RESEARCH PAPER

Diversity of the Astyanax scabripinnis species complex(Teleostei: Characidae) in the Atlantic Forest, Brazil:species limits and evolutionary inferences

Jonathan Pena Castro • Mauricio Osvaldo Moura • Orlando Moreira-Filho •

Oscar Akio Shibatta • Mateus Henrique Santos • Viviane Nogaroto •

Marcelo Ricardo Vicari • Mara Cristina de Almeida • Roberto Ferreira Artoni

Received: 22 April 2014 / Accepted: 4 November 2014

� Springer International Publishing Switzerland 2014

Abstract The Astyanax scabripinnis species com-

plex with its wide geographical distribution is an

excellent model for evolutionary studies. Populations

are usually geographically isolated but also, in some

cases, occur in sympatry. In this study, five allopatric

and/or sympatric populations of A. scabripinnis were

analysed using geometric morphometry, cytogenetic

markers, assays for induced breeding and phyloge-

netic inferences to draw conclusions on species limits

and speciation processes in a natural setting. The

morphometry of individuals indicated that the popu-

lations were well differentiated from each other.

Cytogenetic evidence revealed a more conserved

karyotypic macrostructure; however, molecular cyto-

genetic results obtained by in situ hybridization

indicated 5S and 18S rDNA gene probe locations

specific to each population. The reproduction tests for

three locations suggest isolation between populations

and the phylogenetic analyses suggest that the fish

evaluated cluster in a monophyletic group. The

combined data indicate that individuals are adapted

to different environments in a complex evolutionary

scenario, with linkage of populations during a recent

geological period. However, due to reproductive

isolation, the populations are evolving independently,

reinforcing the existence of distinct cryptic species.

Keywords Fish � Biodiversity � Climate variability �Evolutionary analysis � Geometric morphometry �Cytogenetic

Introduction

The Atlantic Forest, in the Neotropical region, has a

high diversity of habitats and is considered one of the

J. P. Castro � M. H. Santos � V. Nogaroto �M. R. Vicari � M. C. de Almeida � R. F. Artoni (&)

Programa de Pos Graduacao em Biologia Evolutiva,

Departamento de Biologia Estrutural, Molecular e

Genetica, Universidade Estadual de Ponta Grossa,

Avenida Carlos Cavalcanti 4748, Ponta Grossa,

PR 84030-900, Brazil

e-mail: rfartoni@gmail.com

M. O. Moura

Departamento de Zoologia, Centro Politecnico,

Universidade Federal do Parana, Avenida Coronel

Francisco Heraclito dos Santos, 210, Jardim das

Americas, Curitiba, PR 81531-980, Brazil

O. Moreira-Filho

Departamento de Genetica e Evolucao, Universidade

Federal de Sao Carlos, Rodovia Washington Luis, Km

235, Monjolinho, Sao Carlos, SP 13565-905, Brazil

O. A. Shibatta

Departamento de Biologia Animal e Vegetal, Centro de

Ciencias Biologicas, Universidade Estadual de Londrina,

Rodovia Celso Garcia Cid, Campus Universitario,

Londrina, PR 86051-970, Brazil

123

Rev Fish Biol Fisheries

DOI 10.1007/s11160-014-9377-3

most important areas of vegetation in Brazil. The

region has a high percentage of endemic fish species

because of the large number of independent coastal

drainages (Menezes et al. 2007). Approximately 70 %

of the Atlantic Forest freshwater fish can be consid-

ered unique to this biome. The high rate of speciation

and the high degree of geographical endemism are

important factors that must be considered in preser-

vation policies for this habitat (Abilhoa et al. 2011).

Identification at the species level in natural popula-

tions is crucial for evolutionary, biogeographical and

ecological analyses (Agapow et al. 2004), especially

when taxa are problematic on their taxonomy.

The biological species concept considers reproduc-

tively isolated individuals capable of interbreeding as

the same species. Based on this approach, the appli-

cation of different methodologies for interpreting

diversity is necessary because no single method is

capable of providing sufficient data to define species in

the ontological sense (Marshall et al. 2006). Therefore,

in this study, we used several different approaches

(geometric morphometry, molecular cytogenetics,

reproductive data and phylogenetic analysis) to eval-

uate five sympatric and/or allopatric populations of

Astyanax scabripinnis Jenyns (Teleostei: Characidae)

species complex in the Atlantic Forest. Our goal was

to analyse the species limits for these populations,

applying the biological species concept.

Astyanax Baird and Girard 1854 is one of the most

abundant fish taxa in South America, and is distributed

in almost all watercourses in the Neotropical region

(Gery 1977; Lima et al. 2003), comprising 154 valid

species (Eschmeyer 2014). These fishes are consid-

ered a ‘‘species complex’’ that serves as a model for

evolutionary studies because of their morphological

and chromosomal variability, as well as their wide

geographical distribution (Moreira-Filho and Bertollo

1991). Their populations are restricted to small

streams or headwaters of small tributaries (Britski

1972) and are known, among other characteristics, for

the presence of supernumerary or B chromosomes in

21 populations, some of which have been isolated for

millions of years in different watersheds, separated by

hundreds of miles (Moreira-Filho et al. 2004).

According to Shibatta and Artoni (2005), population

isolation due to vicariance is the principal factor

promoting speciation in Astyanax.

The fish populations analysed here cannot be

distinguished morphologically by classical methods,

and therefore require the use of other methods to

assess genetic and morphological variability. Here, we

provide information that supports the conservation of

Astyanax species in the Atlantic Forest (Ryder 1986;

Povh et al. 2008); it sheds light on evolutionary trends

in this taxon and highlights the need for a variety of

tools for species identification.

Material and methods

Characterization of the study subject

A total of 232 Astyanax scabripinnis Jenyns

(1842) specimens from four locations were sampled

in September 2010 (Table 1). Specimens were iden-

tified according to Melo (2001) and Bertaco and

Lucena (2006). All accessed locations (Fig. 1) are part

of the Atlantic Forest in southern Brazil (Roma 2007).

The Ribeirao Grande River (RGA, RGB) is located

on the plateau of the Serra da Mantiqueira, a moun-

tainous region comprised of crystalline rocks. It

originates in the city of Pindamonhangaba at an

altitude of approximately 1,940 m. With several

waterfalls, it descends steeply to a height of 650 m,

crosses a plain, and empties into the Paraıba do Sul

River, at an altitude of approximately 400 m. The

Corrego das Pedras (CP) has its sources in the

municipality of Campos do Jordao at an approximate

altitude of 1,590 m and belongs to the Sapucaı river

basin. The Corrego Tatupeba (CT) is located in the

city of Maringa, Parana and originates at an altitude of

approximately 400 m. It is a tributary of the Ivaı River

that flows into the upper basin of the Parana River.

Geometric morphometry

Images of each individual were obtained using a

digital camera (Canon PowerShot A495; USA, Mel-

ville, New York) with 10 megapixel resolution and a

standard focal length of 30 cm. The software TpsUtil

1.46 (Rohlf 2010b) was used for grouping and

formatting of data (file extension *.tps). Seventeen

anatomical landmarks were selected along the body,

representing its general shape.

The tpsDig 2.16 software (Rohlf 2010a) was used to

scan the anatomical landmarks. To determine marking

errors, the process was repeated three times and

analysed by ANOVA (Hammer et al. 2001).

Rev Fish Biol Fisheries

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Afterwards, Procrustes superimposition was per-

formed to eliminate variations in position, scale, and

orientation (Klingenberg 2002) using the Morpho J

1.02j software (Klingenberg 2011). This method

superimposes all individuals, adjusting and centering

each configuration between homologous landmarks,

thereby generating a reference configuration. This

detects deformations generated by differences in

relation to the position of the anatomical landmarks

caused by variations in morphology (termed partial

deformations Bookstein 1991).

Discriminant function analysis (DFA) was per-

formed with the software Morpho J (Klingenberg

2011). This analysis examined the separation of two

observational groups (male and female). These a priori

groups were confirmed by gonad examination. This

analysis is most useful for comparisons of specific

groups, whereas Canonical Variance Analysis is more

suitable for the general analysis of group structure of

populations.

Differences in body shape among samples were

determined by analysing the canonical variables

associated with Multivariate Analysis of Variance/

Canonical Variance Analysis (MANOVA/CVA)

(Klingenberg, Barluenga and Meyer 2002), where

the response variable represented partial deformations

and the classifying variable represented the population

of origin. Hotelling analysis was also performed to

verify gender dimorphism (Hotelling 1931). All

morphometric analyses were performed in Morpho J

1.02j (Klingenberg 2011) and Past 2.10 (Hammer et al.

2001).

Cytogenetics

The idiogram of each sample was constructed based

on standard karyotypes described by Salvador and

Moreira-Filho (1992), Neo et al. (2000) and Fernandes

and Martins-Santos (2005) using the software Easy

Idio 1.0 (Diniz and Melo 2006). The 5S rDNA and 18S

rDNA site locations in the idiogram were determined

according to fluorescence in situ hybridization (FISH)

in mitotic chromosomes. Mitotic chromosomes were

obtained using the technique described by Bertollo

et al. (1978) and C-banding was performed according

to Sumner (1972), which allowed the verification of B

chromosomes in the karyotype.

For the identification of 5S and 18S rDNA regions

in the double FISH, a labelled probe was used with the

primers NS1 50-GTAGT CATATGCTTGTCTC-30

and NS8 50-TCCGCAGGTTC ACCTACGGA-30

(White et al. 1990). For the 18S and 5S probes, A 50-TACGCCCGATCTCGTCCGATC-30 and B 50-GCTGGTATGGCCGTAGC-30 (Martins and Galetti

1999) primers were used. The used 5S and 18S probes

have been previously submitted to Blast and the

sequences confirmed as the respective probes in

previous work with A. scabripinnis (Vicari et al.

2011). The 18S probe amplified by PCR was marked

with a Biotin Nick Translation Kit (Roche Applied

Science, Germany, Penzberg) and the amplified 5S

probe was marked with a Dig Nick Translation Kit

(Roche Applied Science) following the manufac-

turer’s instructions.

Hybridization was performed under high stringency

conditions (2.5 ng/lL probe, 50 % formamide,

2XSSC, 10 % dextran sulphate) following the general

procedure described by Pinkel et al. (1986). Signal

detection was performed with Alexa Fluor� strepta-

vidin antibodies (Invitrogen, Molecular Probes�, UK,

Paisley) and Anti-Digoxigenin-Rhodamine (Roche

Applied Science). The chromosomes were counter-

stained with DAPI (0.2 lg/mL) in Vectashield mount-

ing medium (Vector Laboratories, USA, Burlingame,

Table 1 Details of sampling sites

Location/adopted legend Altitude Geographical coordinates No #/$ Voucher number

Ribeirao Grande (Pindamonhangaba, Sao Paulo/RGA 662 m 22�46057.3800S and

45�26033.8000W27/32 MZUEL no. 5656

Ribeirao Grande (Pindamonhangaba, Sao Paulo)/RGB 1,850 m 22�43059.2200S and

45�27032.8100W28/35 MZUEL no. 5657

Corrego das Pedras (Campos do Jordao, Sao Paulo)/CP 1,590 m 22�43033.2000S and

45�3307.4000W25/53 MZUEL no. 5655

Corrego Tatupeba (Maringa, Parana)/CT 400 m 23�29059.0000S and

52�1041.0000O19/13 MZUEL no. 5654

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CA) and analysed using an Olympus BX41 epifluo-

rescence microscope connected to a DP 71 capture

system (Olympus, Japan).

Reproduction assays

The reproduction tests were designed to examine

reproductive isolation between locations and were

carried out 24 h after acclimating fish in the labora-

tory. The sex of the specimens was easily identified

because male fish expelled sperm when light pressure

was applied to the animal’s stomach. A total of 51

crosses of individual pairs were made. The randomly

chosen pairs were kept in individual tanks under light

for 24 h at a temperature of 26 �C and fed twice daily

with food for ornamental fish. The crosses were

organized according to Table 2.

Semi-natural induction was performed with the use

of carp pituitary extract diluted in 0.9 % saline at a

concentration of 0.3 mg/mL in a single dose for male

fish. In female fish, a second dose was applied intra-

peritoneally 12 h after the first application in female

fish. A hormone concentration of 0.5 mg/mL (stock

solution) was applied to the female fish in two doses:

the first at the end of egg maturation using a 10 %

stock solution and the second to induce spawning

using the full dosage of the hormone solution. After

spawning, the parents were euthanised and their sex

confirmed by microscopic examination of the gonads

and cytogenetic and morphometric analyses.

Phylogenetic analysis

To study the relationship between populations, we

performed three phylogenetic analyses: neighbour-

joining, maximum likelihood and maximum parsimony

with the program Mega v. 5.05 (Tamura et al. 2007). For

these analyses we used the first portion of the mito-

chondrial COI gene from A. scabripinnis from RGA,

RGB, CP and CT and two sequences from A. altipar-

anae Garutti and Britski as the outgroup (collected from

the Salto Segredo River, Foz do Iguacu—PR). Samples

from RGA individuals could not be used because the

quality of amplified DNA was not sufficient for

sequencing, even after using different procedures. The

700-bp amplification products of the mitochondrial COI

gene were obtained using the forward primer FishF1

(F1) (50-TCAACCAACCACAAAGACATTGGCAC-

30) and reverse FishR1 (R1) (50-TAGACTTCTGGG

TGGCCAAAGAATCA-30) (Ward et al. 2005). The

250-lL reaction tubes contained 2.5 lL 10X PCR

buffer, 1.28 lL MgCl2 in 50 mM, 0.5 lL 10 mM dNTP

mix, 0.2 lL Taq polymerase (1U), 0.26 mL of each

primer, 2.0 lL DNA template, and 18.0 lL ultrapure

water. The thermocycler program consisted of an initial

step of 2 min at 95 �C followed by 35 cycles of 30 s at

94 �C, 30 s at 54 �C and 1 min at 72 �C with a final

extension of 10 min at 72 �C and then kept at 4 �C. All

phylogenetic analyses were performed with 10,000

bootstrap steps, Kimura two parameters as the substi-

tution rate (except for the maximum parsimony analy-

ses) and pairwise deletion in the program Mega v. 5.05

(Tamura et al. 2007).

Results

Morphometric analysis

The analysis of measurement errors (allocation of

marks) indicated a random distribution of errors

(Wilkes’s lambda: 0.9101, p = 0.615) and confirmed

the reliability of the sample.

Discriminant function analysis (DFA) of all indi-

viduals showed the presence of sexual dimorphism at

all locations (Wilks’s lambda = 0.5236; df1 = 170;

f = 2.69, p \ 0.0001), thus requiring separate analy-

ses for male and female fish.

A sample overview (males and female fish

together), using relative deformations as response

b Fig. 1 a Location of sampling sites. b Location details for the

Corrego Tatupeba site (CT), and c Ribeirao Grande (RGA and

RGB) and Corrego das Pedras (CP) sites

Table 2 Crosses performed

Cross

(1–9)

(# x $)

No.

couples

#

Broodstock

origin

No.

couples

$

Broodstock

origin

1 7 CP 7 CP

2 7 RGB 7 RGB

3 7 RGA 7 RGA

4 5 CP 5 RGA

5 5 RGA 5 CP

6 5 RGA 5 RGB

7 5 RGB 5 RGA

8 5 CP 5 RGB

9 5 RGB 5 CP

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variables in the multivariate analyses (MANOVA/

CVA), indicated morphometric differences between

locations (test with 10,000 permutations for Mahalan-

obis distance and Procrustes among groups, all

p \ 0.0001), separating them in four canonical axes,

with the canonical axis CV1 explaining 59.4 % and

CV2 explaining 29.3 % of the variation among groups

(Fig. 2).

For female fish, the canonical axes separated four

locations CT, RGA, RGB, and CP. The first canonical

axis separated RGA and CT (positive scores) and CP

and RGB (negative scores). The CT and RGA individ-

uals, in the positive canonical portion of the first axis,

showed an enlargement of the anal fin region, which

corresponded to marks 14 and 15. They also had a more

fusiform body. The CP and RGB individuals, located in

the negative canonical portion of the first axis, showed a

contraction of the anal fin region and a dilation in the

ventral region near the pectoral fin (Fig. 2).

In the second canonical axis, which explained

29.62 % of the variation, populations were discrimi-

nated only within the positive range of the first

canonical axis. The CP (1,590 m) and RGA samples

were located in the positive range of the second

canonical axis. These had larger head and eye sockets

compared with the populations of the negative range of

the second canonical axis (RGB and CT) (Fig. 2).

The CVA of the male fish also separated all

locations and resulted in a similar pattern of morpho-

metric variation to that of female fish (test with 10,000

permutations for Mahalanobis and Procrustes dis-

tances between groups, all p \ 0.0001), with the first

canonical axis explaining 58.81 % of variation and the

second explaining 32.96 % (Fig. 2).

Morphometry and B chromosome

Only CP female fish could be analysed here,

because the sample size of male B chromosome

carriers was not sufficient in this or any of the other

populations. The CVA grouped individuals accord-

ing to presence and absence of the B chromosome

b Fig. 2 a–d Representation of the deformation (dark blue) of

each CV in relation to the reference configuration (light blue) in

female fish. j Position of the scores of the female fish from the

four locations, in the space of the first and second canonical axis.

e–h Representation of the deformation (dark blue) of each CV in

relation to the reference configuration (light blue) in male fish.

k Position of the scores of the male fish from four locations, in

the space of the first and second canonical axis. i Position of the

scores of the four locations, in the space of the first and second

canonical axes. Note the separation of the four locations in the

canonical axes

Fig. 3 a Representation of the deformation (dark blue) of CV1

in relation to the reference configuration (light blue) of the CP

female fish. In the CV-, female fish with a B, and in the CV?,

female fish with no B chromosome. b Representation of the

deformation (dark blue) of CV1 in relation to the reference

configuration (light blue) of the female CT karyomorphs. In

CV1-, 2n = 50 karyomorph. In CV1?, 2n = 48 karyomorph

Rev Fish Biol Fisheries

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(10,000 permutations for Hotelling and Mahalanobis

distances between groups, p \ 0.0001). Morpholog-

ical variation was observed in the anterior region of

the body and individuals with the B chromosome

had a less dilated ventral region compared to

individuals without B chromosomes (Fig. 3a).

Fig. 4 Idiogram of karyomorphs showing the 18S and 5S

rDNA site locations, highlighting the B chromosome and its

heterochromatin. a CP (second karyotype described by Salvador

and Moreira-Filho 1992), b RGB, c RGA (according to the

karyotype described by Neo et al. 2000), d CT cytotype 2n = 50

and e MG cytotype 2n = 48 (second karyotype described by

Fernandes and Martins-Santos 2005). Scale = 10 lm

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Morphometric analysis of 2n = 48 and 2n = 50

karyomorphs

A specific morphometric analysis was performed for

the CT populations to detect morphometric differ-

ences between them for the two karyomorphs found.

To exclude differences caused by sexual dimorphism,

males and female fish were analysed separately.

Female morphology differed between karyomorphs

(CVA, 10,000 permutations for Procrustes and Maha-

lanobis distances between groups, p \ 0.0001). The

DFA analysis also indicated significant differences

among karyomorphs (1,000 permutations,

p \ 0.0001). The 2n = 50 individuals had a more

fusiform body, compared with individuals with

2n = 48 (Fig. 3b). In male fish, the CVA found

differences (10,000 permutations for Procrustes and

Mahalanobis distances between groups, p = 0.0006)

that were not significant according to DFA (1,000

permutations, p = 0.2800).

Cytogenetics

All populations had a similar karyotypic macrostruc-

ture, as shown by their idiograms (Fig. 4). The diploid

karyomorph, 2n = 50, consisted of six metacentric,

22 submetacentric, 10 subtelocentric and 12 acrocen-

tric chromosomes with a fundamental number (NF, the

number of chromosomal arms) equal to 88. An

additional karyomorph, found in the CT population

only, composed of 48 chromosomes with eight

metacentric, 26 submetacentric, six subtelocentric

and eight acrocentric chromosomes with an NF equal

to 88.

The B chromosomes in three of the samples

analysed (CP, RGB and the 2n = 50 karyomorph

from CT) were similar to each other: being the large

metacentric type, with almost the same size as the first

chromosome pair of the A complement and com-

pletely heterochromatic. Additionally, a partially

heterochromatic B chromosome was found for the

karyomorph 2n = 48 in CT individuals. Only RGA

individuals were lacking this supernumary element

(Fig. 4).

The double FISH analysis revealed distinct rDNA

(18S and 5S) marking sites in all populations analysed.

Four 18S rDNA sites were detected in RGB individ-

uals (Fig. 5a), six in RGA individuals (Fig. 5d) and

eight in the CP population (Fig. 5g). In the 2n = 48

and 2n = 50 karyomorphs from CT, eight sites were

detected in the 18S rDNA (Fig. 5j, m). In the

karyomorphs from CT, 18S rDNA was observed in

the terminal region of the largest metacentric pair and

in submetacentric, subtelocentric and median pairs.

The chromosomes bearing these ribosomal genes in

the other populations were found in submetacentric,

subtelocentric and acrocentric pairs, always in the

terminal region of these chromosomes.

With regard to 5S rDNA in RGB individuals, six

sites were located in the proximal/interstitial region of

the short arm of one medium pair and two small pairs

(Fig. 5b); whereas in RGA individuals, eight sites

were located in subtelocentric and acrocentric pairs

(Fig. 5e). Six 5S rDNA sites were located in CP

individuals. In the 2n = 48 and 2n = 50 karyomorphs

from CT (Fig. 5k, n, respectively), three 5S sites were

found. The B chromosome did not contain any 5S or

18S rDNA sites in any of the populations sampled and

only the RGA and CP populations exhibited synteny

markings for 5S and 18S rDNA (Fig. 5f, i).

Reproduction assays

Offspring were only produced by the 21 crosses

between specimens from the same location. The 30

inter-location crosses did not result in offspring,

suggesting reproductive isolation with an absence of

induced spawning and spermiation, even with hor-

monal stimulus and in the same environmental con-

ditions in which the intralocation crosses were

performed. Additionally, the eggs produced by manual

extrusion and dry fertilisation did not fertilise or

produce embryos, as is normally the case for other fish

(Ihering 1937). It was also observed that female fish

were aggressive in the presence of male fish from other

locations, persecuting and pushing them away.

Phylogenetic analysis

In all trees resulting from the phylogenetic analyses, A.

scabripinnis formed a monophyletic group with high

bootstrap values (Fig. 6). However, with the exception

of the maximum parsimony tree, samples were

grouped by similarity of their geographic location

with moderate to high bootstrap values. The neigh-

bour-joining and maximum parsimony analyses

grouped samples with a completely heterochromatic

B chromosome and the sample from CT with a

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different karyomorph (2n = 50) with high bootstrap

values (Fig. 6).

Discussion

The results of the morphometric analysis indicated

that the A. scabripinnis populations studied were

relatively well differentiated from each other, includ-

ing sexual dimorphism. These inter-populational

variations corroborate the work of Moreira-Filho and

Bertollo (1991), who argue that A. scabripinnis

comprises a species complex that is adapted to

different environments.

The isolated populations of A. scabripinnis from

Serra da Mantiqueira (RGB and CP) found at higher

altitudes, were differentiated by geometric morphom-

etry from those found in lower regions, such as CT and

RGA. The phylogenetic trees also indicated a clear

separation between high- and low-altitude locations.

However, the CT sample with a heterochromatic B

chromosome and different karyomorph (2n = 50)

showed an intermediary position in the analyses,

probably because of ancient genetic ancestry between

the two locations. Populations from higher altitudes had

a more prominent anterior region of the body compared

with the posterior region. This was in contrast to

populations from lower altitudes where the caudal

peduncle was more robust. Additionally, in populations

from lower altitudes, the general shape of the body was

fusiform, possibly indicating better hydrodynamic use

favourable in the presence of predators and when

foraging for food (Sibbing and Nagelkerke 2001). Such

an advantage is also facilitated by the larger muscle

mass of the caudal peduncle.

The morphometric differences of the two kar-

yomorphs from CT (2n = 48 and 2n = 50) reinforced

the cytogenetic data obtained by Fernandes and Mar-

tins-Santos (2005). The absence of natural hybrids and

morphometric and karyotypic differences fixed in these

populations indicate that different species of the A.

scabripinnis complex occur in sympatry in this region.

The diploid karyotype 2n = 50 is considered

ancestral in the A. scabripinnis complex and Robert-

sonian-type translocation rearrangements may be

related to the origin of lower diploid numbers (Vicari

et al. 2008a), as observed in the present study in CT

individuals with 2n = 48. Our phylogenetic results

show a clear separation between the samples from

high and low altitudes and the intermediary position of

CT specimens with a different karyomorph may

support this hypothesis. These results suggest a

possible migratory event from high to low altitudes,

with migration remnants found in the DNA sequences

and chromosomes of individuals from low-altitude

populations. Our data support this hypothesis because

the 2n = 48 karyomorph showed a reduction in

subtelocentric and acrocentric chromosomes in con-

trast to an increase in chromosomes with two arms.

b Fig. 5 Fluorescent in situ hybridisation with 18S (a, d, g, j,m) and 5S rDNA probes (b, e, h, k, n), and superimposition of

images (c, f, i, l, o) for the populations from RGB (a, b, c), RGA

(d, e, f), CP (g, h, i), MG (j, k, l) 2n = 48 and CT (m, n,

o) 2n = 50, respectively. The arrows indicate the ribosomal

DNA site locations identified by FISH. Scale = 10 lm

Fig. 6 Relationship cladograms performed with COI

sequences generated by neighbour-joining (NJ), maximum

likelihood (ML) and maximum parsimony (MP) analyses.

Bootstrap values are represented in the branches. On the right,

chariotic number of the samples and type of chromosome found.

Dashes on the right represent no supranumeric chromosome

found. The species A. altiparanae was used as outgroup (Out)

Rev Fish Biol Fisheries

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Although the number of chromosome arms (NF)

was constant in all populations analysed here

(NF = 88), the location of ribosomal genes showed

a karyotypic inter-populational diversity greater than

that observed in the karyotypic macrostructure. Sev-

eral authors (Neo et al. 2000; Ferro et al. 2001;

Mantovani et al. 2005; Vicari et al. 2008a) have stated

that the number and location of 18S rDNA sites in

Astyanax is variable and multiple, suggesting that

transposition mechanisms are required to explain this

large variation (Fernandes and Martins-Santos 2006;

Vicari et al. 2008b), and their findings are in agree-

ment with the present study.

Unlike for the major DNA (18S), the 5S rDNA sites

in Astyanax tend to be preserved in the region close to

the long arm of one acrocentric pair and one

metacentric pair (Ferro et al. 2001; Almeida-Toledo

et al. 2002; Mantovani et al. 2005; Vicari et al. 2008a).

However, there is evidence of more chromosome pairs

carrying this site in some populations (Ferro et al.

2001). Among the populations of A. scabripinnis

analysed in the present study, only one exhibited the

more conserved pattern, while the other populations

had between three and five chromosomes with 5S

rDNA, suggesting a higher diversity for this marker

than previously described.

In different groups of fish 5S rDNA with 18S rDNA

synteny has been suggested as an ancestral condition

(Jesus and Moreira-Filho 2003; Hatanaka and Galetti

2004; Vicari et al. 2006). Similarly, Almeida-Toledo

et al. (2002) observed a 5S rDNA site co-located with

28S rDNA in one of the acrocentric chromosome pairs

of an A. scabripinnis population from the Tiete River,

SP and a population of A. fasciatus from Mogi Guacu,

SP. We found different synteny conditions between 5S

and 18S rDNA, revealing a more complex pattern of

ribosomal DNA location in Astyanax.

As noted by Kandul et al. (2007), chromosomal

rearrangements and rapid karyotypic diversification

are important factors in post-zygotic reproductive

isolation that can lead to speciation. In addition to

cytogenetic data, our observations on reproduction

suggest prezygotic reproductive isolation and a break

in gene flow between populations. This situation may

favour inbreeding and may result in allopatric speci-

ation (Futuyma 2013).

The combination of several approaches in the present

study satisfactorily demonstrates that the populations

studied represent cryptic species. Considering the

biological species concept, each population is experi-

encing a speciation process at the level of pre-zygotic

reproductive isolation without hybrids. This observation

is supported by the fact that no hybrid karyotype has

been recorded. The morphological similarity between

them, despite the differences shown by our analyses,

suggests that the evolutionary divergence is recent.

Likewise, Marshall et al. (2006) required different

methodologies and analyses to define species in a lizard

species complex of recent evolutionary divergence.

With the data obtained for A. scabripinnis, one can

infer a complex evolutionary scenario, where popula-

tions that were related in a previous geological time

period are at present evolving independently in both

sympatry and in allopatry. The morphometric data

grouped all populations separately with a relationship

between the differentiated body morphology of female

fish and the presence of the B chromosome. The

observed similarity of the karyotype macrostructure

between individuals from CP, RGB and CT (kar-

yomorph 2n = 50), with the exception of the CT

2n = 48 karyomorph, indicates divergence in a geo-

morphological context. Thus, the karyotypic differ-

ences verified by molecular and cytogenetic markers,

together with the observed reproductive isolation,

indicate an evolutionary divergence caused by

restricted gene flow among the populations analysed.

Acknowledgments The authors are grateful to the Instituto

Chico Mendes de Conservacao da Biodiversidade (ICMBio). This

study was financed by the Fundacao de Amparo a Pesquisa do

Estado de Sao Paulo (FAPESP), Conselho Nacional de

Desenvolvimento Cientıfico e Tecnologico (CNPq) Coordenacao

de Aperfeicoamento de Pessoal de Nıvel Superior (CAPES) and

the Fundacao Araucaria (Fundacao Araucaria de Apoio ao

Desenvolvimento Cientıfico e Tecnologico do Estado do Parana).

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