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Phylogeography of the critically endangered neotropical
annual fish, Austrolebias wolterstorffi (Cyprinodontiformes:
Aplocheilidae): genetic and morphometric evidence of a new
species complex
Daiana K. Garcez &Crislaine Barbosa &Marcelo Loureiro &Matheus V. Volcan &
Daniel Loebmann &Fernando M. Quintela &Lizandra J. Robe
Received: 12 September 2017 / Accepted: 8 July 2018
#Springer Nature B.V. 2018
Abstract Austrolebias wolterstorffi is a critically en-
dangered annual fish, occurring in temporary ponds in a
restricted area of Southern Brazil and Uruguay. Here, we
evaluate the levels of genetic diversity and morphometric
differentiation presented by A. wolterstorffi, attempting
to reconstruct the spatiotemporal scenario by which this
species reached their current distribution. Part of the
mitochondrial cytochrome band nuclear rhodopsin
genes were characterized and analysed for a set of 122
and 110 specimens, respectively, collected along the
entire distribution range of the species. Additionally,
shape variations were evaluated for 92 individuals (43
males and 49 females) through geometric morphometric
methods. Our analyses demonstrated several cases of
significantly high levels of genetic differentiation among
individual populations, in an isolation-by-distance pat-
tern of divergence, with at least six different population
groups along the Patos-Mirim lagoon. These groups
differed by a minimum of 0.9% and a maximum of
2.6% of corrected cyt bnucleotide distances and did
not share any mitochondrial haplotype. Such a pattern,
added to the slight morphometric differentiation detected
for most of the groups, suggests the occurrence of incip-
ient speciation as consequence of allopatric fragmenta-
tion. The chronophylogenetic tree performed with the
concatenated dataset supported independent oriental
and occidental colonization routes, with the population
located in the northwest part of the Rio Grande do Sul
coastal plain presenting the most ancient divergence. In
general, the recovered biogeographic patterns are highly
consistent with the records of Quaternary climatic chang-
es and depositional events that have occurred along the
area inhabited by the studied species. This allowed us to
establish a molecular clock calibration system for Neo-
tropical annual fish. Thus, although the taxonomic status
of each of the detected population units needs further
study, it is clear that independent conservation strategies
must be taken in each of the major areas covered by this
study, most of which are located in Brazil.
Keywords Allopatric fragmentation .Cryptic
speciation .Genetic structure .Patos-Mirim lagoon
system
Environ Biol Fish
https://doi.org/10.1007/s10641-018-0795-2
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s10641-018-0795-2) contains
supplementary material, which is available to authorized users.
D. K. Garcez :C. Barbosa :D. Loebmann :F. M. Quintela :
L. J. Robe (*)
Programa de Pós-Graduação em Biologia de Ambientes Aquáticos
Continentais, Universidade Federal do Rio Grande, Rio Grande,
Rio Grande do Sul, Brazil
e-mail: lizbiogen@gmail.com
M. Loureiro
Sección Zoología Vertebrados, Facultad de Ciencias, Universidad
de la República, Montevideo, Uruguay
M. Loureiro
Departamento de Ictiología, Museo Nacional de Historia Natural,
Montevideo, Uruguay
M . V. Vo l c a n
Laboratório de Ictiologia, Instituto Pró-Pampa, Pelotas, Rio
Grande do Sul, Brazil
Introduction
Freshwater annual fish (Cyprinodontiformes:
Aplocheiloidei) possess uncommon developmental, eco-
logical, and physiological adaptations. They have an un-
usually short life cycle of less than one year, which is
entirely correlated to the seasonal ponds they inhabit
(Loureiro and de Sá 2015). Their non-overlapping gener-
ation times and habitats make annual fish an excellent
model for studies of evolution. Also, their patchy distribu-
tion provides ideal conditions for studying the underlying
evolutionary mechanisms (e.g., genetic drift, gene flow,
selection) that usually result in rapid divergence between
populations or even allopatric speciation with or without
morphological differentiation (de Sá et al. 2015).
Among Aplocheiloidei, Aplocheilidae is one of the
most diverse families, with about 350 described species
living in South and Central America and in the southern
United States (Costa 2008). Austrolebias currently en-
compasses 45 annual species, distributed along the
Paraná-La Plata, Amazonas and Patos-Mirim basins
(Costa 2006; Nielsen and Pillet 2015). This genus is
particularly diverse in southern Brazil and Uruguay,
especially in the Patos-Mirim lagoon system (Costa
2006). Nevertheless, several of these species are endan-
gered because of their frequently restricted and patchy
distributions, their characteristic low vagility, and the
loss and fragmentation of their habitats (ICMBio 2013;
Volcan et al. 2015).
Austrolebias wolterstorffi is one of the largest species
of annual fish and has one of the widest distribution
ranges within the genus (~50,000 km
2
), occurring in
temporary ponds from the north of the Patos Lagoon
to the south of the Mirim Lagoon, along Southern Brazil
and in Uruguay (Loureiro et al. 2015). It is considered a
critically endangered (CR) species (Reis et al. 2003;
ICMBio 2013) because of a combination of its peculiar
evolutionary properties and the loss and degradation of
its habitats. Nevertheless, despite the incipient incentive
of studies aiming to enhance the knowledge of annual
fish (ICMBio 2013), even the phylogenetic position of
A. wolterstorffi remains controversial. In this sense,
although classical studies (Costa 2006,2010)tendto
allocate this species as a member of the A. elongatus
group, a recent analysis (García et al. 2014) recovered it
as an early offshoot within its genus.
Although the relatively wider geographical distribu-
tion of A. wolterstorffi suggests it might recover more
easily than other congeners annual fish, the combination
of high levels of genetic drift, frequent bottlenecks,
inbreeding, and low levels of gene flow (de Sá et al.
2015) might have led to population differentiation or
unrecognized cryptic speciation, which could affect its
long-term persistence. Therefore, we aimed to assess the
levels of diversity and the genetic and morphometric
structure within and among populations of
A. wolterstorffi, attempting to help in the reconstruction
of its evolutionary history and in the establishment of
management and conservation strategies.
Materials and methods
Study area
This study includes molecular and/or morphometric data
from a total of 134 individuals of A. wolterstorffi collect-
ed between 2014 and 2015 in 22 sampling locations
distributed in the entire known distribution range of the
species (Loureiro et al. 2015; Volcan et al. 2015), which
comprises the Patos-Mirim lagoon system, in the south-
ernmost Brazilian state of Rio Grande do Sul and Uru-
guay (Fig. 1; Supporting Information Table 1Sand2S).
With the exception of Eldorado do Sul, all of the
sampled sites in the Rio Grande do Sul Brazilian state
are within Quaternary sedimentary deposits of the coastal
plain. This region was intensely reworked during the
paleoclimatic alternations of Quaternary, which caused
variations in sea level, thereby opening and closing areas
of communication with the Atlantic Ocean, and building
a system referred to as the Multiple Barrier (Villwock and
Tomazelli 2007). This system is formed by four major
depositional events (Barrier I–IV) that extended from
400 to 5 thousand years ago (kyr) and led to the forma-
tion of the Patos, Mirim, and Mangueira lagoons. The
locality of Eldorado do Sul is inserted in the western
portion of the Permian-Triassic sedimentary deposits,
locally named the Peripheral Depression, which is part
of the Paraná geological basin. Sampled localities in
Uruguay are distributed along the Precambrian Shield,
Paraná basin (Permian sedimentary and Mesozoic volca-
nic formation), and Quaternary sedimentary deposits
(Rocha department) (Bossi and Navarro 1988).
Samplings
The fishes were collected in temporary ponds with the
help of hand nets, euthanized with an overdose of
Environ Biol Fish
3000 mg/L of eugenol anesthetic and then fixed in 95%
ethanol. For the molecular procedures, a piece of the
caudal peduncle was dissected from a set of 83 and 39
Brazilian and Uruguayan specimens, respectively
(Table 2S). For geometric morphometry, 43 males and
49 females were photographed on their left side with a
digital câmera (Olympus VG-120, supermacro mode).
DNA manipulation
Total DNA was extracted from each individual from
approximately 30 mg of muscular tissue, using a
phenol/chloroform protocol (Sambrook et al. 1989). Ap-
proximately 800 bp of the mitochondrial cytochrome b
(cyt b) gene and 820 bp of the nuclear rhodopsin (RHO)
were amplified from each sample using the primers
L14735 (5′-AAAAACCACCGTTGTTATTCAACTA-
3′) and CB3-H (5′-GGCAAATAGGAARTATCATTC-
3′)(Palumbietal.1991;Wolfetal.1999) and Rh193
(5′-CNTATGAATAYCCTCAGTACTACC-3′)and
Rh1039r (5′-TGCTTGTTCATGCAGATGTAGA-3′)
(Chen et al. 2003), respectively. PCR reactions were
carried out using 100 ng of DNA in 25 μL reactions,
containing 1× buffer, 0.5–1μM of each primer, 0.25 mM
of each dNTP, 2.5–3mMofMgCl
2
and 1–1.5 U of Taq
DNA polymerase, with the aid of 5% Dimethyl Sulfox-
ide in the amplification of RHO. PCR conditions for cyt b
consisted of an initial stage of denaturation at 94 °C for
5min,followedby35cyclesofdenaturationat94°Cfor
45 s, annealing at 55 °C for 45 s and extension at 72 °C
for 60 s, and a final extension stage at 72 °C for 10 min;
for RHO, cycling consisted of denaturation at 94 °C for
Fig. 1 Median-joining networks of the 39 cyt bhaplotypes (a)
and of the four RHO alleles (b). The size of each circle is propor-
tional to haplotype frequencies, and colours refer to the main
population groups recovered in this study (see Results) in relation
to their geographic distribution (presented in the map)
Environ Biol Fish
30 s, annealing at 58 °C for 30 s and extension at 72 °C
for 30 s. To check whether the amplification was suc-
cessful, 5 μL of the PCR product were separated by
agarose (0.8%) gel electrophoresis and stained with
GelRed (Biotium). The amplified fragments were then
purified with a solution of 7.5 M ammonium acetate
(C
2
H
7
NO
2
) and directly sequenced. Sequencing was per-
formed in a Perkin-Elmer ABI Prism 377 Automated
Sequencer (MACROGEN, Seoul, Korea) using the same
amplification primers.
Data analysis
First, electropherograms were assembled and edited in
the Gap4 software of the Staden package (Staden 1996).
The consensus sequences thus obtained had their iden-
tity confirmed using BLASTN (NCBI website) and
were then aligned using the ClustalW algorithm, as
implemented in Mega 6 software (Tamura et al. 2013).
Each polymorphic site encountered along this alignment
was individually checked and manually corrected, if
necessary. For RHO, heterozygous sites were coded
according to the nucleotide degeneracy/redundancy ta-
ble and after unphased in DnaSP 5.10 software (Librado
and Rozas 2009). This software was also employed to
measure the minimum number of recombination events
that best explains the diploid dataset.
The DnaSP 5.10 software was used to calculate the
levels of genetic diversity within populations or popula-
tion groups individually for each gene, as estimated from
the average number of different haplotypes (H) or alleles
(A) (Nei 1987), the average number of nucleotide differ-
ences between haplotypes or alleles (k) (Tajima 1983),
the haplotype diversity (Hd) or expected heterozigosity
(He) (Nei 1987), and the nucleotide (π) diversities (Nei
1987). The neutrality tests Tajima’s(1989), and Fu’s
(1997) were performed in Arlequin 3.5 (Excoffier and
Lischer 2010), with significance measured through
10,000 random permutations for both genes.
The relationships between haplotypes or alleles were
inferred for both genes individually from networks gen-
erated by median-joining in the Network v.4.510 soft-
ware (Bandelt et al. 1999). The levels of genetic differ-
entiation among populations were measured for cyt b
and RHO by the fixation index (FST) in Arlequin 3.5
using pairwise differences with 10,000 random permu-
tations. A Mantel test was finally performed using these
measures to check the correlation between the genetic
(FST) and geographic distances.
For the concatenated dataset, different hypotheses of
population groupings were assessed through spatial
analysis of molecular variance (SAMOVA)
(Dupanloup et al. 2002), in order to define the number
and structure of the groups that are geographically ho-
mogeneous and maximally differentiated from each oth-
er. In this case, different hypotheses were evaluated
through hierarchical analysis of molecular variance
(AMOVA), as performed in Arlequin, with 10,000 per-
mutations. The population structure and the most likely
number of clusters of individuals were also assessed
using the Bayesian Population Structure analysis per-
formed in BAPS 6 (Corander et al. 2008).
The supermatrix phylogenetic tree was reconstructed
using Bayesian analysis and maximum likelihood esti-
mates in MrBayes 3.2.6 (Ronquist et al. 2012)and
RaxMLHPC (Stamatakis 2014), respectively, under
the partitioning scheme and substitution models sug-
gested by PartitionFinder 1.1.1 (Lanfear et al. 2012).
The Markov Chain Monte Carlo (MCMC) of the BA
was run for 10,000,000 generations, sampling trees
every 1000 generations, and burning 25% of the initial
results. The ML search was performed under the new
rapid hill-climbing algorithm, with 1000 bootstrap rep-
licates. These analyses were performed without the use
of outgroups, and the phylogenetic trees were further
visualized and edited in FigTree 1.4.3 (Rambaut and
Drummond 2009) as radial phylograms.
Additionally, a chronophylogenetic tree was recon-
structed under a Bayesian approach (BA) in Beast 1.5.4
(Drummond and Rambaut 2007), using cyt band RHO
sequences with unlinked substitution and clock models.
In this case, an uncorrelated lognormal relaxed molecu-
lar clock analysis was performed with a mean rate of 2%
per million years (SD = 0.5%) for cyt b,asadjustedfor
ectotherms mitochondrial genomes (Brown et al. 1979).
This prior was further complemented by the dating of
325 kyr (SD = 12.5 kyr) to the major split between
oriental and occidental lineages, as recovered in the first
Bayesian search conducted with MrBayes (see results).
The analysis encompassed two independent runs of
20,000,000 generations, with trees and parameters sam-
pled every 2000 iterations, and a burn-in of 25%. Re-
sults of each run were visualized in Tracer 1.7 (Rambaut
et al. 2018) to ensure that stationarity was achieved and
that convergence was reached. Posterior probabilities
and the maximum credibility tree were inferred using
TreeAnnotator 1.5.4 (Drummond and Rambaut 2007)
and further visualized and edited in FigTree 1.4.3.
Environ Biol Fish
Finally, to estimate the effective number of females
of each population group, a Bayesian analysis based on
coalescence was implemented in LAMARC 2.1.10
(Kuhner 2006) under the GTR nucleotide substitution
model, with effective population sizes set to 1 and 4, and
relative mutation rates set 1 and 0.5 for the mitochon-
drial and nuclear partitions, respectively. Runs consisted
of four simultaneous searches, each with 100 initial and
four final chains, with a minimum of 1000 and 10,000
recorded parameter sets, respectively, and sampling ev-
ery 20 generations after a burn-in of 1000 genealogies.
Only population groups with more than 10 sampled
individuals were used in this analysis.
Morphometry analysis
A set of 14 landmarks were defined and digitalized
using TpsDig2 software ver. 2.26 (Rohlf 2016)
(Supporting Information Fig. 1S). The coordinates were
aligned using a Generalized Procrustes Analysis (GPA),
where non-shape variability was removed after mini-
mizing the differences in translation, scaling and rota-
tion. Due to sexual dimorphism, the analyses were per-
formed independently for males and females.
Shape variations among individuals of the different
groups were first evaluated through an exploratory Prin-
cipal Component Analysis (PCA), followed by
MANOVA and pairwise MANOVA. Afterwards, a Ca-
nonical Variate Analysis (CVA) was employed to find
the linear combination of predictors that best discrimi-
nates among groups. Cross validation test was used to
measure the accuracy of correct classifications. These
analyses were permormed in R (R Core Team 2017),
using Geomorph (Adams and Otarola-Castillo 2013)
and Vegan packages (Oksanen et al. 2017).
Results
In this study, sequences spanning 798 bp of the mito-
chondrial cyt bgene and 821 bp of the nuclear RHO
gene were characterized, respectively, for 122 and 110
individuals of A. wolterstorffi collected at 22 different
localities (Fig. 1) (Supporting Information Tables 1S
and 2S). The intraspecific matrix of cyt bencompassed
39 different haplotypes presenting 65 variable sites, of
which 48 were parsimoniously informative. For RHO,
only four alleles were detected, and variability was
restricted to three sites.
Molecular analyses
Cyt b dataset
Concerning cyt bdiversity estimates, the number of
haplotypes per sampling locality ranged from 1 (Sal,
33-INIA, 33-Pas, SJN-4, Buj, Pel-S, PN-RG and TAIM)
to 6 (Tav and IL-RG), with minimum Hd and πof 0
(Sal, 33-INIA, 33-Pas, SJN-4, Buj, Pel-S, PN-RG and
TAIM), and maximum Hd and πvalues of 0.889 (IL-
RG) and 0.006 (SJN-3), respectively (Table 3S). None
locality presented significant deviations from neutrality
(Table 3S). A total of 111 cases of significant genetic
structure were detected in the pairwise comparisons
between populations, and these Fst values varied from
0.20 (in the comparison between IL-RG and Pel-P) to
1.00 (in the comparison between SJN-4 and Sal, 33-
INIA, Buj and between Buj and 33-INIA) (Table 4S).
The Mantel test indicated a significant correlation be-
tween genetic and geographic distances (r= 0.52;
p<0.000).
The haplotype network reconstructed with cyt bse-
quences revealed the presence of six independent
haplogroups (Fig. 1a): (1) Eldorado, with haplotypes
sampled at the municipality of Eldorado do Sul, RS,
Brazil; (2) Pelotas, clustering haplotypes sampled in
seven localities of Pelotas and Rio Grande, RS, Brazil;
(3) Uruguay, grouping together the haplotypes sampled
in the nine localities of Uruguay; (4) Tavares, with
haplotypes sampled at Tavares, RS, Brazil; (5) São José
do Norte (SJN), with haplotypes sampled at
Cachoeirinha, RS, Brazil and in three localities of SJN,
RS, Brazil; and (6) São José do Norte 2, with haplotypes
sampled at one locality of SJN, where haplogroup 5 was
also collected. No haplotype was shared between these
haplogroups, and a star-like pattern of ramification
could be evidenced for Uruguay (Fig. 1a).
RHO dataset
The pattern detected for RHO is less informative, since
this marker revealed to be highly conserved in
A. wolterstorffi. In this case, more than a single allele
was detected only for SJN-3, SJN-4, Buj, Tav, Pel-P,
PN-RG, IL-RG, IL2-RG and MP-RG, and the higher
values of He and πwere 0.644 (SJN-4) and 0.0009
(SJN-4), respectively (Table 5S). None locality present-
ed significant deviations from neutrality (Table 5S), and
significant Fst values were restricted to the comparisons
Environ Biol Fish
involving some Uruguayan and Brazilian localities
(Table 6S), which resulted in a significant correlation
between genetic and geographic distances in the Mantel
Tes t (r=0.20;p< 0.018). Even so, in the Network, none
clear subdivision could be detected, and a starlike pat-
tern was recovered for the species considered as a whole
(Fig. 1b).
Concatenated dataset
Despite the differences in variability levels, as cyt band
RHO presented similar divergence patterns in the target
species, they were jointly considered in the remaining
analyses. The population structure suggested by
SAMOVA for the supermatrix dataset lead to a plateau
in FCT values when k was set to six, which subdivided
A. wolterstorffi into the same population groups previ-
ously recovered in the cyt bNetwork: Pelotas, Uruguay,
Tavares, São José do Norte, Eldorado and São José do
Norte 2. In fact, the AMOVA performed with this struc-
ture revealed that the six groups of populations were
able to explain 75.95% of the variation encountered in
the concatenated dataset (Table 1). Bayesian clustering
approaches implemented through the spatial BAPS
model also supported the same structure.
Accordingly, the topology of the radial phylogram
also recovered a similar structure, and all the population
groups were recovered as reciprocally monophyletic in
both BA and ML trees, with support values higher than
0.9 and 63, respectively (Fig. 2). These lineages grouped
into two main clusters encompassing populations
inhabiting drainages at the western (population groups
of Pelotas, Uruguay, and Eldorado) and eastern
(Tavares, São José do Norte and São José do Norte 2)
margins of the Patos Lagoon. After setting this diver-
gence to a time prior of 325 kyr (SD = 12.5 kyr) in a
chronophylogenetic analysis, diversification of these
clades was dated to approximately 222 and 186 kyr,
respectively (Fig. 3). In this case, the Eldorado and
Tavares population groups seem to have encompassed
the most ancient divergences, respectively, although
support for this position was tenuous, especially for
the last. Diversifications of each of the six population
groups were set between 104 and 7 kyr (Fig. 3).
Morphometric analyses
Applying the subdivision in six population groups, sig-
nificant differences were found in the shape patterns for
both, males and females (p< 0.01). Among females,
most pairwise comparisons involving the population
groups of Eldorado and Uruguay evidenced significant
differentiation, although for males such a result was
restricted to two comparisons involving Uruguayan
specimens (Table 7S). In this sense, the PCA for males
and females also suggested the existence of some dif-
ferences between groups, especially in regard to the
insertion of the caudal fin and to the position of the
dorsal fin, respectively (Fig. 4a, b). For males, the PC1
explained 29.5% of shape variation, whereas PC2 ex-
plained 27.1%. For females, PC1 and PC2 explained 22
and 19.2% of shape variation, respectively. Neverthe-
less, when a CVA was used with the four population
groups that had at least six individuals sampled, mor-
phological differentiation between the different popula-
tion groups became more evident (Fig. 4c, d). In this
case, the overall classification accuracy reached 59.5
and 60.9% for males and females, respectively.
Diversity and differentiation evaluations in face
of the new subdivision
As the subdivision among six population groups
was consistently defined by the network of cyt b,
and by SAMOVA, spatial BAPS, and the phyloge-
netic analyses of the concatenated dataset, this
Tabl e 1 Analysis ofmolecular variance (AMOVA) performed with the concatenated dataset, following the subdivision proposed by spatial
analysis of molecular variance (SAMOVA) with k set to six
Source of variation Degrees of freedom Sum of squares Variance components Percentage of variation
Among groups 5 879.955 4.47445 Va 75.95
Among populations, within groups 16 77.258 0.45024 Vb 7.64
Within populations 222 214.558 0.96648 Vc 16.41
Total 243 1171.770 5.89117
Environ Biol Fish
structure was further employed in new neutrality,
diversity, and genetic differentiation tests. In gen-
eral, at least two haplotypes were sampled for cyt
bin each of the six population groups, which
always presented haplotype diversity (Hd) values
higher than 0.60 for this marker (Table 2). For
rhodopsin, the population groups of Eldorado,
SJN2 and Uruguay were fixed for a single allele,
and the highest value of He was 0.54 for Tavares
(Table 2). Conversely, the nucleotide diversity
values (π) were generally moderate to low, ranging
from 0.010 to 0.058 and 0.0004 to 0.0007 for cyt
band RHO, respectively. Regarding the neutrality
tests, significant negative results were only obtain-
ed by cyt bfor the populations of Pelotas and
Uruguay (Table 2).
When testing genetic differentiation levels for cyt b,
most comparisons between population groups presented
significant genetic differences (Table 3,belowthe
diagonal). The sole comparison that did not present a
significant FST value was between Eldorado and SJN 2,
which is probably an outcome of reduced sampling size.
In all the significant comparisons, FST values between
groups were higher than 0.7, indicating the presence of
high levels of genetic differentiation (Table 3, below the
diagonal). For RHO, significant genetic differences
were only detected between the Uruguayan and Brazil-
ian groups (Table 4, below the diagonal). The net
Tamura 3-parameters distances also supported such a
pattern, and the six lineages differed by a minimum of
0.9% (as seen between Tavares and SJN) and a maxi-
mum of 2.6% (as seen between groups Pelotas and SJN
2) for cyt b(Table 3, above the diagonal). Nevertheless,
net distances between groups were zero when only RHO
sequences were analysed (Table 4, above the diagonal).
Population parameters inferred for the concatenated
dataset in LAMARC revealed theta values ~12.4-fold
higher for Pelotas than for SJN, whereas the populations
of Uruguay and Tavares presented similar intermediate
theta values (Table 5). Even so, Bayesian analysis re-
covered effective numbers larger than 11,000 for all
populations (Table 5).
Discussion
It is generally assumed that annual fish are subject to the
concomitant action of a set of environmental, demo-
graphic, and metabolic conditions commonly associated
with high evolutionary rates (Whitlock 2000;Loureiro
2004), which can lead to a great variability among
populations. In agreement with this, our analyses have
shown that, in addition to several cases of significantly
high differentiation levels between individual popula-
tions, in an isolation-by-distance divergence pattern,
A. wolterstorffi is subdivided into at least six different
groups of populations. Such a structure was evidenced
by the Network of cyt b, and by SAMOVA, BAPS, and
the phylogenetic analyses of the concatenated dataset,
and further supported by shape variation. So, despite the
differences in evolutionary rates presented by the mito-
chondrial and nuclear markers, which can be attributed
to distinct mutation rates, modes of inheritance and
population sizes (Avise 2004; Templeton 2006), this
subdivision was able to explain more than 75% of the
variation detected for this species in both of the sampled
Fig. 2 Radial phylogram of the
concatenated sequences, with
internal node labels representing
support values, given by posterior
probabilities and bootstrap values
in BA and ML evaluations,
respectively. Branch lengths are
proportional to the number of
substitutions per site, and colours
refer to population groups (see
Fig. 1)
Environ Biol Fish
loci. Moreover, cross validation tests performed with
discriminant CVA resulted in overall classifications ac-
curacies ranging 60%, evidencing an incipient morpho-
metric differentiation between at least some of the eval-
uated groups. This suggests that vicariance has played
an important role in the diversification of
A. wolterstorffi, as also reported for several other
Aplocheilidae species (Jowers et al. 2008;Garcíaetal.
2009,2012,2015; Bartáková et al. 2013; Ponce de León
et al. 2014;Loureiroetal.2015).
Although allopatric fragmentation seems to be an
ongoing process within A. wolterstorffi, some level of
gene flow seems to occur, especially at short distances,
encompassing mainly populations located within each
of the population groups. In fact, all FST values recov-
ered in pairwise comparisons involving the six groups
were significant and high, and they differed by a mini-
mum of 0.9% corrected cyt bdistances (with a maxi-
mum of 2.6%). These results, added by the shape diver-
gence patterns and the evidence of recent diversification
(see below), lead us to conclude that at least some of
these groups may constitute incipient species. In fact,
distances as small as 1.4% were previously reported for
cyt bbetween different species of Austrolebias (García
Fig. 3 Bayesian
chronophylogenetic tree based on
cyt band RHO sequences
sampled for A. wolterstorffi.
Values above internal branches
represent support values, given by
posterior probabilities values in
BA analysis. Values below nodes
indicate divergence time
estimates, with the highest
posterior density (HPD) interval
containing 95% of the sampled
values presented within brackets
for some of the detached clades.
Groups of populations were
represented by their respective
names and colours (see Fig. 1).
This tree was rooted with cyt b
and RHO sequences of
A. nigrofasciatus,A. minuano,
and A. adloffi
Environ Biol Fish
Fig. 4 Plots of Principal Component Analysis (PCA) (aand b)
and Canonical Variate Analysis (cand d) showing the distribution
of variation in A. wolterstorffi morphometric patterns: (aand c)
males, (b and d) females. For the PCA plots, extreme PC values are
represented by fishes’warped drawings on grid deformations.
Colours refer to the different population groups (see Fig. 1)
Tabl e 2 Genetic diversity estimates and neutrality tests of each of the six population clusters suggested in this study (see Results) for
A. wolterstorffi
Cyt bRHO
Groups N H Hd πNeutrality tests N A He πNeutrality tests
Taj ima ’s
D
Fu’sF Tajima’s
D
Fu’sF
Pelotas 40 15 0.888 ± 0.0321 0.058 ± 0.033 −1.5353 −3.584 70 2 0.358 ± 0.055 0.0004 ± 0.0001 0.89601 1.417
Uruguay 39 9 0.789± 0.046 0.019 ± 0.014 −1.3892 −3.526 72 1 0 0 NC NC
Tavares 17 6 0.713 ± 0.108 0.032 ± 0.023 0.4712 −0.128 28 3 0.542 ± 0.086 0.0007 ± 0.0001 0.38476 0.412
SJN 20 5 0.600 ± 0.101 0.016± 0.013 −0.1381 −0.933 40 4 0.477 ± 0.082 0.0006 ± 0.0001 −0.56317 −0.886
Eldorado 3 2 0.677 ± 0.314 0.020 ± 0.021 NC 1.061 6 1 0 0 NC NC
SJN 2 3 2 0.667 ± 0.314 0.010 ± 0.013 NC 0.201 4 1 0 0 NC NC
Total 122 39 0.951 ± 0.008 0.149 ± 0.076 −0.6296 −6.667 220 4 0.299 ± 0.037 0.00038 ± 0.00005 −0.6059 −1.126
N, the number of sequences; H, number of haplotypes; A, number of alleles; Hd, haplotype (gene) diversity; He, expected heterozygosity; π,
nucleotide diversity (per site); Tajima’s D and Fu’s Fs, neutrality tests. The values in bold indicate significant measures (P<0.05)
Environ Biol Fish
et al. 2000), with even lower distance ranges (0 to 1.8%)
being recently reported within the A. bellottii species
complex (García et al. 2015). Considering that DNA-
based approaches previously unravelled several unrec-
ognized lineages of Neotropical annual fishes (Costa
and Amorim 2011;Costa2013; Costa et al. 2014,
2016,2017), we argue here that A. wolterstorffi may in
fact constitute a species complex.Nevertheless, it is
likely that this complex presents a continuum of differ-
entiation, in which at least some of the lineages might
have yet reached a speciation endpoint, given by estab-
lishment of complete reproductive isolation.
Independent of the taxonomic status assigned to each
of the six evolutionary units detected here, it is impor-
tant that independent conservation strategies are applied
to each these major sampling areas, four of which are
located in Brazil. Although the levels of genetic diver-
sity encountered for A. wolterstorffi as a whole (or for
each of the individual population groups) were relative-
ly high, this is likely explained by small-scale events of
gene flow within population groups or even by the
putative higher mutational rates previously assigned to
mitochondrial genes in Austrolebias (García et al.
2015). In fact, similar levels of intrapopulation diversity
were previously reported for several other species of
annual fishes inhabiting different regions (García et al.
2000,2015; Bartáková et al. 2013).
Nevertheless, the fact that each of these areas em-
braces an independent genetic stock calls attention to the
need for rapid interventions, principally in the face of
the rapid fragmentation and degradation of wetlands
within the Brazilian territory (Volcan et al. 2015). This
situation might decrease effective population sizes and
gene flow, enhancing the action of random genetic drift
and inbreeding and increasing the risk of mutational
meltdown and inbreeding depression, respectively
(Frankham et al. 2013). Likewise, the periodic flooding
of rivers and lagoons within the distribution range of
A. wolterstorffi might also threaten the persistence of
each of these independent evolutionary units. In this
sense, signs of population admixture were encountered
for the population of São José do Norte, which presents
two different haplogroups differing by a minimum of 11
mutational steps and assigned to different population
groups. This result led us to hypothesize that the SJN2
individuals might represent immigrants from a distinct
unknown population. Secondary colonisations might
also be invoked to explain the presence of individuals
Tabl e 3 Pairwise fixation indices (FST) (below the diagonal) and net Tamura 3-parameters distances (above the diagonal) of cyt bbetween
the six population groups suggested in this study for A. wolterstorffi
Groups Pelotas Uruguay Tavares SJN Eldorado SJN 2
Pelotas 0 0.013 0.019 0.019 0.015 0.026
Uruguay 0.74075* 0 0.017 0.016 0.013 0.020
Tavares 0.77765* 0.87547* 0 0.009 0.018 0.017
SJN 0.81055* 0.90051* 0.75729* 0 0.017 0.014
Eldorado 0.70103* 0.86887* 0.83745* 0.91507* 0 0.023
SJN 2 0.82251* 0.92041* 0.83515* 0.90392* 0.9434 0
The asterisks indicate significant differences (P<0.05)
Tabl e 4 Pairwise fixation indices (FST) (below the diagonal) and net Tamura 3-parameters distances (above the diagonal) for rhodopsin
between the six population groups suggested in this study for A. wolterstorffi
Groups Pelotas Uruguay Tavares SJN Eldorado SJN 2
Pelotas 0 0 0 0 0
Uruguay 0.22010* 0 0 0 0
Tavares 0.05240 0.26234* 0 0 0
SJN −0.00574 0.18247* 0.00455 0 0
Eldorado 0.06203 0 0.02251 0.00122 0
SJN 2 0.01984 0 −0.02711 −0.04696 0
The asterisks indicate significant differences (P<0.05)
Environ Biol Fish
from the SJN haplogroup in Cachoeirinha, a municipal-
ity located northwest of the Patos Lagoon. So, although
current estimates of the effective population sizes do not
suggest an incipient risk of extinction for any of the
sampled population groups (see Frankham et al. 2004),
and none of the populations presented significant signs
of genetic bottlenecks, this could changerapidly and it is
important that interventions are performed before the
evolutionary potential of the populations is lost.
Finally, regarding the spatio-temporal evolutionary
scenario, it is possible to infer that the six groups of
populations encompass two distinct lineages, whose
distribution coincides with the eastern and western mar-
gins of the Patos Lagoon, and suggest a north-south
colonization route. Using the onset of the formation of
the Patos Lagoon, related to the occurrence of the sec-
ond Pleistocene Lagoon Barrier Depositional System
and dated to approximately 325 kyr (Villwock and
Tom azel li 2007) as prior to the divergence time between
Oriental and Occidental lineages, together with the
mean mitochondrial ectothermic rate of 2% per million
years (Brown et al. 1979; Avise 1994), it was possible to
infer that the first divergence within these two clades
occurred around 222 (western) and 186 (eastern) kyr,
compatible with range expansions enabled by the sec-
ond Pleistocene Lagoon Barrier Depositional System
related to the paleogeographical evolution of the South
American Coastal Plain (Montaña and Bossi 1995;
Tomazelli and Villwock 2005; Villwock and Tomazelli
2007). Interestingly, for the western clade, the lineage
with the further north distribution encompasses the early
offshoot, which is also in agreement with the paleogeo-
graphic history of the region (Villwock and Tomazelli
2007). Furthermore, the diversification of each of the six
population groups seem to have occurred around 104
kyr (Pelotas population group) and 7 kyr (SJN2), and it
is tempting to speculate that at least some of these events
were related to the third or fourth Lagoon Barrier De-
positional Systems, dated to approximate 120 kyr and
18 kyr to near the present, respectively (Villwock and
Tom azel li 2007). The Uruguayan population group, in
particular, inhabits a geologically older formation,
which seems to have been recently colonized (about
63 kyr). In agreement with this, this lineage presents
significant signs of population expansion, as given by
the Fu’s FS test and by the star-like network pattern
recovered with the cyt bdataset, and by the lower
diversity values revealed by RHO. More subtle signals
of demographic or spatial expansions were also detected
for the species complex taken as a whole (as given by
the star-like pattern recovered with RHO), and for the
Pelotas population group (as given the Tajima’s D test
performed with cyt b).
The well-registered paleogeoclimatic history of the
South American Coastal Plain (Montaña and Bossi
1995; Tomazelli and Villwock 2000,2005; Villwock
and Tomazelli 2007) and the clear vicariance between
Oriental and Occidental lineages of A. wolterstorffi also
allowed us to use this divergence to calibrate our relaxed
molecular clock analysis and to establish a new
calibration system to the group. Until now, the most
widely used method to date divergence events in the
group employed the mean substitution rate for
mitochondrial genes that was established by Brown
et al. (1979) and adjusted by Avise (1994)for
ectothermal species taken as a whole (García et al.
2012,2014,2015). As molecular clock substitution
rates need to be calibrated for each gene in each studied
lineage, the use of this molecular clock calibration sys-
tem will allow the estimation of substitution rates for
several other genes within the genus, enabling refine-
ment of several questions related to the spatio-temporal
evolutionary history of this and several other species of
annual fish.
So, although this study clearly helped to enhance
knowledge about the evolution of A. wolterstorffi,
evidencing some hidden taxonomic and conservation
problems, it should be followed by additional studies
and initiatives aimed at the conservation of annual fish,
which encompass one of the most threatened vertebrate
Table 5 Bayesian posterior distribution of population genetic
parameters inferred for the six population groups of
A. wolterstorffi with cyt band rhodopsin
MPE 90% CI
Theta
Pelotas 0.0087 0.0001–0.0108
Uruguay 0.0020 0.0001–0.0032
Tavares 0.0011 0.0006–0.0016
SJN 0.0007 0.0003–0.0012
Nef
Pelotas 143,279 977–177,787
Uruguay 32,623 215–53,066
Tavares 18,311 9869–25,541
SJN 11,230 5344–20,197
MPE, most likely estimate; Nef, effective number of females; CI,
confidence interval
Environ Biol Fish
groups in Brazil (ICMBio 2013; Volcan et al. 2015). It is
important that the knowledge generated here be used to
help in the promotion and implementation of environ-
mental education programs and to encourage the crea-
tion of protected areas. Only the adoption of a whole set
of actions can help in the conservation of this fascinating
and biologically unique group of species.
Acknowledgements Thisstudy was funded by Fundação Grupo
Boticário de Proteção à Natureza as part of the BPeixes Anuais do
Pampa^and the BPadrões micro e macroevolutivos em peixes
anuais de Cynopoecilus eAustrolebias (Cyprinodontiformes:
Rivulidae) ao longo do Sistema de Drenagens Patos-Mirim: um
enfoque comparativo com aplicações para a conservação –
1090_20171^projects. We thank CAPES and CNPq for providing
fellowships to DKG and CB, respectively. All the collections were
authorized by the Brazilian Ministério do Meio Ambiente (MMA),
in the form of the Sistema de Autorização e Informação em
Biodiversidade (SISBIO) (process number 55651-1). This study
was also approved by the Ethics Comittee on Animal Use of the
Universidade Federal do Rio Grande (process number
23116.008163/2015-23).
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