNA in the piracema fish Prochilodus lineatus (Characiformes)." Genetics and Molecular Research 6(4): 1026-1034.