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Comparative Cytogenetics of Seven Ctenidae

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 Comparative Cytogenetics of Seven Ctenidae Species (Araneae)
Author(s): Douglas Araujo , Edson Gabriel de Oliveira , Andr Marsola Giroti , Viviane Fagundes
Mattos , Emygdio Paula-Neto , Antonio Domingos Brescovit , Marielle Cristina Schneider , and
Doralice Maria Cella
Source: Zoological Science, 31(2):83-88. 2014.
Published By: Zoological Society of Japan
DOI: http://dx.doi.org/10.2108/zsj.31.83
URL: http://www.bioone.org/doi/full/10.2108/zsj.31.83

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ZOOLOGICAL SCIENCE 31: 8388 (2014) 2014 Zoological Society of Japan

Comparative Cytogenetics of Seven Ctenidae Species (Araneae)

Douglas Araujo1*, Edson Gabriel de Oliveira2, Andr Marsola Giroti3,
Viviane Fagundes Mattos2, Emygdio Paula-Neto2, Antonio Domingos Brescovit3,
Marielle Cristina Schneider4, and Doralice Maria Cella2
1
Universidade Federal de Mato Grosso do Sul, UFMS, Setor de Biologia Geral, Centro de Cincias
Biolgicas e da Sade, Cidade Universitria, Bairro Universitrio, 79070-900,
Campo Grande, Mato Grosso do Sul, Brazil
2
Universidade Estadual Paulista, UNESP, Instituto de Biocincias, Departamento de Biologia,
Avenida 24-A, 1515, Caixa Postal 199, 13506-900, Rio Claro, So Paulo, Brazil
3
Instituto Butantan, Laboratrio Especial de Colees Zoolgicas, Av. Vital Brasil, 1500,
05503-900, So Paulo, So Paulo, Brazil
4
Universidade Federal de So Paulo, UNIFESP, Departamento de Cincias Biolgicas,
Av. Prof. Artur Riedel, 275, 09972-270, Diadema, So Paulo, Brazil

The present study elevates the number of cytogenetically analyzed ctenid species and genera from
two to eight and six, respectively, presenting comparisons between chromosomal data obtained
and the phylogenetic hypothesis proposed in the literature. Six ctenid species presented 13 auto-
somal pairs, exhibiting either X1X20 (Ctenus ornatus, Ctenus sp., Parabatinga brevipes and Phoneu-
tria nigriventer) or X1X2X30 sex chromosome systems (Nothroctenus sp. and Viracucha andicola).
Asthenoctenus borellii showed 2n = 20 + X1X20. In all species, the chromosomes were telocentric.
Some cells of one C. ornatus specimen exhibited one extra chromosome that, considering the
behavioral similarities between the two chromosomes, can be considered to be supernumerary,
derived from or giving rise to a sex chromosome. Silver impregnation revealed nucleolar organizer
regions on one autosomal pair of C. ornatus and P. nigriventer (Cteninae) and two pairs of V. andi-
cola (Acanthocteninae). Chromosomal data suggests that the X1X2X30 system arose several times
in the evolution of entelegyne spiders, and that conversion of an X1X20 system into an X1X2X30 sys-
tem and vice-versa has been a relatively common event in spiders. All the chromosomal data cor-
roborate the close relationship between Ctenus and Phoneutria, the placement of P. brevipes within
Cteninae, the placement of Anahita in a separated branch within Cteninae, and the inclusion of A.
borellii in a distinct group within the ctenids (Viridasiinae), all of which are as proposed by phylo-
genetic hypotheses available in the literature.

Key words: diploid number, karyotype, meiosis, nucleolar organizer region, sex chromosome system,
GST clade, Cteninae, Acanthocteninae, Viridasiinae

grate-shaped tapetum (GST) clade, which includes at least

INTRODUCTION
Within the infraorder Araneomorphae, the Entelegynae
lineage includes the highest number of species described
taxonomically (Platnick, 2013) and cytogenetically (for a
review, see Araujo et al., 2013). Nevertheless, considering
the wide diversity of spiders belonging to this group, which
includes more than 36,000 species (Platnick, 2013), this
chromosome information comprises less than 2% of the rep-
resentatives known, with around 600 species karyotyped to
date (Araujo et al., 2013).
Containing 40 genera and 480 species distributed world-
wide (Platnick, 2013), the family Ctenidae belongs to the
* Corresponding author. Tel. : +55-67-3345-7311;
Fax : +55-67-3345-7311;
E-mail: d.araujo@ufms.br
eight other families. Ctenidae can be subdivided into eight
lineages, five of which correspond partially to the classical
subfamilies Viridasiinae, Acanthocteninae, Acantheinae,
Calocteninae, and Cteninae, as described in the phyloge-
netic hypothesis of Silva (2003) and Polotow and Brescovit
(in press). The acanthoctenines Nothroctenus Badcock,
1932 and Viracucha Lehtinen, 1967 are composed of nine
and six species, respectively, and restricted to Brazil,
Bolivia, Paraguay, and Argentina. Within the Cteninae,
Ctenus Walckenaer, 1805 is the largest genus, including
235 species distributed worldwide; Phoneutria Perty, 1833
and Parabatinga Polotow & Brescovit, 2009 possess eight
and one species, respectively, distributed in Central and
South America. The Viridasiinae genus Asthenoctenus
Simon, 1897 is composed of only three species restricted to
Brazil, Uruguay, Paraguay, Argentina, and Guyana (Silva,

In memoriam 2003; Platnick, 2013; Polotow and Brescovit, in press).

doi:10.2108/zsj.31.83 To date, only two ctenid species have been cytogeneti-
84 D. Araujo et al.

cally analyzed: Ctenus ornatus (Keyserling, 1877), with 2n
= 28 (Oliveira et al., 1996), and Anahita fauna Karsch,
1879, possessing 2n = 29 and telocentric chromosomes
(Chen, 1999); these species presented the same number of
autosomes, but differed with regard to the type of sex chro-
mosome system, which were X1X20 and X1X2X30, respec-
tively. Supernumerary chromosomes are important elements
involved in the origin of some sex chromosome systems in
spiders (Araujo et al., 2012); however, the presence of these
chromosomes in spiders of the GST clade has been
reported only by Montgomery (1905), for one representative
of Lycosidae, Gladicosa pulchra (Keyserling, 1877), which
showed two very small extra elements in some mitotic cells,
without mentioning the morphology, pycnotic behavior, or
origin of these supernumeraries.
In the present study, the mitotic and meiotic chromo-
somes of seven ctenid species belonging to six genera were
analyzed, with the aim of increasing our knowledge of ctenid
cytogenetics and contributing to the understanding of the
chromosomal evolution of this group. It should be noted that
five of the six genera studied here were chromosomally ana-
lyzed for the first time.
MATERIALS AND METHODS
The number of individuals and collection locality of the species
analyzed in this work are mentioned in Table 1, along with a sum-
marized karyotype constitution for all ctenids that have been chro-
mosomally analyzed. The voucher specimens were deposited in the
arachnological collection of the Laboratrio Especial de Colees
Zoolgicas, Instituto Butantan (IBSP, curator D. M. Barros-Battesti),
So Paulo, state of So Paulo, Brazil. The chromosome prepara-
tions were obtained following Araujo et al. (2008), standard stained
with 3% Giemsa solution (47 ml distilled water, 1.5 ml phosphate
buffer pH 6.8, 1.5 ml Giemsa) for 12 min, and impregnated with silver
nitrate to detect active nucleolar organizer regions (NOR), following
Howell and Black (1980). The chromosome morphology was deter-
mined following the nomenclature proposed by Levan et al. (1964).
RESULTS
Giemsa staining
Male karyotypes of six ctenid species exhibited 13 auto-
somal pairs gradually decreasing in size. Nothroctenus sp.
and Viracucha andicola showed sex chromosome system of
the X1X2X30 type (Fig. 1A, B) while Ctenus ornatus, Ctenus
sp., Phoneutria nigriventer and Parabatinga brevipes
revealed X1X20 system (Fig. 1CF). In Asthenoctenus borel-
lii, the diploid number 2n = 20 + X1X20 was verified in
mitotic metaphase nuclei; however, in the sample of cells
studied, the chromosomes appeared superposed, making it
impossible to carry out the karyotype analysis. In all species,
the autosomes and sex chromosomes demonstrated telo-
centric morphology. With respect to size, the X1 and X2 sex
chromosomes were, respectively, similar to the large and
small chromosomes of the complement, while the X1, X2,
and X3 sex chromosomes were classified as large, medium,
and small-sized (Fig. 1). In some mitotic metaphase cells, a
prominent secondary constriction was visualized in pair 3
distal region of Nothroctenus sp. (Fig. 1A) and pair 6 proxi-
mal region of Nothroctenus sp., V. andicola and Ctenus sp.
(Fig. 1A, B, D). In one male of C. ornatus, the study of
mitotic cells revealed an intraindividual variability of diploid
number, which occurred due to the presence of some cells
with a supernumerary chromosome (2n = 26 + X1X20 +
1S) and other cells with the typical 2n = 26 + X1X20. The

Table 1. Ctenidae species cytogenetically studied, including those presented in this work with their respective samples, basic karyotype data,
and collection localities in Brazil. PR = state of Paran; SP = state of So Paulo.

Species Sample Karyotype (2n ) Collection locality Reference

Acanthocteninae
Nothroctenus sp.* 6,2 29, X1X2X30 Rio Claro (2224S, 4734W), SP Present work
Margem da Lagoa Xambr, Parque Nacional
de Ilha Grande, Altnia (2352S, 5400W),
PR
Viracucha andicola (Simon, 1906)* 1 29, X1X2X30 Carapicuiba (2331S, 4650W), SP Present work
Cteninae
Anahita fauna*** 1 29, X1X2X30 Taiwan Chen, 1999
Ctenus ornatus (Keyserling, 1877)* 3 28, X1X20 Embu-Guau (2349S, 4648W), SP Present work,
So Paulo (2332S, 4638W), SP Oliveira et al.,
Vargem Grande Paulista (2336S, 4701W), 1996
SP
Ctenus sp.* 1 28, X1X20 So Paulo (2332S, 4638W), SP Present work
Phoneutria nigriventer (Keyserling, 1891)* 5 , 6 embryos 28, X1X20 Rio Claro (2224S, 4734W), SP Present work
Taubat (2301S, 4532W), SP
Parabatinga brevipes (Keyserling, 1891)** 1 28, X1X20 Altnia (2352S, 540W), PR Present work
Viridasiinae
Asthenoctenus borellii Simon, 1987*** 2 22, X1X20 Ilha Grande, Parque Nacional de Ilha Grande, Present work
Guara (2401S, 5410W), PR
Ilha So Francisco, Parque Nacional de Ilha
Grande, Guara (2400S, 5409W), PR
* Phylogenetic position considering Silva (2003) and Polotow and Brescovit (in press).
** Phylogenetic placement considering Polotow and Brescovit (2009, in press).
*** Phylogenetic position according to Polotow and Brescovit (in press).
Phylogenetic placement of Anahita considering only the generic level, the species listed here are not the same included in the papers of Silva
(2003) and Polotow and Brescovit (in press).
 Chromosome analysis in ctenid spiders 85

valents were maintained until late

prophase I substages (Fig. 2EJ).
However, the sex chromosomes
appeared distant from each
other (Fig. 2K) and heteropycno-
sis was in some cases less
clearly evident (Fig. 2L). More-
over, conspicuous secondary
constrictions were visualized in
two autosomal bivalents of C.
ornatus (Fig. 2H) and Ctenus sp.
(Fig. 2I). Metaphase II cells of all
species showed haploid numbers
that confirmed the type of sex
chromosome system and the
regular segregation of all chro-
mosomes, i.e., n = 13 or n = 16
in species with 2n = 29, n = 13
or n = 15 in species with 2n =
28, and n = 10 or n = 12 (Fig. 2M)
in the species with 2n = 22.
The supernumerary chromosome
found in some cells of one male
of C. ornatus exhibited the same
pattern of condensation/heteropy-
cnosis and behavior observed to
the X1 and X2 chromosomes (Fig.
2B, G). In this individual, some
metaphase II cells demonstrated
n = 13 and n = 13 + X1X2 + 1S
(Fig. 2NO), indicating co-segre-
gation of the two sex chromo-
somes and the supernumerary
element.

Fig. 1. Male karyotypes of six ctenid species stained with Giemsa. (A) Nothroctenus sp., 2n = 26 + Silver nitrate impregnation
X1X2X30. In detail, pairs 3 and 6 with conspicuous distal and proximal secondary constrictions Chromosome preparations of
(arrowheads), respectively. (B) Viracucha andicola, 2n = 26 + X1X2X30. (C) Ctenus ornatus, 2n = 26 +nearly all spiders analyzed here
X1X20 + 1S. S = supernumerary chromosome. (D) Ctenus sp., 2n = 26 + X1X20. (E) Phoneutria were impregnated with silver
nigriventer, 2n = 26 + X1X20. (F) Parabatinga brevipes, 2n = 26 + X1X20. Scale bar = 10 m. nitrate, but the NOR distribution
pattern was only established in
three species. In V. andicola, C.
supernumerary element was intermediate in length between ornatus and P. nigriventer, two NORs on terminal region of
the X1 and X2 chromosomes (Fig. 1C). pair 10 were observed (Fig. 3). Additionally, some cells of V.
In early prophase I spermatocytes of the seven ctenid andicola also showed NORs on terminal region of the 6th
species studied here, the sex chromosomes of both autosomal pair (Fig. 3AB, insert).
X1X2X30 (Fig. 2A) and X1X20 (Fig. 2BD) systems appeared
DISCUSSION
as univalents highly condensed, positively heteropycnotic
and arranged side by side. The diplotene nuclei showed the To date, knowledge of ctenid cytogenetics has been
meiotic formula 13II + X1X2X30 in Nothroctenus sp. and V. restricted to only two species (Oliveira et al., 1996; Chen,
andicola (Fig. 2EF), 13II + X1X20 + 1S, with a supernumer- 1999), comprising less than 0.5% of the 490 species taxo-
ary chromosome, in some cells of one specimen of C. orna- nomically described (Platnick, 2013). The present paper
tus (Fig. 2G), 13II + X1X20 in C. ornatus, Ctenus sp., P. shows the cytogenetic data of seven ctenid species, six of
nigriventer and P. brevipes (Fig. 2HK), and 10II + X1X20 in which had not previously been karyotyped, increasing the
A. borellii (Fig. 2L). In all species, one interstitial or terminal percentage of ctenid species chromosomally studied to
chiasma per bivalent was the pattern most frequently nearly 1.5%. Additionally, this is the first chromosomal study
observed in the majority of the elements; but two interstitial in five of the six ctenid genera presented here, increasing
and/or terminal chiasmata in one bivalent were eventually the proportion of ctenid genera cytogenetically analyzed
verified in all ctenid species, with the exception of V. andi- from 5% to 17.5%. Previous studies on ctenid chromosomes
cola, P. brevipes and A. borellii (Fig. 2EL). In general, the focused only on Anahita (Chen, 1999) and Ctenus (Oliveira
positive heteropycnosis and close association of the sex uni- et al., 1996) both belonging to the subfamily Cteninae (Silva,
86 D. Araujo et al.

Despite the low number of entel-

egyne species presenting the sex
chromosome system of the
X1X2X30 type, this system was
observed in 16 of 36 entelegyne
families chromosomally analyzed
thus far (~45%) (Araujo et al.,
2012, 2013). It is noteworthy that
in 40% of the cases, the X1X2X30
sex chromosome system coexists
with the X1X20 system in conge-
neric species and, even more
noteworthy, is that in nearly 20%
of the records, these two systems
coexist in a same species (Araujo
et al., 2013). These facts suggest
that: 1) The X1X2X30 system
arose several times in the evolu-
tion of entelegyne spiders; 2) The
conversion of an X1X20 system
into an X1X2X30 system and vice-
versa is a relatively common
event in spiders.
The mechanisms that underlie
the origin of an X1X2X30 system
from an X1X20 system in spiders
have been proposed by several
authors, involving only the sex
chromosomes (Ptau, 1948;
Postiglioni and Brum-Zorrilla,
1981; Parida and Sharma, 1986),
or the sex chromosomes and a
supernumerary element (Bole-
Gowda, 1952). The origin of the
X1X20 system from X1X2X30 in
Fig. 2. Spermatocytes of ctenid species stained with Giemsa. (AD) Early prophase I cells. (A) spiders was proposed by Krl
Nothroctenus sp., (B) Ctenus ornatus. (C) Ctenus ornatus. (D) Phoneutria nigriventer. Note the high (2007), involving only the sex
degree of condensation of the sex chromosomes and supernumerary chromosome (S) in these chromosomes (for a review see
cells. (EL) Diplotene nuclei. (E) Nothroctenus sp., 13II + X1X2X30. (F) Viracucha andicola, 13II + Araujo et al., 2012). These
X1X2X30. (G) Ctenus ornatus, 13II + X1X20 + 1S. (H) Ctenus ornatus, 13II + X1X20. (I) Ctenus sp.,
hypotheses regarding to the ori-
13II + X1X20. (J) Phoneutria nigriventer, 13II + X1X20. (K) Parabatinga brevipes, 13II + X1X20. (L)
Asthenoctenus borellii, 10II + X1X20. Large arrow = interstitial chiasma. Small arrow = terminal chi-
gin of sex chromosome systems
asma. (MO) Metaphase II cells. (M) Asthenoctenus borellii, with n = 10 (left) and n = 12 (right). (N appear to be applicable in the
O) Ctenus ornatus, with n = 13 and n= 13 + X1X2 + 1S, respectively. Scale bar = 10 m. case of ctenids, in which some
species have 2n = 26 + X1X20
and others have 2n = 26 +
2003; Polotow and Brescovit, in press). The present study X1X2X30.
includes genera of the subfamilies Cteninae (Ctenus, In spiders belonging to several families (Agelenidae,
Phoneutria and Parabatinga), and, for the first time, Acan- Araneidae, Eresidae, Linyphiidae, Lycosidae, Miturgidae,
thocteninae (Nothroctenus and Viracucha), and Viridasiinae Oecobiidae, Philodromidae, Sparassidae and Uloboridae),
(Asthenoctenus), (Silva, 2003; Polotow and Brescovit, in congeneric species presenting X1X20 and X1X2X30 sex chro-
press). mosome systems also showed differences in the number of
One interesting characteristic observed in both Acan- autosomes. However, these differences are probably not
thocteninae species analyzed here (Nothroctenus and Vira- related to the conversion of an X1X20 system into an
cucha) was the presence of an X1X2X30 sex chromosome X1X2X30 system and vice versa, considering that: 1) in many
system. This type of system was previously described in cases, the species with an X1X2X30 system possessed a
Anahita fauna (Cteninae) by Chen (1999). higher number of autosomes than did the species with an
Since its first description by Soklska (1925) in Tege- X1X20 system; and 2) even in cases in which the species
naria domestica (Clerk, 1757) (Agelenidae), the X1X2X30 with an X1X2X30 exhibited a lower number of autosomes
sex chromosome system was recorded in only 59 (9.8%) of than species with an X1X20 system, the difference almost
the 604 entelegyne spider species that have been cytoge- always involved more than one autosome pair.
netically analyzed (see review in Araujo et al., 2013). Despite the fact that the X1X2X30 system is observed in
 Chromosome analysis in ctenid spiders
Fig. 3. Mitotic metaphase cells stained with Giemsa (A, C, E) and
silver-impregnated (B, D, F). The arrows indicate the nucleolar orga-
nizer regions (NORs). (AB) Viracucha andicola, 2n = 26 +
X1X2X30 and NORs on pair 10. The detail shows NORs on pair 6
and 10. (CD) Ctenus ornatus, 2n = 26 + X1X20 and NORs on pair
10. (EF) Phoneutria nigriventer, 2n = 26 + X1X1X2X2 and NORs
on pair 10. Scale bar = 10 m.
8.7% of all spiders chromosomally analyzed, the chromosome
formula 2n = 26 + X1X2X30 was recorded only in eight spe-
cies (13% of the 59 entelegynes with an X1X2X30 system and
only 1.3% of all 604 entelegynes cytogenetically studied) and
is restricted to the families Salticidae (Dionycha) (Datta and
Chatterjee, 1983), Selenopidae (Dionycha) (Sharma et al.,
1959; Mittal, 1961, 1966; Srivastava and Shukla, 1986),
Ctenidae (Ctenoidea) (Chen, 1999; present work) and
Pisauridae (higher or true lycosoid) (Srivastava and Shukla,
1986). Except by Selenopidae, with only three species ana-
lyzed, in the other three families that possess species with
2n = 26 + X1X2X30 there is a predominance of species
with the karyotype 2n = 26 + X1X20. In Pisauridae, the
species possessing 2n = 26 + X1X20 are congeneric to the
species presenting 2n = 26 + X1X2X30. On the other hand,
in Salticidae and Ctenidae, these chromosome formulae are
not found in the same genus. Surprisingly, the 2n = 26 +
X1X2X30 had not previously been reported within Lycosidae,
the family with the highest number of chromosomally studied
87
species (115) and also with predominance of representa-
tives with the karyotype 2n = 26 + X1X20 (Araujo et al.,
2013).
Although chromosome numbers and sex chromosome
systems are highly homoplasious in many circumstances,
the phylogenetic value of these characters should not be
rejected when used together with other types of data. Rep-
resentatives of only three ctenid subfamilies (Acanthocteni-
nae, Cteninae and Viridasiinae) had been cytogenetically
analyzed to present. The close relationship between the
genera Ctenus and Phoneutria - Cteninae or higher ctenids
(Griswold et al., 1993; Silva, 2003; Raven and Stumkat,
2005; Polotow and Brescovit, 2009; Polotow and Brescovit,
in press), as well as the placement of Parabatinga brevipes
(transferred from Ctenus brevipes) within the ctenines
(Polotow and Brescovit, 2009; Polotow and Brescovit, in
press), are reinforced by the fact that all these three genera
presented 2n = 26 + X1X20. According to Silva (2003),
Anahita is placed in a more basal branch, separated from
Ctenus and Phoneutria. Interestingly, A. fauna presents a
different sex chromosome system, with 2n = 26 + X1X2X30
(Chen, 1999), equal to that found in the basal ctenids of the
subfamily Achantocteninae (Nothroctenus and Viracucha).
Asthenoctenus borellii, with 2n = 20 + X1X20, is the spe-
cies chromosomally more distinct from the other ctenids
karyotyped up to now, what is in agreement with its place-
ment in a separate clade, the Viridasiinae, by Polotow and
Brescovit (in press).
The presence of extra chromosomal elements, such as
verified in C. ornatus has been sporadically reported in spiders
(Montgomery, 1905; Painter, 1914; Avils and Maddison,
1991; Rowell and Main, 1992). In the species studied here,
the supernumerary element showed a pattern of heteropyc-
nosis/condensation, behavior, and segregation similar to the
sex chromosomes, and could constitute a transient state in
the origin of an X3 chromosome from a B chromosome.
According to Camacho et al. (2000), similarities between
supernumerary and sex chromosomes are more than just
coincidental, there are certain reasons for similarities
between these types of chromosomes, one of which is a real
homology, indicating that one have originated from the
other. The origin of supernumerary elements from sex chro-
mosomes and vice-versa has been proposed in several
organisms (Camacho et al., 2000; Yoshida et al., 2011).
Thus, the occurrence of two cellular types (28, X1X20 and
29, X1X20 + 1S) in the gonads of one male individual of C.
ornatus, the similar behavior of the supernumerary element
and sex chromosomes, and the presence of other species
of Ctenidae with 2n = 28, X1X20 and 2n = 29, X1X2X30, allow
us to hypothesize that the X3 chromosome in this group may
have arisen from a supernumerary element, as proposed by
Bole-Gowda (1952), to the X3 chromosome of the Sparassi-
dae spider Heteropoda venatoria (Linnaeus, 1767).
The NORs visualized in three ctenid species (C. ornatus,
P. nigriventer and V. andicola) were invariably located on
autosomal pairs. According to Oliveira et al. (2007), auto-
somal NOR could constitute a common characteristic to
Entelegynae spiders, differing from Haplogynae, in which
this region was also located on the sex chromosomes. The
results obtained with the use of silver nitrate impregnation
revealed that in addition to the karyotype features, the
88 D. Araujo et al.
NOR distribution pattern is conserved in ctenid species
examined here, considering that this region appeared on
pair 10 of C. ornatus, P. nigriventer (Cteninae) and V. andicola
(Acanthocteninae). Furthermore, the majority of the spider
species revealed multiple NORs localized on two autosomal
pairs. Thus, the presence of NORs on a single autosome
pair of C. ornatus and P. nigriventer may represent a
derived character, or occur due to the methodological pro-
cedure used for detecting the NORs, which register only
active rDNA sites.
ACKNOWLEDGMENTS
We are indebted to two anonymous reviewers for critical reading
and valuable suggestions for the manuscript. This research was sup-
ported by Fundao de Amparo Pesquisa do Estado de So Paulo,
FAPESP (2010/14193-7), Conselho Nacional de Desenvolvimento
Cientfico e Tecnolgico, CNPq (ADB, DA 471821-2008), and
CAPES (EGO). The collection permit for Parque Nacional de Ilha
Grande specimens was granted by the Instituto Brasileiro do Meio
Ambiente e dos Recursos Renovveis IBAMA and Instituto Chico
Mendes de Conservao da Biodiversidade (ICMBio) (#15157-1).
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(Received July 2, 2013 / Accepted October 9, 2013)