Biological Journal of the Linnean Society, 2021, 134, 198–213. With 3 figures.

Fine-scale genetic structure of suckermouth Hypostomus

ancistroides populations: the importance of Neotropical
streams for fish conservation
CAROLINE APOLINÁRIO-SILVA1, BRUNO AMBROZIO GALINDO2,

Downloaded from https://academic.oup.com/biolinnean/article/134/1/198/6284360 by guest on 20 August 2023

RAUL HENRIQUE CARDOSO NASCIMENTO1, WILSON FRANTINE-SILVA3, ,
THAIS KOTELOK-DINIZ1, SILVIA HELENA SOFIA1,*, and DHIEGO GOMES FERREIRA2
1
Departamento de Biologia Geral, Centro de Ciências Biológicas, Universidade Estadual de Londrina,
Londrina, PR 86.057-970, Brazil
2
Universidade Estadual do Norte do Paraná, Campus Cornélio Procópio, Cornélio Procópio, PR 86.300-
000, Brazil
3
Laboratório de Ciências Ambientais, Centro de Biociências e Biotecnolgia, Universidade Estadual do
Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, RJ 28.013-602, Brazil

Received 4 December 2020; revised 18 February 2021; accepted for publication 18 February 2021

In the Neotropics, freshwater streams harbour high fish diversity and are constantly threatened by anthropogenic
disturbance. However, there are few studies on the genetic diversity of fish populations inhabiting these streams.
We aimed to assess, based on microsatellite and mitochondrial DNA markers, the population structure and genetic
diversity of the suckermouth catfish, Hypostomus ancistroides, a Neotropical species widely distributed across the
Upper Paraná River Basin in South America. Twenty-five locations were sampled, distributed across 18 sites in six
tributary streams and another seven sites in the main river channel. Our analyses revealed a spatial heterogeneity
in genetic diversity within the basin, indicating fine-scale genetic structuring. Samples from all streams showed
exclusive haplotypes and private alleles, reinforcing the importance of preserving the tributaries for the conservation
of the genetic diversity of the studied populations. The fine-scale genetic structuring of H. ancistroides is probably
related to the limited displacement capacity of this species.

ADDITIONAL KEYWORDS: D-loop – genetic diversity – microsatellite – Neotropical streams – Paraná River
Basin – population structure.

INTRODUCTION plenty of evidence that this rich species diversity

is experiencing strong anthropogenic impacts
The freshwater fish fauna of South America is the
(Pelicice et al., 2017; Castro & Polaz, 2020), with some
most diverse on the planet, with more than 5000
of the species facing some degree of extinction risk in
species already catalogued and recent estimates
recent decades (Reis et al., 2016).
surpassing 8000 species (Reis et al., 2016). Among
Harbouring an important component of the South
the species already known, approximately 2500 are
American freshwater fish diversity, the Paraná River
distributed along Brazilian drainage basins, which
Basin, the second largest drainage basin in South
form the most extensive hydrographic network in the
America, represents an important example of constant
world (Ota et al., 2018). Approximately half of this
human impacts on Neotropical rivers and streams
fish fauna is composed of small species, which mainly
(Agostinho et al., 2007). In fact, the Paraná River
inhabit small bodies of water such as streams (Castro
is one of the most dammed rivers in South America
& Polaz, 2020). The current literature provides
(Reis et al., 2016). In addition, the main tributaries
of the Paraná Basin have also been impacted by
the construction of several dams and other forms
*Corresponding author. E-mail: shsofia@uel.br of anthropogenic interference, such as intense

© The Author(s) 2021. Published by Oxford University Press on behalf of The Linnean Society of London.
198
All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
 POPULATION STRUCTURE OF SUCKERMOUTHS
agriculture and cattle-raising activities, elimination
of riparian vegetation, and species introductions,
which have degraded water quality (Agostinho et al.,
2008). According to these authors, this is exactly the
scenario describing the Upper Paraná River Basin
(UPRB), which harbours approximately 10% of the
Neotropical freshwater fish species and is entirely
located in the Brazilian territory. In addition, the
inadequate national policies employed by Latin
American governments constitute another concern
regarding freshwater fish conservation throughout
the Neotropics (Pelicice et al., 2017).
Regarding freshwater fish fauna from the UPRB,
while great efforts have been made in recent decades to
characterize its ichthyofauna (Ota et al., 2018), studies
aiming to characterize how the genetic diversity in
native fish species is distributed among populations
inhabiting rivers and tributaries of this basin are
still fragmentary (Ferreira et al., 2017). In addition,
existing genetic studies rely mostly on fish populations
from the main rivers in the UPRB (Garcez et al., 2011;
Ferreira et al., 2017), while genetic investigations
on a micro-geographical scale involving populations
inhabiting small tributaries are scarce (Ferreira et al.,
2015, 2016).
Studies on streams from temperate regions have
revealed fine-scale genetic differentiation among fish
populations (Carlsson et al., 1999) and maintenance
of portions of the total genetic diversity in different
drainages from a watershed (Adamson et al., 2012).
In Neotropical streams, however, there are few
studies concerning the importance of conservation
of the genetic diversity of populations of fish species
that inhabit these small drainages (Sofia et al., 2008;
Ferreira et al., 2016). Furthermore, considering that
Neotropical streams are recognized as distinct from
temperate drainage basins in various physical-
chemical and biological aspects (Winemiller et al.,
2008), it is difficult to infer whether patterns described
for temperate drainages are equivalent to those of
Neotropical drainages. In this context, there is a need
for more genetic studies on fish fauna inhabiting
streams across the Neotropics, especially if we consider
that watersheds from this region are frequently at risk
from anthropic activities. Moreover, these studies could
disclose patterns in the diversity and genetic structure
of native fish populations which are also facing these
anthropic risks.
Considering the characteristics of hydrographic
systems, robust and relevant information about
genetic distribution patterns in widely distributed
species could be obtained from population genetic
studies, including several streams and the main
channel of a watershed. This approach may contribute
to understanding the influences of geological and
© 2021 The Linnean Society of London, Biological Journal of the Linnean Society, 2021, 134, 198–213
199
behavioural aspects, recognized as important in genetic
distribution of freshwater fish (Allan & Flecker, 1993;
Wu et al., 2020), as well as aiding the understanding
of other factors, such as colonization patterns along
the basin or even anthropic interferences, which may
also influence genetic organization of fish populations
in streams (Heithaus & Laushman, 1997; Blum
et al., 2012, Splendiani et al., 2020). Furthermore,
information on the patterns of genetic diversity
distribution within and between populations, gene flow
levels, influences of demographic events, and other
Downloaded from https://academic.oup.com/biolinnean/article/134/1/198/6284360 by guest on 20 August 2023
processes in the evolutionary history of species are
considered essential for understanding evolutionary
aspects and biodiversity conservation in freshwater
ecosystems (Frankham et al., 2010).
In the Paranapanema River Basin (belonging to
the Upper Paraná River system), the microbasin
of the Laranjinha River exhibits interconnectivity
and many small tributaries, presenting appropriate
conditions for the design of an experimental study
aiming to understand the genetic distribution of a
species throughout a watershed. Previous studies
on the ichthyofauna of these drainage basins have
highlighted fish species with great potential for this
type of study, such as the suckermouth armoured catfish
Hypostomus ancistroides (Ihering, 1911), present
in both tributaries, streams and the main channel
(Sofia et al., 2008; Galindo et al., 2020). H. ancistroides
(Siluriformes: Loricariidae: Hypostominae) was first
described at UPRB (Weber, 2003), presenting wide
distribution and a phylogeographic history that
probably includes different colonization events for
new drainages, population isolation and subsequent
secondary contacts (Hollanda-Carvalho et al., 2016).
Since H. ancistroides is a benthic detritivorous species
(bottom scraping), it is among the fish species that
act as important premineralizers of organic material
in freshwater ecosystems (Angelescu & Gneri, 1949),
which reproduces from October to January through
external fertilization and spawning in instalments,
and does not perform reproductive migrations
(Britto et al., 2003). Due to the reproductive and
morphological characteristics of H. ancistroides (Britto
et al., 2003), as well as the indicative low gene flow
(Sofia et al., 2008; Hollanda-Carvalho et al., 2016), it
has been suggested that this species exhibits limited
displacement along drainages (Zawadzki et al., 2005;
Hollanda-Carvalho et al., 2016), which may result in
some degree of genetic divergence between co-specific
populations (Endo et al., 2012).
In this context, considering the limited displacement
capacity of H. ancistroides, despite the particularities
of the Neotropical streams compared to temperate
streams, our main prediction was that we would expect
to find a similar pattern of genetic structuring in short
200 C. APOLINÁRIO-SILVA ET AL.

geographical distances in populations of this species Study area and sampling

from Neotropical streams. Furthermore, the current
study aimed to assess the genetic diversity, gene flow
and population structure of H. ancistroides sampled
across a microbasin of the Upper Paraná River system,
to bring to light information concerning the important
role of Neotropical streams in fish fauna conservation.
MATERIAL AND METHODS
The source of the Laranjinha River is in the
municipality of Ventania, state of Paraná (southern AQ5
Brazil), and extends for approximately 350 km, flowing
into the Cinzas River, an important sub-basin on the
left of the Paranapanema River. Representatives of
H. ancistroides have been studied in the main channel
and six tributaries of the Laranjinha River. Thus,
H. ancistroides samples were collected from seven
sites along the Laranjinha River, from the headwaters
to the mouth (south-north direction), spaced at

Downloaded from https://academic.oup.com/biolinnean/article/134/1/198/6284360 by guest on 20 August 2023

All animal experiments were approved by the
Universidade Estadual de Londrina Animal Ethics
Committee (protocol CEUA no. 8756.2016.30). Voucher
specimens are deposited at Museu de Zoologia da
Universidade Estadual de Londrina (catalogue
numbers MZUEL: 19310 to 19339).
distances of around 50 km and denoted as L1, L2, L3,
L4, L5, L6 and L7. The streams sampled were selected
according to their position in the river, two streams
near to the source, two streams near to the middle and
two streams near to the mouth, one on each riverbank.
Each stream was sampled in three distinct sections:

Figure 1. Distribution and sampling locations for H. ancistroides in the Laranjinha River (L) and tributaries. Circles
indicate sampling locations in the river; geometric shapes indicate sampling locations in streams and black bars indicate
dams. (Source: modified from Google Earth, 2018. https://www.google.com.br/maps).

© 2021 The Linnean Society of London, Biological Journal of the Linnean Society, 2021, 134, 198–213
 POPULATION STRUCTURE OF SUCKERMOUTHS
Upper section (U) near source, Middle section (M) near
middle and Lower section (L) near mouth. (Fig. 1).
In total, 731 H. ancistroides specimens were
sampled from 25 localities along the Laranjinha River
and tributaries (Fig. 1). Fish sampling was conducted
using multi-mesh gill nets and fishing sieves, with a
minimum collection effort of 1 h per site, sampling
approximately 300 m. Collections were conducted in
different seasons, ranging from two to six collections
per site, capturing fish with total lengths from 20 mm
(juveniles) to 250 mm (adults). For each sampled site,
we recorded geographical coordinates and distance
between sections in the drainage system (Supporting
Information, Table S1).
DNA extraction and PCR
Total genomic DNA was isolated from muscle tissue
using a phenol-chloroform protocol. Polymerase Chain
Reactions (PCRs) for microsatellite loci (Simple
Sequence Repeat—SSR) were performed in 10 µL
reaction volumes using nine primers specifically
designed for H. ancistroides (Galindo et al., 2015) and
reactions were carried out as described in Ferreira
et al. (2015). Products were genotyped following the
method of Schuelke (2000) and the microsatellite
polymorphism was analysed on ABI-PRISM 3500
xL automated sequencer (Applied Biosystems),
using GeneScan 600 Liz (Applied Biosystems) as a
molecular weight marker. Allele sizes were assigned
manually using GeneMarker 1.85 (SoftGenetics, State
College, PA).
A partial mitochondrial DNA control region
(D-loop) was amplified with the primers DLA-
III 5’-TATTTAAAGRCATAATCTCTTGAC-3’ and
HygDL-R 5’-WTGCKARTATGTGCCGYYTG-3’
(Cardoso et al., 2011). Reactions were performed in a
total volume of 25 µL, with 12.5 µL 10× GoTaq Green
Master Mix (Promega), 1.25 µL of each primer at
10 µM, 7 µL water and 3 µL DNA template at 15 ng.
The PCR program was performed with an initial
denaturation at 94 °C for 4 min, followed by 40 cycles
at 94 °C for 30 s, 56 °C for 30 s and 72 °C for 2 min, and
a final extension at 72 °C for 10 min.
PCR products were purified with IllustraExoStar
IT (GE) enzyme and prepared for sequencing
using a Big Dye Terminator (Applied Biosystems)
kit, which was then used for bidirectional Sanger
sequencing on an ABI-PRISM 3500 xL, following
the manufacturer’s recommendations. Sequence
data were edited and aligned using MEGA 5.0
(Tamura et al., 2011). The haplotypes found were
deposited in the GenBank [KJ126727 to KJ126743
and MN310717 to MN310724 (http://www.ncbi.nlm.
nih.gov/genbank/)].
© 2021 The Linnean Society of London, Biological Journal of the Linnean Society, 2021, 134, 198–213
201
Molecular data analysis
The software Micro-Checker 2.2.1 (van Oosterhout
et al., 2004) was used on the microsatellite data to
identify possible null alleles, large allele dropout
and scoring errors. We estimated population means
of genetic diversity parameters such as number of
alleles (A), number of rare alleles (RA) with frequency
< 0.05, number of private alleles (PA), mean of alleles
( N A ), effective alleles ( N E ), and observed (H O) and
expected (H E ) heterozygosity using GenAlEx 6.5
(Peakall & Smouse, 2012). The number of alleles per
Downloaded from https://academic.oup.com/biolinnean/article/134/1/198/6284360 by guest on 20 August 2023
locus [allelic richness (AR)] using rarefaction and the
test for significant inbreeding (F IS) (P < 0.05) were
estimated in Fstat v.2.9.3 (Goudet, 2001) with 10 000
permutations. Genepop v.1.2 (Raymond & Rousset,
1995) was used to test for deviations in distributions
from Hardy–Weinberg expectations and genotypic
linkage disequilibrium applying a sequential
Bonferroni correction with 1000 iterations and 1000
dememorization. The effective population size (Ne) for
each population was calculated based on the Linkage
Disequilibrium (LD) method (Waples & Do, 2008)
using the program NeEstimator v.2.0 (Do et al., 2014),
considering a random mating model and PCrit of 0.02 to
allele frequencies.
Bottleneck 1.2.02 (Piry et al., 1999) was used to
test evidence of recent population bottlenecks using
the “Sign test” (Cornuet & Luikart, 1996) and the
Wilcoxon signed rank test (Luikart & Cornuet, 1998)
of heterozygosity excess under the three mutational
models: the Infinite Alleles Model (IAM), the Two-
Phase Model (TPM—with 90% SMM and 10% IAM)
and the Stepwise Mutation Model (SMM).
BayesAss 3 (Wilson & Rannala, 2003) was used to
test for and estimate recent migration rates between
populations. Structure v.2.3.3 (Pritchard et al.,
2000) was used to conduct 20 replicate runs of each
K, from K = 1–28, according to Evanno et al. (2005)
with a burn-in of 10 000 Markov Chain Monte Carlo
(MCMC) iterations, followed by 100 000 MCMC of
data collection. The optimal K was estimated using
Structure Harvester (Earl, 2012).
On mtDNA data, haplotypes were generated by
DnaSP v.5 (Librado & Rozas, 2009) and haplotypic
and nucleotide diversity were evaluated using
Arlequin v.3.5.1.3 (Excoffier et al., 2005). Demographic
analyses were investigated by Tajima’s D (Tajima,
1989) and Fu’s Fs (Fu, 1997) statistics with Arlequin
v.3.5.1.3 (Excoffier et al., 2005) and pairwise mismatch
distributions in DnaSP v.5 (Librado & Rozas, 2009) to
detect the demographic history of H. ancistroides.
Network 4.6.1.1 [Fluxus Technology Ltd (http://
www.fluxus-engineering.com)], was used to build
and investigate the relationship between haplotypes,
based on a median-joining algorithm (Bandelt et al.,
202 C. APOLINÁRIO-SILVA ET AL.
1999). The Tamura & Nei (1993) model was selected
as the best-fit model of evolution by ModelTest v.3.7
(Posada & Crandall, 1998).
Arlequin v.3.5.1.3 (Excoffier et al., 2005) was used to
conduct the Analysis of Molecular Variance (AMOVA)
for both markers. Using this approach, the samples
were clustered in seven groups, for which each stream
(Upper, Middle and Lower samples) was considered
a group and another group was formed by all the
Laranjinha River samples. The same software was
used to estimate levels of genetic differentiation using
pairwise Φ ST (Weir & Cockerham, 1984), analogous
to Wright’s F-statistics. GenAlEx 6.5 (Peakall &
Smouse, 2012) was employed to measure Jost’s (2008)
differentiation (Dest) and pairwise GST (Nei, 1973) for
microsatellites. In addition, the correlation between
genetic differentiation and geographical distance was
calculated in GenAlEx 6.5 (Peakall & Smouse, 2012)
using a Mantel test that compared linearized Φ ST and
D est values and geographical distance with 10 000
permutations.
Data availability
The mtDNA haplotype data are available from
GenBank (see above) and further data and analyses
are given in the Supporting Information.
RESULTS
Genetic diversity
We obtained 193 alleles across nine microsatellite
loci. Null allele frequency estimates were found
in loci Hanc1 (SFr U, Ara L and Gde L), Hanc6 (SFr M)
and Hanc32 (Bar M , Ara L and Gde L ). Linkage
disequilibrium was detected in a few pairs of loci after
sequential Bonferroni correction (α = 0.05, k = 36) for
multiple comparisons: Hanc32 × Hanc184 (L 1 and
SFrU); Hanc2 × Hanc184 and Hanc2 × Hanc32 (L1);
Hanc7 × Hanc72 (L 5); Hanc6 × Hanc1 (SFr M). Some
loci deviated from Hardy–Weinberg proportions after
sequential Bonferroni correction (α = 0.05, k = 9):
Hanc2 (SFrM); Hanc6 (AleL); Hanc9 (L1, L2, L3, L4 and
L5); Hanc32 (L 1, L 5, L 7, Bar M, Ara M, Ara L, Gde M and
GdeL); Hanc143 (AleU) (Supporting Information, Table
S2). Inbreeding coefficient values (FIS) per population
were significant (P value < 0.05) only in Ara L and
GdeL (Table 1). In general, the proportion of loci with
negative or positive F IS was similar between most
samples and most FIS values ranged from -0.1 to 0.1.
However, the only significant FIS values were positive
and found for Hanc2 (SFr U, Bar M, Ara L, Gde U and
GdeM); Hanc6 (L7, SFrM); Hanc72 (AleL); and Hanc32
(L 7, Bar M, Ara L, Gde U, Gde M and Gde L) (Supporting
© 2021 The Linnean Society of London, Biological Journal of the Linnean Society, 2021, 134, 198–213
Information, Table S2). Nine independent runs were
performed, each removing a different locus (eight loci
per run), which showed similar results in all runs,
thus all the nine loci were considered for analysis.
Furthermore, no indications were found that large
allele dropout or stuttering affected our results.
Most of the observed heterozygosity values found
for microsatellite markers were above 0.5 (HO = 0.511
to 0.748) and differed subtly from the expected
heterozygosity values (HE = 0.481 to 0.741). The mean
number of alleles per locus ( N A ) ranged from 4.556
Downloaded from https://academic.oup.com/biolinnean/article/134/1/198/6284360 by guest on 20 August 2023
(AgrU) to 9.889 (SFrM), the number of rare alleles (RA)
ranged from 11 (AgrL) to 43 (SFrM and GdeL), and the
mean number of effective alleles ( N E ) ranged from
2.410 (Ale M) to 5.760 (L 6). The highest number of
private alleles was found in the SFrM (PA = 10). Allelic
richness (AR) was corrected for a minimum sample size
of 17 diploid individuals and values ranging from 4.223
(AgrU) to 8.502 (SFrM) (Table 1). The lowest effective
population size was 16.4 (AleU), while AleL, SFrL, BarM
and GdeL showed infinite size estimates (Table 1).
In contrast, haplotype diversity values were lower
than 0.5 for most samples (Table 1). Comparatively,
the mean values of diversity were generally lower
in samples from sites near the headwater of the
Laranjinha River main channel, whereas the highest
genetic diversity values were observed in samples close
to the Middle portion of this river (Table 1). Complete
diversity measures for all samples are shown in Table
1. A total of 48 variable sites, including six indels,
constituting 26 haplotypes were detected among the
mtDNA control region sequences (556 bp) (Supporting
Information, Table S3). Haplotype diversity ranged
from 0 to 0.805 and nucleotide diversity ranged from 0
to 0.0108 (Table 1). H2 was the most widely distributed
haplotype, occurring in 24 out of 25 sites. However, nine
haplotypes were singletons, of which six were sampled
only in the Laranjinha River and four were found only
in streams. In addition, another four haplotypes were
restricted to stream samples (Supporting Information,
Table S3; Fig. 2). The network haplotype shows a star-
like structure. Most haplotypes were closely related
to the common central haplotype, H2. However, some
haplotypes were shown to be several mutations
away, with more than 20 mutation steps identified,
suggesting other mitochondrial lineages (Fig. 2).
Demographic analyses
Similar to the haplotype network, the demographic
analyses also suggested recent expansion of
H. ancistroides populations throughout the Laranjinha
River Basin, since mtDNA sequences showed
significantly negative values for the Tajima neutrality
test (D) in 11 samples and seven were significantly
 Table 1. Estimates of genetic diversity for 25 populations of H. ancistroides using nine microsatellite loci and mtDNA. N = number of samples, Ne = effective
population size [confidence interval (CI) 95%], A = number of total alleles/ RA = rare alleles (frequency < 5%), PA = number of private alleles, AR = allelic
richness (adjusted by rarefaction to 17 diploid individuals), N A = mean number of alleles, N E = mean number of effective alleles, HO = observed heterozygosity,
HE = expected heterozygosity, FIS = rate of inbreeding. Nh = number of haplotypes found, h = haplotype diversity, π = nucleotide diversity, D = Tajima’s neutrality
test (Tajima, 1989), Fs = Fu’s neutrality test (Fu, 1997)

Sample Microsatellites mtDNA (D-loop)

N Ne (CI 95%) A/RA PA AR NA NE HO HE FIS N Nh h π D Fs

L1 31 16.5 (11.9–23.6) 63/30 5 6.013 7 3.651 0.606 0.594 -0.003 31 4 0.187 0.0004 -1.731* -3.436*
L2 30 3607.2 (91.8–∞) 65/19 0 6.563 7.222 4.124 0.689 0.632 -0.074 30 3 0.131 0.0002 -1.507* -2.355*
L3 28 82.7 (42.6–446.2) 78/29 1 7.807 8.667 4.895 0.710 0.684 -0.020 28 4 0.267 0.0005 -1.527* -2.610*
L4 30 124.2 (56.1–∞) 81/29 1 7.911 9 5.003 0.748 0.686 -0.073 30 4 0.434 0.0036 -2.439* 2.561
L5 39 97.7 (58.8–235.2) 83/39 0 7.693 9.222 4.947 0.690 0.668 -0.020 39 5 0.435 0.0010 -1.079 -1.855
L6 24 240.4 (68.2–∞) 79/29 0 8.263 8.778 5.760 0.746 0.727 -0.006 24 7 0.736 0.0081 -1.580* 1.994
L7 33 341.8 (94.4–∞) 82/39 2 7.826 9.111 4.812 0.656 0.686 0.060 33 10 0.805 0.0108 -1.199 1.640
AleU 30 16.4 (11.5–24.5) 56/19 2 5.522 6.222 3.742 0.619 0.597 -0.019 30 2 0.067 0.0001 -1.147* -1.211
AleM 30 53.9 (24.9 -518.9) 47/15 1 4.601 5.222 2.410 0.511 0.481 -0.045 30 1 0 0 0 0
AleL 25 Infinite (92.6–∞) 81/38 6 8.135 9 5.016 0.676 0.664 0.004 25 3 0.227 0.0004 -1.214 -1.454*
AgrU 32 37.2 (19.9–103.7) 41/12 0 4.223 4.556 2.615 0.521 0.529 0.032 32 3 0.234 0.0006 -0.606 -0.595
AgrM 30 76.9 (27.5–∞) 48/19 0 4.631 5.330 2.801 0.589 0.563 -0.028 30 4 0.251 0.0007 -1.196 -1.885*
AgrL 30 176.8 (42–∞) 44/11 0 4.540 4.889 2.697 0.526 0.556 0.071 30 4 0.407 0.0011 -0.403 -0.694
SFrU 30 65.2 (35.6–220.7) 69/20 1 6.879 7.667 4.519 0.733 0.704 -0.025 30 2 0.287 0.0042 0.42 6.881
SFrM 28 17.8 (13.6–23.9) 89/43 10 8.502 9.889 4.694 0.718 0.741 0.048 24 2 0.228 0.0033 -0.474 5.29
SFrL 25 Infinite (103.9–∞) 74/31 2 7.409 8.222 4.311 0.653 0.653 0.020 25 4 0.230 0.0004 -1.733* -3.084*
BarU 29 62.6 (21.5–∞) 83/36 4 7.906 9.222 4.623 0.693 0.672 -0.014 29 5 0.466 0.0013 -1.769* -1.518
BarM 31 Infinite (224.4–∞) 80/39 1 7.796 8.889 4.779 0.659 0.678 0.044 30 1 0 0 0 0
BarL 30 280.5 (72.7–∞) 74/27 0 7.263 8.222 4.630 0.674 0.637 -0.041 30 2 0.331 0.0006 0.485 0.891
AraU 18 57.5 (23.7–∞) 56/14 0 6.222 6.222 3.601 0.605 0.623 0.058 18 4 0.523 0.0038 2.395 5.323

© 2021 The Linnean Society of London, Biological Journal of the Linnean Society, 2021, 134, 198–213
AraM 31 169.7 (59.3–∞) 78/36 3 7.444 8.667 4.411 0.684 0.694 0.031 28 5 0.690 0.0068 -1.726* 3.695
AraL 30 162.4 (59–∞) 80/36 1 7.573 8.889 4.781 0.658 0.699 0.077* 30 7 0.766 0.0054 -1.895* 1.186
GdeU 32 292.4 (61.2–∞) 53/21 0 5.239 5.889 2.951 0.552 0.567 0.042 32 4 0.286 0.0007 -1.602* -1.894*
GdeM 29 126.7 (50.8–∞) 74/29 2 7.239 8.222 4.658 0.659 0.686 0.056 29 5 0.470 0.0022 -0.632 -0.205
GdeL 26 Infinite (83.9–∞) 85/43 4 8.228 9.444 5.109 0.662 0.708 0.084* 26 5 0.702 0.0039 1.054 1.303

*Significant values: P ≤ 0.05.

POPULATION STRUCTURE OF SUCKERMOUTHS
203

Downloaded from https://academic.oup.com/biolinnean/article/134/1/198/6284360 by guest on 20 August 2023

204 C. APOLINÁRIO-SILVA ET AL.

Downloaded from https://academic.oup.com/biolinnean/article/134/1/198/6284360 by guest on 20 August 2023

Figure 2. Haplotype network obtained from the sequencing of the D-loop region (mtDNA) for 723 H. ancistroides from 25
sites. Circle sizes are proportional to haplotype frequency. Black traces represent mutation steps; white squares represent
unsampled or predicted haplotypes.

negative for the Fu neutrality test (Fs). In mismatch Table 2. AMOVA of H. ancistroides populations studied in
distributions, sequence data indicated expectations of 25 localities
a population expansion model for most samples that
revealed unimodal patterns (Supporting Information, Source of variation % of variation
Fig. S1).
The test for recent bottlenecks in effective population Microsatellite D-loop
size detected a heterozygosity excess (bottleneck Among groups 7.63% 54.10%
signal) in eight samples under the IAM test, deficiency Among populations 5.01% 4.7%
heterozygosity in two samples under the TPM test within groups
(Two-Phase Model, 30% IAM + 70% SMM), and Within population 86.71% 40.93%
deficiency heterozygosity in 14 samples under the
SSM (Supporting Information, Table S4). Fixation index

Genetic differen- 0.133* 0.591*

tiation between
Gene flow and genetic structure populations (ФST)
The estimates of recent gene flow through Bayesian Genetic differen- 0.054* 0.108*
analysis indicated high levels of non-migrants, ranging tiation between
populations within
from 67% to 86%. In contrast, migrant rates were less
groups (ФSC)
than 0.6% for most samples and the highest migrant
Genetic differen- 0.076* 0.541*
value was 21% (BarL to L4) (Supporting Information,
tiation between
Fig. S3).
groups (ФCT)
AMOVA results for microsatellite data showed that
the majority of molecular variation occurred within
*Significant values P ≤ 0.05.
populations (86.71%). At the same time, mtDNA data
revealed a large and significant percentage of variation
among groups (54.10%). The global Φ ST values were seven pairs of comparisons did not show statistically
0.133 for microsatellite and 0.591 for D-loop (Table 2). significant values for all three indices, Ф ST, GST and Dest.
Our analysis based on microsatellite markers Significant values ranged from 0.007 to 0.267 for Ф ST,
revealed that out of 300 pairwise comparisons, only 0.018 and 0.669 for Dest (Table 3), and 0.004 to 0.154 for

© 2021 The Linnean Society of London, Biological Journal of the Linnean Society, 2021, 134, 198–213
 Table 3. Pairwise genetic differentiation estimated from microsatellites in Hypostomus ancistroides specimens. Below diagonal = Φ ST values. Above
diagonal = Dest values. Each significant pairwise comparison is color based on the range estimate: light grey: 0.001 to 0.05; medium grey: 0.051 to 0.15; dark grey:
0.151 to 0.300; darkest grey: > 0.301

L1 L2 L3 L4 L5 L6 L7 AleU AleM AleL AgrU AgrM AgrL SFrU SFrM SFrL BarU BarM BarL AraU AraM AraL GdeU GdeM GdeL
L1 0.062* 0.142* 0.129* 0.133* 0.332* 0.439* 0.189* 0.303* 0.117* 0.260* 0.260* 0.261* 0.213* 0.315* 0.229* 0.148* 0.182* 0.172* 0.387* 0.354* 0.330* 0.360* 0.334* 0.303*
L2 0.036* 0.071* 0.076* 0.086* 0.320* 0.423* 0.129* 0.214* 0.046* 0.209* 0.198* 0.193* 0.161* 0.265* 0.158* 0.086* 0.109* 0.107* 0.351* 0.330* 0.299* 0.322* 0.289* 0.312*
L3 0.071* 0.033* 0.019* 0.043* 0.322* 0.445* 0.098* 0.177* 0.026* 0.150* 0.131* 0.129* 0.105* 0.157* 0.031* 0.021* 0.032* 0.047* 0.386* 0.347* 0.312* 0.387* 0.312* 0.350*
L4 0.064* 0.035* 0.007* 0.026* 0.263* 0.413* 0.105* 0.227* 0.041* 0.211* 0.180* 0.203* 0.125* 0.240* 0.079* 0.033* 0.027* 0.055* 0.341* 0.301* 0.273* 0.338* 0.278* 0.298*
5 0.069* 0.042* 0.018* 0.011* 0.203* 0.328* 0.094* 0.206* 0.059* 0.200* 0.171* 0.208* 0.163* 0.270* 0.128* 0.024* 0.052* 0.060* 0.269* 0.232* 0.203* 0.296* 0.213* 0.233*
L
Fo
6
L 0.192* 0.125* 0.111* 0.091* 0.076* 0.067* 0.385* 0.412* 0.329* 0.438* 0.431* 0.444* 0.423* 0.523* 0.395* 0.299* 0.346* 0.352* 0.074* 0.051* 0.025* 0.196* 0.040* 0.015
0.494* 0.531* 0.456* 0.565* 0.563* 0.572* 0.572* 0.669* 0.541* 0.446* 0.514* 0.514* 0.096* 0.087* 0.069* 0.266* 0.175* 0.108*
L7 0.192* 0.174* 0.164* 0.153* 0.131* 0.027*
rP
AleU 0.109* 0.071* 0.050* 0.052* 0.049* 0.158* 0.210* 0.136* 0.064* 0.190* 0.180* 0.184* 0.177* 0.287* 0.134* 0.074* 0.082* 0.096* 0.476* 0.419* 0.387* 0.392* 0.366* 0.394*
0.154* 0.262* 0.276* 0.240* 0.284* 0.420* 0.244* 0.205* 0.214* 0.220* 0.452* 0.450* 0.418* 0.381* 0.358* 0.440*
AleM 0.200* 0.140* 0.109* 0.134* 0.126* 0.209* 0.267* 0.101*
ee
AleL 0.062* 0.023* 0.011* 0.018* 0.027* 0.118* 0.173* 0.035* 0.101* 0.160* 0.149* 0.146* 0.109* 0.200* 0.080* 0.033* 0.043* 0.059* 0.384* 0.358* 0.317* 0.351* 0.294* 0.320*
U 0.164* 0.127* 0.086* 0.116* 0.113* 0.202* 0.261* 0.125* 0.198* 0.095* 0.005 0.003 0.242* 0.266* 0.181* 0.150* 0.182* 0.168* 0.485* 0.479* 0.447* 0.376* 0.352* 0.403*
rR
Agr
M
Agr 0.154* 0.113* 0.070* 0.093* 0.092* 0.185* 0.245* 0.111* 0.195* 0.082* 0.004 0.018* 0.249* 0.255* 0.166* 0.127* 0.154* 0.143* 0.484* 0.472* 0.432* 0.388* 0.351* 0.400*
0.217* 0.255* 0.154* 0.150* 0.172* 0.170* 0.503* 0.490* 0.449* 0.380* 0.350* 0.409*
ev
AgrL 0.157* 0.112* 0.071* 0.106* 0.111* 0.192* 0.251* 0.115* 0.177* 0.083* 0.003 0.014*
SFrU 0.099* 0.070* 0.042* 0.049* 0.066* 0.134* 0.193* 0.083* 0.157* 0.045* 0.127* 0.121* 0.109* 0.163* 0.116* 0.111* 0.106* 0.115* 0.516* 0.467* 0.444* 0.483* 0.415* 0.432*
SFrM 0.131* 0.103* 0.057* 0.083* 0.097* 0.147* 0.203* 0.119* 0.205* 0.074* 0.129* 0.115* 0.118* 0.056* 0.135* 0.183* 0.187* 0.204* 0.623* 0.557* 0.542* 0.583* 0.499* 0.507*
iew
SFrL 0.117* 0.077* 0.015* 0.035* 0.058* 0.141* 0.203* 0.072* 0.153* 0.038* 0.109* 0.093* 0.089* 0.049* 0.053* 0.047* 0.038* 0.044* 0.492* 0.446* 0.430* 0.477* 0.381* 0.403*
BarU 0.076* 0.041* 0.009* 0.014* 0.010* 0.107* 0.168* 0.039* 0.126* 0.015* 0.088* 0.070* 0.083* 0.045* 0.067* 0.022* 0.008 0.008 0.375* 0.331* 0.309* 0.369* 0.286* 0.307*
M
Bar 0.091* 0.052* 0.014* 0.012* 0.023* 0.120* 0.187* 0.043* 0.130* 0.020* 0.103* 0.082* 0.093* 0.043* 0.068* 0.018* 0.004 -0.003 0.444* 0.376* 0.356* 0.420* 0.335* 0.364*
BarL 0.093* 0.055* 0.022* 0.025* 0.029* 0.133* 0.201* 0.053* 0.142* 0.029* 0.104* 0.083* 0.099* 0.051* 0.080* 0.022* 0.003 -0.001 0.442* 0.382* 0.368* 0.419* 0.342* 0.353*
AraU 0.192* 0.164* 0.160* 0.142* 0.121* 0.033* 0.047* 0.224* 0.266* 0.165* 0.258* 0.242* 0.252* 0.194* 0.210* 0.207* 0.160* 0.182* 0.196* 0.024* 0.051* 0.225* 0.115* 0.116*
AraM 0.158* 0.138* 0.130* 0.112* 0.093* 0.019* 0.036* 0.181* 0.236* 0.138* 0.228* 0.212* 0.222* 0.160* 0.172* 0.171* 0.127* 0.140* 0.155* 0.012* 0.015 0.215* 0.097* 0.085*
AraL 0.147* 0.125* 0.117* 0.103* 0.083* 0.010* 0.029* 0.167* 0.219* 0.123* 0.214* 0.195* 0.204* 0.152* 0.166* 0.163* 0.119* 0.133* 0.148* 0.025* 0.006 0.186* 0.062* 0.063*
GdeU 0.200* 0.171* 0.183* 0.162* 0.150* 0.095* 0.134* 0.213* 0.250* 0.175* 0.230* 0.223* 0.223* 0.210* 0.229* 0.227* 0.179* 0.197* 0.210* 0.129* 0.110* 0.095* 0.129* 0.130*

© 2021 The Linnean Society of London, Biological Journal of the Linnean Society, 2021, 134, 198–213
M
Gde 0.153* 0.125* 0.120* 0.107* 0.089* 0.016* 0.072* 0.164* 0.197* 0.118* 0.180* 0.168* 0.170* 0.147* 0.159* 0.151* 0.114* 0.129* 0.143* 0.054* 0.041* 0.026* 0.069* 0.035*
GdeL 0.135* 0.127* 0.127* 0.109* 0.092* 0.007 0.044* 0.167* 0.226* 0.121* 0.195* 0.181* 0.187* 0.145* 0.153* 0.151* 0.115* 0.133* 0.140* 0.052* 0.033* 0.026* 0.068* 0.015*
*Significant values P ≤ 0.05.
POPULATION STRUCTURE OF SUCKERMOUTHS
205

Downloaded from https://academic.oup.com/biolinnean/article/134/1/198/6284360 by guest on 20 August 2023

206 C. APOLINÁRIO-SILVA ET AL.
GST (Supporting Information, Table S5). For mtDNA
sequences, mt-Ф ST values differed significantly from
zero to 194 pairwise comparisons with values of mt-Ф ST
ranging from 0.062 to 0.933 (Supporting Information,
Table S5). The average genetic differentiation along
the river was: SSR-Ф ST = 0.086, SSR-Dest = 0.207, SSR-
G ST = 0.045 and mt-Ф ST = 0.291; while the average
genetic differentiation along streams was: SSR-
Ф ST = 0.130, SSR-D est = 0.282, SSR-G ST = 0.070 and
mt-Ф ST = 0.604; and the average genetic differentiation
among streams and the river was: SSR-Ф ST = 0.107,
SSR-Dest = 0.246, SSR-GST = 0.058 and mt-Ф ST = 0.498.
The Mantel test showed positive and significant
correlations between genetic differentiation and
geographical distances for both microsatellite (SSR-
Ф ST: R = 0.69 and Dest: R = 0.72, P = 0.01) and mtDNA
data (mt-Ф ST: R = 0.219, P = 0.01) (Supporting
Information, Fig. S2).
The first structure from microsatellites, including
all samples data, indicated that the most probable
K value (cluster number) K = 2. The graphic
representing this analysis shows a group formed
by samples in the Lower section of the watershed,
including L 6 , L 7 (in main channel), Araras and
Grande Streams, and another formed by samples in
the Upper and Middle sections, including the main
channel and streams. From that result, new runs
were conducted for each of these groups, obtaining
K = 3 among Upper and Middle section samples and
K = 2 for the Lower section samples (Fig. 3).
Figure 3. Graphical representation of genetic ancestry using the Structure software. A. Result of all 731 Hypostomus
ancistroides sampled in the 25 locations (K = 2), B. Result of the Structure analysis considering only Upper and Middle
section sites. C. Result of the Structure analysis considering only the locations of the Lower section.
© 2021 The Linnean Society of London, Biological Journal of the Linnean Society, 2021, 134, 198–213
DISCUSSION
Both nuclear DNA (microsatellite) and mtDNA data
showed that the genetic diversity of H. ancistroides
is heterogeneously distributed along the Laranjinha
River Basin. Patterns of genetic structure over short
geographical distances were found in most of the
streams analysed. In addition, data indicated that
part of the genetic diversity is restricted to different
drainages from the watershed. In general, the
patterns obtained may demonstrate relationships
with structures of the environments, processes of
Downloaded from https://academic.oup.com/biolinnean/article/134/1/198/6284360 by guest on 20 August 2023
colonization of the basin, and limited displacement of
species.
Fine-scale genetic structure
A significant genetic structure was obtained among
almost all samples studied, occurring even between
samples from the same stream, separated by short
geographical distances. Indeed, the differentiation
estimates of the present study (Ф ST, Dest, GST and some
Structure results) indicate similar patterns to those
previously reported for subpopulations of this species
(Sofia et al., 2008) and other fish species in Neotropical
(Ferreira et al., 2015) and non-Neotropical streams
(Carlsson et al., 1999,), which suggested genetic
differentiation over short geographical distances along
small drainages. When comparing distances between
sampling sites along the river (min.: 37.9 km—max.:
348.8 km) and sampling sites along each stream,
 POPULATION STRUCTURE OF SUCKERMOUTHS
samples analysed in streams are separated by much
smaller geographical distances, ranging from 8.9 km
to 27.2 km between sites. Even so, microsatellite and
mtDNA data showed that a large part of the significant
genetic structure along streams occurs between
samples separated by less than 15 km (e.g. 8.3 km
between SFrU × SFrM: SSR-GST = 0.053; 14 km between
Ara U × Ara L: mt-Ф ST = 0.260). However, mtDNA is
often unable to detect more recent population changes
as efficiently as nuclear DNA (McCusker & Bentzen,
2010), which could explain why mtDNA presented less
significant differentiation values along the streams.
In fact, the differences between nuclear and mtDNA
results suggest that the differentiation patterns
obtained may reflect current processes, including
influences of landscape features and biology of the
species. Streams can present spatial and temporal
variations that influence distribution of fish
populations along the flow (Winemiller et al., 2008).
These variations include, for example, the presence of
barriers which provide obstacles to dispersion, such as
evidenced in the São Francisco Stream in our study,
where there is a waterfall of more than 5 m between
Upper and Middle sections. Artificial barriers, such
as the dam found in the main channel, could also
influence the genetic distribution of fish (Agostinho
et al., 2008). However, this was not evidenced in the
present study, since the genetic differences between
samples separated by the dam (L4 and L5) were not
greater than those between samples without barriers
upstream in the main channel. Other important
stream characteristics could also influence the
genetic distribution of fish species, such as altitude,
precipitation, temperature, riparian forest cover, water
chemistry (Reis et al., 2016), dendritic organization
of the watershed (Hughes et al., 2009), and even
anthropogenic interferences, to which streams are
highly sensitive (Luiz et al., 1998).
At the same time, the biology of the studied species
could also influence the results. The present study
detected very low gene flow levels (< 0.6%) among
most samples and significant values of genetic
differentiation (Ф ST, GST and Dest) were found even for
those samples with estimates of migrants above 0.10,
including a sample with 21% for this estimate (BarL in
L4). Thus, it seems plausible that gene flow rates may
not have been sufficient to avoid the population genetic
structuring, even for geographically close populations.
These results suggest a limited displacement capacity
for the species studied, a feature that could be related
to the biology of the species, which is corroborated
by the differentiation indices (Ф ST-GST and Dest), the
significant correlation between geographical distances
and genetic differentiation estimates in the Mantel
test, and also by mtDNA data, since some haplotypes
were limited to only one site in some streams, a scenario
© 2021 The Linnean Society of London, Biological Journal of the Linnean Society, 2021, 134, 198–213
207
that would be more difficult if the displacement was
not limited.
Although population boundaries for a fish species
seem unrealistic across continuous-flow systems such
as streams, the low displacement of H. ancistroides
and the fine-scale genetic structure herein reported
are also supported by the low number of loci exhibiting
significant FIS and Hardy–Weinberg disequilibrium.
Indeed, in another scenario, including greater
movement and individuals exchanged, it would be
expected that a sample from any site would include a
Downloaded from https://academic.oup.com/biolinnean/article/134/1/198/6284360 by guest on 20 August 2023
greater number of individuals from different genetic
units, favouring the characteristic patterns of the
Wahlund effect, such as several loci with significant FIS
and Hardy–Weinberg disequilibrium (Frankham et al.,
2010). Previous studies have already suggested limited
displacement for H. ancistroides (Zawadzki et al.,
2005; Sofia et al., 2008). However, the present study
is one of the first to specifically address this issue and
provide robust evidence on this feature. In fact, some
studies on the genus Hypostomus highlight that most
of the members of this group are species that do not
perform migratory movements, including species with
parental care (Agostinho et al., 2007). Nevertheless,
until now, information suggesting limited mobility for
H. ancistroides along the drainage basins has mainly
been obtained indirectly, from results of genetic
structuring or considering characteristics of the group
(Zawadzki et al., 2005; Sofia et al., 2008).
Population distribution patterns
The evolutionary history of H. ancistroides in the
Upper Paraná drainage basins includes successive
cycles of geographical isolation, differentiation and
population expansions, so that secondary contacts
are quite plausible (Hollanda-Carvalho et al., 2016).
Indeed, most of our findings reinforce this statement.
For instance, significant levels of genetic differentiation
between sample pairs were detected for both markers
analysed, while Bayesian analysis also revealed a set
of subpopulations distributed in two large genetic
clusters, one comprising Middle and Upper sections
and the other formed by the Lower section (with the
separation of these two groups occurring precisely
between sites L5 and L6).
Concerning population expansion, a recent
(evolutionary) colonization process of this species
throughout the watershed studied could be suggested
by the relationship between haplotypic and nucleotide
diversity values (h > 0.5 and π < 0.5%, according to
Grant & Bowen, 1998) and the star-shaped haplotype
network, which may indicate population expansion
(Slatkin & Hudson, 1991). In addition, the majority
of stream samples presented a unimodal distribution
pattern in the mismatch distribution test and some of
208 C. APOLINÁRIO-SILVA ET AL.
them presented significant and negative values in the
Fu and Tajima tests, which also commonly indicates
the abovementioned pattern (Excoffier et al., 2005;).
Our study also suggests evidence of secondary
contact between distinct mitochondrial lineages,
mainly in the Lower section of the basin (L4, L6, L7,
Ara M and Ara L), where haplotypes (H8, H11, H12,
H15, H16 and H17) from another lineage (possibly
originating in another drainage in the Paranapanema
River Basin) entered the mouth of the Laranjinha
River and appear to be dispersed upstream (along
the main channel and tributaries); although this
process also seems to have been influenced by the
limiting factor between L 5 and L 6. As mentioned
above, dispersion of H. ancistroides in the direction
from the mouth to the source is corroborated by the
data of the present study, mainly from colonization
patterns of the basin, where H. ancistroides seems
to have occupied the Lower section of the basin in
periods prior to dispersal to Upper section drainage
basins. Corroborating this pattern, genetic diversity
indices (Nh, h and π) were higher in streams from
the Lower section of the basin, while some samples
from the Middle and Upper sections presented only
one haplotype. Furthermore, unimodal patterns in
mismatch distribution were more common among
samples from the Upper section, while drainage
basins from the Lower section displayed multimodal
distributions, suggesting that subpopulations in
streams closer to the river mouth were established
before those in Upper section streams.
In this context, mtDNA and microsatellite data also
showed that genetic diversity tends to increase from
the source towards the river mouth (source-mouth
direction), both along the river (HE from 0.594 to 0.727)
and the streams (e.g. Gde: HE from 0.567 to 0.708), and
even across watershed sections. Interestingly, a similar
tendency has been reported for species diversity along
freshwater drainages (increasing in source-mouth
direction), following the River Continuum Concept
(Vannote et al., 1980). Considering the intraspecific
genetic diversity distribution, some authors have
also shown data agreeing with this pattern, including
studies of fish species in Neotropical drainage basins
(Sofia et al., 2008; Ferreira et al., 2016) and other river
systems worldwide (Kelson et al., 2015).
Streams generally undergo gradual changes in their
features along the flow downstream, including more
stable habitats, a lower number of dispersion barriers
in lower stretches, larger meso-habitats, and an
increase in species numbers (Winemiller et al., 2008).
In addition, the tributary junctions (confluences)
within a river network have been pointed out as
biological hotspots, in part due to the heterogeneity
present in these locations (Benda et al., 2004). Thus, it
is not by chance that such places have been loci of core
© 2021 The Linnean Society of London, Biological Journal of the Linnean Society, 2021, 134, 198–213
populations, providing stable sources of dispersers
to recolonize peripheral habitats (Petts et al., 2015).
Therefore, although further studies are needed,
we cannot rule out that the increase in the level of
genetic diversity detected towards downstream could
be a pattern for these drainages, especially due to
the possibility of larger effective population sizes in
downstream sites, as well as in the main stream in
relation to the tributary streams. It is acknowledged
that larger effective population sizes minimize the
effects of genetic drift in within-population genetic
Downloaded from https://academic.oup.com/biolinnean/article/134/1/198/6284360 by guest on 20 August 2023
diversity, for example, decreasing the chances of losing
rare alleles (Frankham et al., 2010). Although the Ne
results of the present study were not conclusive, the
presence of larger populations in Lower sections would
be plausible. Downstream populations would be more
susceptible to migrants from other genetic units, since
downstream areas may be prone to the influence from
adjacent drainages (e.g. stream confluence receiving
migrants from the main channel or river confluence
receiving migrants from a larger downstream river),
while upstream populations would be more isolated
(Hughes et al., 2009; Frankham et al., 2010).
Genetic diversity
Different analyses in our study, including genetic
diversity estimates (mtDNA and microsatellites),
haplotype networks, genetic differentiation levels
and Structure results (K = 3 among Upper and
Middle section samples and K = 2 among Lower
section samples), indicated that genetic diversity of
H. ancistroides is heterogeneously distributed along
the Laranjinha River Basin. Moreover, it is noticeable
that part of the genetic diversity is restricted to
samples from different streams, suggesting a limited
displacement behaviour of H. ancistroides through
the Laranjinha hydrographic basin. This behaviour of
H. ancistroides was already suggested by other authors
(Zawadzki et al., 2005; Sofia et al., 2008). In fact, when
we consider both the distribution of haplotypes and
the fact that most were exclusive haplotypes, with few
mutational steps in relation to the main haplotype, it
is reasonable to suppose a limited displacement for
the studied species. In fact, eight haplotypes were
restricted to streams and five of the six studied streams
contained at least one exclusive haplotype, which
seems to have arisen within the respective occurrence
stream and not yet have been distributed to other
drainage basins. Microsatellite data also corroborate
these results, displaying private alleles along five of
the six studied streams.
The spatial heterogeneity in genetic diversity, with
potions of the variations found restricted to each
stream, was also corroborated by Bayesian clustering
analysis (Structure), which showed sub-structuring
 POPULATION STRUCTURE OF SUCKERMOUTHS
both among Upper and Middle samples and Lower
samples. Considering these analyses, most of the
studied streams seem to represent different genetic
units. In fact, despite possible connectivity between
some of these streams, it is plausible that little
gene flow has occurred between these drainages
since each was colonized, contributing to population
differentiation. Thus, non-migratory behaviour
described for the genus Hypostomus (Agostinho et al.,
2007) may be one of the main factors favouring genetic
structuring among streams of the Laranjinha River
Basin. Additionally, this differentiation tendency
is also commonly reported for other species in the
same genus (Zawadzki et al., 2008) and other non-
migratory freshwater fishes (Adamson et al., 2012;
Ferreira et al., 2015).
In addition to limited displacement of
H. ancistroides and characteristics of streams, the
connectivity patterns and geographical distances
could also have important influences on the results
of the present study. When considering the basin, the
genetic distribution of H. ancistroides throughout the
Laranjinha River Basin seems to match the Stream
Hierarchy Model (SHM) (Meffe & Vrigenhoek, 1988).
According to this model, watersheds have a dendritic
organization where genetic differences between
samples in the same drainage basin tend to be lower
than those observed between samples from different
drainage basins. Furthermore, even though gene
flow seems to be greater over the same drainage
basin, for non-migratory species like H. ancistroides
this process ends up being slower, possibly occurring
according to the Stepping-Stone Model, where allele
exchanges between closer groups over the generations
allow distribution of new variants to more distant
populations (Kimura & Weiss, 1964). In fact, the
Stepping-Stone Model and Stream Hierarchy Model
are corroborated by a significant correlation in the
Mantel test, which indicates that differentiation
patterns may have been established from isolation
by distance (IBD), something expected for a non-
migratory species with wide distribution (Slatkin,
1993). A similar result was found for subpopulations
of Geophagus brasiliensis, a non-migratory species
exhibiting parental care, along the same basin studied
herein, which presented mitochondrial haplotype and
microsatellite alleles restricted to one stream but
absent in the other five sites sampled along the main
stem (Ferreira et al., 2015). Moreover, the significant
genetic differentiation between samples from
streams and the main stem described here has also
been reported for other fish species in river systems
in Europe (Carlsson et al., 1999), North America
(Kelson et al., 2015) and South-east Asia (Adamson
et al., 2012), indicating a similar pattern of genetic
distribution for different fish species across the world.
© 2021 The Linnean Society of London, Biological Journal of the Linnean Society, 2021, 134, 198–213
209
Thus, our genetic findings on the population structure
of H. ancistroides are in line with those described for
fish populations in watersheds in temperate regions
(Carlsson et al., 1999; Adamson et al., 2012; Kelson
et al., 2015).
Furthermore, the information from our study
demonstrates the great importance of understanding
evolutionary aspects related to streams, as well as
preservation of these drainage basins. The genetic
differences found in the present study are in accordance
with the biology of the studied species and corroborate
Downloaded from https://academic.oup.com/biolinnean/article/134/1/198/6284360 by guest on 20 August 2023
information that attributes a high degree of endemism
to small drainage basins, such as streams (Castro
& Polaz, 2020). In addition, if these small drainages
are impacted, their recovery may not be effective
and important portions of genetic diversity of species
along the watershed could be lost permanently. In the
current scenario, microsatellite data indicated that all
sampled subpopulations have maintained satisfactory
levels of genetic diversity ( H O = 0.649). In contrast,
genetic diversity levels in the mtDNA data were low
for most of the studied samples. However, as previously
discussed, the distribution and variation levels of
mtDNA are likely influences from an older scenario,
including a founder effect during new colonization
events, which would justify less variation. In fact, like
mtDNA, other markers with a mutation rate lower
than microsatellites also demonstrated less variation
within the genus Hypostomus. Low levels of genetic
diversity for H. ancistroides and other Hypostomus
species have also been reported in different studies
(Paiva et al., 2005; Zawadzki et al., 2005).
In terms of genetic conservation of species, efforts
commonly seek to conserve as much variation as
possible within and between subpopulations of a species
(Frankham et al., 2010). Therefore, the heterogeneous
distribution of genetic diversity of H. ancistroides from
drainages along the Laranjinha River Basin highlights
that any preservation measure aimed at maintaining
genetic diversity of widely distributed species must
consider both rivers and their streams. As observed in
the present study, degradation of any of the drainages
could culminate in loss of exclusive portions of genetic
diversity. Thus, our data could be further evidence
that exclusive preservation of large drainages is not
sufficient for preservation of freshwater ichthyofauna
in the region.
However, the situation of small Neotropical drainages
has become increasingly alarming. In the Laranjinha
River Basin, for example, different forms of anthropic
interference in streams are evident, including pollution,
deforestation, silting and small barriers, especially in
sites close to urban regions. Unfortunately, today, little
attention is given to these small drainages, which does
not match their richness, both ecological and genetic.
The same can be said in relation to the species under
210 C. APOLINÁRIO-SILVA ET AL.
study, H. ancistroides, which despite being abundant
and having no significant commercial value, has great
biological importance (Angelescu & Gneri, 1949).
It is very important to highlight that so many
particularities restricted to streams, such as those
evidenced in our study, make the measures developed
for larger drainages inappropriate for streams.
Therefore, specific measures must be created, including
the preservation and restoration of riparian forest,
absence of any type of pollution, and non-insertion of
obstacles (e.g. dams) along these drainages, so that
protection of the streams and all their biology can be
effective. Finally, although the focus of the current
study was the suckermouth catfish, H. ancistroides,
effective conservation and preservation of streams
would benefit all types of organisms present in them.
ACKNOWLEDGEMENTS
This study was financed in part by the Coordenação
de Aperfeiçoamento de Pessoal de Nível Superior—
Brazil (CAPES) —Finance Code 001, and Fundação
Araucária. C.A-.S. thanks CAPES for the scholarship
awarded; S.H.S. receives a productivity research
fellowship from Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq) (PQ 305343/2018-1),
W.F-.S. is a researcher on a Programa Nacional de Pós-
Doutorado (PNPD) - Universidade Estadual do Norte
Fluminense (UENF) (CAPES) fellowship. We would
like to thank Robson Rockembacher for his valuable
help during the fieldwork and the Universidade Norte
do Paraná and Universidade Estadual de Londrina
for all their support during the field study. We thank
the Editor and three anonymous reviewers for their
constructive comments, which helped to improve
the manuscript quality. C.A-.S., D.G.F., T.K-.D. and
R.H.C.N. carried out the field activities. C.A-.S., D.G.F.,
B.A.G. and S.H.S. participated in the study sample
design. C.A-.S., W.F-.S., D.G.F. and S.H.S. carried
out the interpretation of the final data. All authors
participated in the writing of the final document. The
authors declare that they have no conflicts of interest.
REFERENCES
Adamson EAS, Hurwood DA, Mather PB. 2012. Insights into
historical drainage evolution based on the phylogeography of
the chevron snakehead fish (Channa striata) in the Mekong
Basin. Freshwater Biology 57: 2211–2229.
Agostinho AA, Pelicice FM, Gomes LC. 2008. Dams
and the fish fauna of the Neotropical region: impacts and
management related to diversity and fisheries. Revista
Brasleira de Biologia 68: 1119–1132.
© 2021 The Linnean Society of London, Biological Journal of the Linnean Society, 2021, 134, 198–213
Agostinho AA, Pelicice FM, Petry AC, Gomes LC, Júlio HF
Jr. 2007. Fish diversity in the upper Paraná River basin:
habitats, fisheries, management and conservation. Aquatic
Ecosystems and Health Management 10: 174–186.
Allan JD, Flecker AS. 1993. Biodiversity conservation in
running waters. BioScience 43: 32–43.
Angelescu V, Gneri FS. 1949. Adaptaciones del aparato
digestivo al regimesalimenticios en algunos peces del Rio
Uruguay y del Rio de La Plata. Revista del Instituto Nacional
de Ciencias Naturales 1: 161–261.
Bandelt HJ, Forster P, Röhl A. 1999. Median-joining
networks for inferring intraspecific phylogenies. Molecular
Downloaded from https://academic.oup.com/biolinnean/article/134/1/198/6284360 by guest on 20 August 2023
Biology and Evolution 16: 37–48.
Benda L, Poff NL, Miller D, Dunne T, Reeves G, Press G,
Pollock MM. 2004. The network dynamics hypothesis: how
channel networks structure riverine habitats. BioScience 54:
413–427.
Blum MJ, Bagley MJ, Walters DM, Jackson SA, Daniel FB,
Chaloud DJ, Cade BS. 2012. Genetic diversity and species
diversity of stream fishes covary across a land-use gradient.
Oecologia 168: 83–95.
Britto SGC, Sirol RN, Vianna NC, Jardim SM, Santos JD,
Pelisari E. 2003. Peixes do Rio Paranapanema. São Paulo:
Duke Energy Internacional Geração Paranapanema.
Cardoso YP, Brancolini F, Protogino L, Lizarralde M.
2011. Actinopterygii, Siluriformes, Loricariidae, Hypostomus
aspilogaster (Cope, 1894). Distribution extension and first
record for Argentina. Check List 7: 596–598.
Carlsson J, Olsen KH, Nilsson J, Øverli Ø, Stabell OB.
1999. Microsatellites reveal fine-scale genetic structure
in stream-living brown trout. Journal of Fish Biology 55:
1290–1303.
Castro RMC, Polaz CNM. 2020. Small-sized fish: the largest
and most threatened portion of the megadiverse Neotropical
freshwater fish fauna. Biota Neotropica 20: e20180683.
Cornuet JM, Luikart G. 1996. Description and power analysis
of two tests for detecting recent population bottlenecks from
allele frequency data. Genetics 144: 2001–2014.
Do C, Waples RS, Peel D, Macbeth GM, Tillett BJ,
Ovenden JR. 2014. NeEstimator v2: re-implementation
of software for the estimation of contemporary effective
population size (Ne) from genetic data. Molecular Ecology
Resources 14: 209–214.
Earl DA, VonHoldt BM. 2012. Structure Harvester: a
website and program for visualizing structure output and
implementing the Evanno method. Conservation Genetics
Resources 4: 359–361.
Endo KS, Martinez ERM, Zawadzki CH, de Souza Paiva LR,
Júlio HF Jr. 2012. Karyotype description of possible new
species of the Hypostomus ancistroides complex (Teleostei:
Loricariidae) and other Hypostominae. Acta Scientiarum
Biological Science 34: 181–189.
Evanno G, Regnaut S, Goudet J. 2005. Detecting the number
of clusters of individuals using the software STRUCTURE: a
simulation study. Molecular Ecology 14: 2611–2620.
Excoffier LG, Laval A, Scheneider S. 2005. Arlequin ver.
3.1: an integrated software package for population genetics
data analysis. Evolutionary Bioinformatics Online 1: 47–50.
 POPULATION STRUCTURE OF SUCKERMOUTHS
Ferreira DG, Galindo BA, Frantine-Silva W, Almeida FS,
Sofia SH. 2015. Genetic structure of a Neotropical sedentary
fish revealed by AFLP, microsatellite and mtDNA markers: a
case study. Conservation Genetics 16: 151–166.
Ferreira DG, Lima SC, Frantine-Silva W, Silva JF,
Apolinário-Silva C, Sofia SH, Carvalho S, Galindo BA.
2016. Fine-scale genetic structure patterns in two freshwater
fish species, Geophagus brasiliensis (Osteichthyes, Cichlidae)
and Astyanax altiparanae (Osteichthyes, Characidae)
throughout a Neotropical stream. Genetics and Molecular
Research 15: gmr15048124.
Ferreira DG, Souza-Shibatta L, Shibatta OA, Sofia SH,
Carlsson J, Dias JHP, Makrakis MC. 2017. Genetic
structure and diversity of migratory freshwater fish in
a fragmented Neotropical river system. Reviews in Fish
Biology and Fisheries 27: 209–231.
Frankham R, Briscoe DA, Ballou JD. 2010. Introduction to
conservation genetics. Cambridge: Cambridge University Press.
Fu YX. 1997. Statistical tests of neutrality of mutations
against population growth, hitchhiking and background
selection. Genetics 147: 915–925.
Galindo BA, Ferreira DG, Almeida FS, Carlsson J,
Sofia SH. 2015. Isolation and characterization of 13
polymorphic microsatellite loci in Hypostomus ancistroides
(Teleostei, Loricariidae) and cross-amplification in related
species. Journal of Fish Biology 86: 1860–1866.
Galindo BA, Ota RR, Garcia TD, Nascsofiaimento RHC,
Ohara WM, Zanatta AS, Ferreira DG, Apolinário-
Silva C, Frantine-Silva W, Carvalho S, Costa ADA,
Sofia SH, Shibatta OA. 2020. Inventory of the fish fauna
from Laranjinha River, Paranapanema River system, Brazil.
Biota Neotropica 20: e20200962.
Garcez R, Calcagnotto D, Almeida-Toledo LFD. 2011.
Population structure of the migratory fish Prochilodus
lineatus (Characiformes) from Rio Grande basin (Brazil), an
area fragmented by dams. Aquatic Conservation 21: 268–275.
Goudet J. 2001. FSTAT, a program to estimate and test gene
diversities and fixation indices (version 2.9.3). Available at:
http://www.unil.ch/izea/softwares/fstat.html
Grant WS, Bowen BW. 1998. Shallow population histories
in deep evolutionary lineages of marine fishes: insights
from sardines and anchovies and lessons for conservation.
Journal of Heredity 89: 415–426.
Heithaus MR, Laushman RH. 1997. Genetic variation
and conservation of stream fishes: influence of ecology, life
history, and water quality. Canadian Journal of Fisheries
and Aquatic Sciences 54: 1822–1836.
Hollanda Carvalho P, Maia Queiroz Lima S,
Henrique Zawadzki C, Oliveira C, de Pinna M.
2016. Phylogeographic patterns in suckermouth catfish
Hypostomus ancistroides (Loricariidae): dispersion,
vicariance and species complexity across a Neotropical
biogeographic region. Mitochondrial DNA. Part A, DNA
Mapping, Sequencing, and Analysis 27: 3590–3596.
Hughes JM, Schmidt DJ, Finn DS. 2009. Genes in
streams: using DNA to understand the movement of
© 2021 The Linnean Society of London, Biological Journal of the Linnean Society, 2021, 134, 198–213
211
freshwater fauna and their riverine habitat. BioScience
59: 573–583.
Jost L. 2008. G ST and its relatives do not measure
differentiation. Molecular Ecology 17: 4015–4026.
Kelson SJ, Kapuscinski AR, Timmins D, Ardren WR. 2015.
Fine-scale genetic structure of brook trout in a dendritic
stream network. Conservation Genetics 16: 31–42.
Kimura M, Weiss GH. 1964. The stepping stone model of
population structure and the decrease of genetic correlation
with distance. Genetics 49: 561–576.
Librado P, Rozas J. 2009. DnaSP v5: a software for
comprehensive analysis of DNA polymorphism data.
Downloaded from https://academic.oup.com/biolinnean/article/134/1/198/6284360 by guest on 20 August 2023
Bioinformatics 25: 1451–1452.
Luikart G, Cornuet JM. 1998. Empirical evaluation of a test
for identifying recently bottlenecked populations from allele
frequency data. Conservation Biology 12: 228–237.
Luiz EA, Agostinho AA, Gomes LC, Hahn NS. 1998.
Ecologia trófica de peixes em dois riachos da bacia do rio
Paraná. Revista Brasileira de Biologia 58: 273–285.
McCusker MR, Bentzen P. 2010. Positive relationships
between genetic diversity and abundance in fishes. Molecular
Ecology 19: 4852–4862.
Meffe GK, Vrijenhoek RC. 1988. Conservation genetics in the
management of desert fishes. Conservation Biology 2: 157–169.
Nei M. 1973. Analysis of gene diversity in subdivided
populations. Proceedings of the National Academy of Sciences
of the United States of America 70: 3321–3323.
van Oosterhout C, Hutchinson WF, Wills DP, Shipley P.
2004. Micro-Checker: software for identifying and correcting
genotyping errors in microsatellite data. Molecular Ecology
Notes 4: 535–538.
Ota RR, Deprá GDC, Graça WJD, Pavanelli CS. 2018.
Peixes da planície de inundação do alto rio Paraná e áreas
adjacentes: revised, annotated and updated. Neotropical
Ichthyology 16: e170094.
Paiva SD, Renesto E, Zawadzki CH. 2005. Genetic
variability of Hypostomus (Teleostei, Loricariidae)
from the Ribeirão Maringá, a stream of the upper Rio
Paraná basin, Brazil. Genetics and Molecular Biology 28:
370–375.
Peakall R, Smouse PE. 2012. GenAlEx 6.5: genetic analysis
in Excel. Population genetic software for teaching and
research–an update. Bioinformatics 28: 2537–2539.
Pelicice FM, Azevedo-Santos VM, Vitule JR, Orsi ML,
Lima Junior DP, Magalhães AL, Agostinho AA. 2017.
Neotropical freshwater fishes imperilled by unsustainable
policies. Fish and Fisheries 18: 1119–1133.
Petts G, Grosselin M-P, Gray J. 2015. The dynamics of
rivers in relation to fishes and fisheries. In: Craig JF, ed.
Freshwater fisheries ecology. Oxford: Wiley, 9–30.
Piry S, Luikart G, Cornuet JM. 1999. Bottleneck: a
computer program for detecting recent reductions in the
effective population size using allele frequency data. Journal
of Heredity 90: 502–503.
Posada D, Crandall KA. 1998. MODELTEST: testing the
model of DNA substitution. Bioinformatics 14: 817–818.
212 C. APOLINÁRIO-SILVA ET AL.

Pritchard JK, Stephens M, Donnelly P. 2000. Inference
of population structure using multilocus genotype data.
Genetics 155: 945–959.
Raymond M, Rousset F. 1995. Genepop (version 12):
population genetics software for exact tests and ecumenicism.
Journal of Heredity 86: 248–249.
Reis RE, Albert JS, Di Dario F, Mincarone MM, Petry P,
Rocha LA. 2016. Fish biodiversity and conservation in
South America. Journal of Fish Biology 89: 12–47.
Schuelke M. 2000. An economic method for the fluorescent
labelling of PCR fragments. Nature Biotechnologies 18:
233–234.
and maximum parsimony methods. Molecular Biology and
Evolution 28: 2731–2739.
Vannote RL, Minshall GW, Cummins KW, Sedell JR,
Cushing CE. 1980. The river continuum concept. Canadian
Journal of Fisheries and Aquatic Sciences 37: 130–137.
Waples RS, Do C. 2008. ldne: a program for estimating
effective population size from data on linkage disequilibrium.
Molecular Ecology Resources 8: 753–756.
Weber C. 2003. Subfamily Hypostominae (Armored catfishes).
In: Reis RE, Kullander SO, Ferraris CJ Jr, eds. Check list
of the freshwater fishes of South and Central America. Porto
Alegre: EDIPUCRS, 351–372.

Downloaded from https://academic.oup.com/biolinnean/article/134/1/198/6284360 by guest on 20 August 2023

Slatkin M. 1993. Isolation by distance in equilibrium and non-
equilibrium populations. Evolution 47: 264–279.
Slatkin M, Hudson RR. 1991. Pairwise comparisons of
mitochondrial DNA sequences in stable and exponentially
growing populations. Genetics 129: 555–562.
Sofia SH, Galindo BA, Paula FM, Sodré LM, Martinez CB.
2008. Genetic diversity of Hypostomus ancistroides
(Teleostei, Loricariidae) from an urban stream. Genetics and
Molecular Biology 31: 317–323.
Splendiani A, Berrebi P, Tougard C, Righi T, Reynaud N,
Fioravanti T, Conte PL, Delmastro GB, Baltieri M,
Ciuffardi L, Candiotto A, Sabatini A, Barucchi VC.
2020. The role of the south-western Alps as a unidirectional
corridor for Mediterranean brown trout (Salmo trutta
complex) lineages. Biological Journal of the Linnean Society
131: 909–926.
Tajima F. 1989. Statistical method for testing the neutral mutation
hypothesis by DNA polymorphism. Genetics 123: 585–595.
Tamura K, Nei M. 1993. Estimation of the number of nucleotide
substitutions in the control region of mitochondrial DNA in
humans and chimpanzees. Molecular Biology and Evolution
10: 512–526.
Tamura K, Peterson D, Peterson N, Stecher G, Nei M,
Kumar S. 2011. MEGA5: molecular evolutionary genetics
analysis using maximum likelihood, evolutionary distance,
Weir BS, Cockerham CC. 1984. Estimating F-statistics for
the analysis of population structure. Evolution; International
Journal of Organic Evolution 38: 1358–1370.
Wilson GA, Rannala B. 2003. Bayesian inference of recent
migration rates using multilocus genotypes. Genetics 163:
1177–1191.
Winemiller KO, Agostinho AA, Caramaschi EP. 2008.
Fish ecology in tropical streams. In: Dudgeon D, ed. Tropical
stream ecology. London: Academic Press, 107–146.
Wu H, Gu Q, Zhou C, Tang Y, Husemann M, Meng X,
Zhang J, Nie G, Li X. 2020. Molecular phylogeny and
biogeography of Triplophysa stone loaches in the Central
Chinese Mountains. Biological Journal of the Linnean
Society 130: 563–577.
Zawadzki CH, Renesto E, dos Reis RE, Moura MO,
M a t e u s R P. 2 0 0 5 . A l l o z y m e r e l a t i o n s h i p s i n
hypostomines (Teleostei: Loricariidae) from the Itaipu
Reservoir, Upper Rio Paraná basin, Brazil. Genetica 123:
271–283.
Z a w a d z k i C H , R e n e s t o E , Pe r e s M D , Pa i v a S .
2008. Allozyme variation among three populations
of the armored catfish Hypostomus regani (Ihering,
1905) (Siluriformes, Loricariidae) from the Paraná and
Paraguay River basins, Brazil. Genetics and Molecular
Biology 31: 767–771.

SUPPORTING INFORMATION
Additional Supporting Information may be found in the online version of this article at the publisher’s web-site:
Table S1. Geographic coordinates of the study area on the Laranjinha River and six of its tributaries.
Code = drainage name abbreviated and sampled section in subscript (U = Upper, M = Middle and L = Lower).
Table S2. Summary statistics for nine microsatellite loci among H. ancistroides collections. N = number of
individuals; NA = number of alleles; RA = number of rare alleles; HE = expected heterozygosity; HO = observed
heterozygosity; HW = probability values of concordance with Hardy–Weinberg expectations; FIS = computed as in
Weir & Cockerham (1984); - = only one allele; NE = not estimated. *Significant values: P < 0.05 after Bonferroni
correction.
Table S3. Distribution of 26 haplotypes and variable sites found in 723 H. ancistroides from 25 sites in the
Laranjinha Basin.
Table S4. Bottleneck tests for H. ancistroides from 25 sites. Wilcoxon sign-rank test for heterozygosity excess and
Mode-shift test for patterns of allele frequency distribution. N = number of individuals analysed, Hd = number
of loci with heterozygosity deficiency, He = number of loci with heterozygosity excess. *d = Significant values for
heterozygosity deficiency, *e = significant values for heterozygosity excess (P ≤ 0.05). Normal L-shaped distribution,
a
= Infinite Allele Model, b = Two-Phase Model (90% SSM-10% IAM), c = Stepwise Mutation Model.

© 2021 The Linnean Society of London, Biological Journal of the Linnean Society, 2021, 134, 198–213
 POPULATION STRUCTURE OF SUCKERMOUTHS 213

Table S5. Pairwise genetic differentiation estimated from microsatellites in H. ancistroides specimens. Below
diagonal = GST estimated SSR markers. Above diagonal = Ф ST estimated mtDNA marker. *Significant values
P ≤ 0.05. Each significant pairwise comparison is colour based on the range estimate: light grey: 0.001 to 0.05;
medium grey: 0.051 to 0.15; dark grey: 0.16 to 0.300; darkest grey: > 0.301.
Figure S1. Mismatch distributions for each H. ancistroides population. The x-axis indicates the number of
pairwise differences between compared haplotypes. The y-axis indicates the frequency for each value. In the
histograms, (- - -) indicates the observed frequencies of pairwise divergences among haplotypes, and (—) indicates
the expectation under the model of population expansion.
Figure S2. Relationship between genetic and geographic distance by the Mantel test with 10 000 permutations
of H. ancistroides from 25 sites in the Laranjinha Basin. A, comparing SSR-Ф ST values and geographic distance.
B, comparing SSR-Dest values and geographic distance. C, comparing mtDNA-Ф ST values and geographic distance.

Downloaded from https://academic.oup.com/biolinnean/article/134/1/198/6284360 by guest on 20 August 2023

Figure S3. Gene flow estimates based on Bayesian inferences of contemporary migration rates using BayesAss.
Only values within a 95% confidence interval are shown. Non-migrant proportion is indicated by values in
parentheses near the name of each site. Migration estimates and the direction of gene flow are represented by
arrows. Values below 5% (almost all smaller than 0.6%) are not shown.

© 2021 The Linnean Society of London, Biological Journal of the Linnean Society, 2021, 134, 198–213