CompCytogen 5(2): 133-142 (2011) COMPARATIVE Apeerssiewed onenaccessoural

doi: 10.3897/CompCytogen.v5i2.964 vA Cyto genetics

www.pensoft.net/journals/compcytogen International Journal of Plant & Animal Cytogenetic,

Karyosystematics, and Molecular Systematics

Description of karyotype in Hypostomus regani (Ihering,
1905) (Teleostei, Loricariidae) from the Piumhi river
in Brazil with comments on karyotype variation
found in Hypostomus

Ernani de Oliveira Mendes-Neto', Marcelo Ricardo Vicari’,
Roberto Ferreira Artoni?, Orlando Moreira-Filho!

| Universidade Federal de Sao Carlos. Depto. Genética e Evolucgéo. Séo Carlos, SPR Brazil 2. Universidade
Estadual de Ponta Grossa. Depto. Biologia Estrutural, Molecular e Genética. Ponta Grossa, PR, Brazil

Corresponding author: Roberto Ferreira Artoni (rfartoni@pq.cnpq.br)

Academic editor: VA. Lukhtanov| Received 27 January 2011 | Accepted 25 April 2011 | Published 30 June 2011

Citation: Mendes-Neto EO, Vicari MR, Artoni RE Moreira-Filho O (2011) Description of karyotype in Hypostomus
regani (Ihering, 1905) (Teleostei: Loricariidae) from the Piumhi river in Brazil with comments on karyotype variation

found in Hypostomus. Comparative Cytogenetics 5(2): 133-142. doi: 10.3897/JHR.v5i2.964

Abstract

‘The paper represents a comparative cytogenetic analysis of three populations of Hypostomus regani in Bra-
zil. Two populations belong to the Upper Parana River Basin and the third one, the karyotype of which is
described for the first time, was probably introduced into the Sao Francisco River Basin through transposi-
tion from the Piumhi River. Karyotype features of populations of H. regani from the Piracicaba and Tieté
River Basins are also discussed. The occurrence of H. regani in the Sao Francisco River Basin is reported
for the first time here. The study also revealed distinct differences in the location of the Ag-NORs between
the analyzed populations that enable individuals from the Piumhi River, Mogi-Guacu River and Tieté
River to be distinguished from one another. Thus, the data obtained indicate the possibility of geographic
variation fixing different karyotypes even in the same basin of origin.

Keywords
cytotaxonomy, karyotype diversification, rDNA

Introduction

The family Loricariidae is the second most numerous among fish, with 716 species
distributed among 96 genera (Ferraris 2007). These fish are endemic to the Neotropics,
occurring from Costa Rica to Argentina (Reis et al. 2003). This considerable diversity

Copyright Ernani de Oliveira Mendes-Neto et al. This is an open access article distributed under the terms of the Creative Commons Attribution
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134 Ernani de Oliveira Mendes-Neto et al. / Comparative Cytogenetics 5(2): 133-142 (2011)

has resulted in constant identification problems and new species have frequently been
described (Pereira and Oyakawa 2003, Cardoso and Silva 2004). Recent studies have
revealed that the taxonomy of Loricariidae remains poorly resolved (Armbruster 2004),
but six subfamilies are recognized: Loricariinae, Hypoptopomatinae, Hypostominae,
Neoplecostominae, Lithogeneinae and Delturinae (Reis et al. 2006). Although Reis et
al. (2003) consider Loricariidae to be the largest family of catfish in the world, little is
known regarding the constitution and organization of the karyotype in this group, which
exhibits a tendency to show quite divergent karyotypes (Artoni and Bertollo 2001).

Hypostomus Lacépéde, 1803 is the largest genus of the armored catfish family Lo-
ricariidae, with approximately 120 nominal species (Weber 2003). It is also one of the
better characterized genera among the loricariids from the cytogenetic standpoint, re-
vealing a variation in diploid number from 2n = 54 in H. plecostomus (Linnaeus, 1758)
(Muramoto et al. 1968, cited in Artoni and Bertollo 2001) to 2n = 84 in Hypostomus sp.
2 (Cereali et al. 2008). Although some trends of the karyotype evolution have been de-
scribed in Hypostomus (Artoni and Bertollo 2001, Kavalco et al. 2005, Alves et al. 2006,
Kavalco et al. 2005, Milhomem et al. 2010), especially such features as increase in the
diploid number by centric fission (Artoni and Bertollo 1996), the number of karyo-
typed species is still very small compared to the diversity of species known in this genus.

In the present study, a comparative cytogenetic analysis was carried out on three
different populations of H. regani (Ihering, 1905). Two populations are from the Up-
per Parana River Basin and the other, the karyotype of which is described for the
first time, was probably introduced into the Sao Francisco River Basin through the
transposition of the Piumhi River. The aim was to investigate the karyotype in these
populations, seeking chromosomal characters potentially important for understanding
the taxonomy and biogeography of the species.

Material and methods

Sixteen specimens of H. regani were examined (8 males and 8 females), collected from the
mouth of the Piumhi River at the Sao Francisco River in the region of the municipality of
Piumhi — MG, Brazil (20°20'31.0" S; 45°59'03.4" W, Alt.: 640 m), (Fig. 1, detail). Ac-
cording to C.H. Zawadzki (personal communication), Hypostomus regani is characterized
as a species with a body covered by small, round, light-colored and generally well-defined
spots. It has a high body and relatively long (narrow) head, large eyes and long dorsal fins,
generally with rays reaching the spine of the adipose fin when adpressed. It has plates on
the abdomen, except for very young specimens. Although distributed throughout the Par-
ana-Paraguay Basin, its type locality is the Piracicaba River in the state of Sao Paulo, Brazil.

The specimens were identified and deposited in the Museu Nacional do Rio de
Janeiro (MNRJ 32778; MNRJ 32782; MNRJ 32787). The collection authorization
(number 472897) was granted by IBAMA [Brazilian Environmental Protection Agen-
cy] and the fishing license (number 091/07) was granted by the Instituto Estadual de
Floresta de Minas Gerais, Brazil.

Description of karyotype in Aypostomus regani... 135

2.5
inal Piumhi
oP

Figure |. Map of Brazil highlighting the large hydrographic basin of the Parana-Paraguai system, area of
natural distribution of Hypostomus regain. Detail: area of the divider of the waters of the Upper Parana and
Sao Francisco River Basins, altered by the transposition of the Piumhi River (with original drainage to the
Grande River in the Upper Parana Basin) to the Upper Sao Francisco Basin through an artificial channel
that links it to the Sujo River (tributary of the Sao Francisco River). Star (*) and triangle (W) indicate
sampling sites for the H. regani populations studied by Artoni and Bertollo (1996, 2001) and Alves et al.
(2006) in the Mogi-Guacu and Tieté River Basins, respectively; circle (@) indicates the sampling site for
the 1. regani specimens analyzed in the present study in the confluence of the Rio Piumhi with to Rio Sao

Francisco, upper Rio Sao Francisco basin.

Karyotype data on the H. regani populations studied by Artoni and Bertollo
(1996), Artoni and Bertollo (2001) from the Mogi-Guagu River Basin and by Alves et
al. (2006) from the Tieté River Basin (Fig. 1) were also accessed (Table 1).

Chromosome preparations were obtained from cells from the anterior portion of
the kidney, using iz vivo treatment with colchicine (Bertollo et al. 1978). Nucleolus

136 Ernani de Oliveira Mendes-Neto et al. / Comparative Cytogenetics 5(2): 133-142 (2011)

organizer regions (NORs) were detected using silver nitrate (Ag-NORs), based on the
method described by Howell and Black (1980). C-positive heterochromatin was ana-
lyzed using the C-banding method (Sumner 1972).

Fluorescent in situ hybridization (FISH) was employed to locate ribosomal genes
in the chromosomes. An 18S rDNA probe from the fish Prochilodus argenteus (Agassiz,
1829) (Hatanaka and Galetti Jr. 2004) and a 5S rDNA probe from the fish Leporinus
elongatus Valenciennes, 1849 (Martins and Galetti Jr. 1999) were used to map the
rDNA sites on the chromosomes. Both probes were labeled with 14-dATP biotin by
nick translation, following the manufacturer’s instructions (Bionick Labeling System
— Invitrogen). Amplification and detection of the hybridization signals was carried out
using the avidin-FITC and anti-avidin biotin complex system (Sigma). FISH signals
were viewed based on the method described by Pinkel et al. (1986) and analyzed un-
der an epifluorescence microscope (Olympus BX51). The images of the chromosomes
were captured using the CoolSNAP-Pro software program (Media Cybernetics).

Approximately 30 metaphases from each specimen were analyzed in order to de-
termine the modal diploid number (2n), fundamental number (FN) and karyotype
formula. The chromosomes were identified based on the approach described by Levan
et al. (1964) and classified in the karyotype as metacentric (m), submetacentric (sm),
subtelocentric (st) and acrocentric (a).

Results

All the Hypostomus regani specimens analyzed in the present study had 2n = 72 chro-
mosomes with a karyotype formula 8m+16sm+20st+28a. The number of arms was FN
=")16;@labler1, Fig.2).

Constitutive heterochromatin was distributed in small blocks (Fig. 2,b). The inter-
stitial region of the long arms of subtelocentric chromosomes pairs 14, 18 and 20 as
well as acrocentric pairs 23, 26, 27, 28 and 33 had quite evident blocks. Metacentric
chromosome pair 1 had fainter labeling in the interstitial region of the short arm.
Chromosome pairs 13, 15 and 36 had heterochromatic blocks in the centromeric re-

Table |. Chromosomal data of the Hypostomus regani populations; from Artoni and Bertollo (2001) (Ref.
1), Alves et al. (2006) (Ref. 2) and new data from the population introduced into the Sao Francisco River
Basin (Ref. 3).

Locality 2n/FN Ag-NOR

Rio Mogi-Guagu, Rio Mogi- 72/116 10m+20sm+42st/a Multiple
Guacu basin 1 pair “a” large
1 par “st” small

Rio Araqua, Rio Tiete basin 72/116 12m+18sm+26st+16a | Multiple
2 pairs “a” largies

Confluence of the Rio Piumhi 72/116 8m+16sm+20st+28a Simple
with to Rio Sao Francisco, upper 1 pair “st” large
Rio Sao Francisco basin

Description of karyotype in Aypostomus regani... 137

gion. Heterochromatin was located in the telomeric region of the long arm in chromo-
some pair 31. Nucleolar organizing regions (NORs) labeled by silver nitrate were only
evident in the short arm of subtelocentric chromosome pair 15 (Fig. 2,c and 4,a). All
cells analyzed exhibited heteromorphism in relation to the size of the Ag-NORs.

Fluorescent in situ hybridization confirmed the presence of 18S rDNA coinciding
with the Ag-NORs as well as the size heteromorphism of the sites (Figs 3,a-c). The 5s
rDNA sites were located in four chromosome pairs: in the terminal region of the short
arm of two acrocentric pairs; in the centromeric region of one submetacentric pair; and
on another chromosome with no evident homologous labeling (Figs 3,b-d).

Discussion

The Piracicaba River, which is a tributary of the Upper Parana River, is the type locality
of 1. regani, although the natural distribution of the species is related to the Parana, Para-
guay and Uruguay River Basins (Carvalho and Bockmann 2007). The occurrence of H.
regani in the Sao Francisco River Basin is reported for the first time here and is added to
the data from the available taxonomic sources (Isbriicker 1980, Montoya-Burgos 2003,
Weber 2003, Armbruster 2004, Ferraris 2007). H. regani likely invaded the Sao Fran-
cisco River Basin after the transposition of the Piumhi River (Moreira-Filho and Buckup
2005). Although the field and taxonomic data confirm the origin of this species in the
Upper Parana River Basin, the data presented here indicate a divergence in the karyotype
macrostructure that involves the possibility of geographic variation. The different cyto-
types may have been isolated in allopatry approximately six million years ago (this dating
is based on the origin time of the basin of San Francisco river) (Montoya-Burgos 2003).
On the other hand, we must also consider the possibility of karyotype diversification oc-
curring within the same hydrographic basin, like the Upper Parana River Basin.
Karyotype differences in natural fish populations that inhabit the same hydro-
graphic basin have been found, for instance, in the genus Astyanax Baird et Girard,
1854. E.g. there are at least three different cytotypes of A. prope fasciatus (Cuvier,
1819) living in sympatry in the Upper Tibagi River, which is a tributary of the
Parana River (Artoni et al. 2006). According to Artoni et al. (2009), events of geo-
graphic vicariance stemming from the history of the South American continent are
among the factors to be considered in the karyotype diversification of Neotropical
freshwater fish. The authors also consider the evolutionary time for the fixation of
chromosome rearrangements and the biology of species. We must stress here that
H. regani is not a great migrator, as with the majority of Loricariidae, the anatomy
of which imposes difficulties on the movement of these fish in overcoming physical
barriers, such as waterfalls (Paiva et al. 2005). This imposition favors the formation
of more restricted population demes, which may accelerate the formation of new
cytotypes within geographically isolated areas through the action of genetic drift and
the restriction of gene flow. In Ancistrus Kner, 1854, for example, the great karyo-
typic variability may be related to biological and behavioral characteristics of these

138 Ernani de Oliveira Mendes-Neto et al. / Comparative Cytogenetics 5(2): 133-142 (2011)

in ai UT
bah aa a bi hd A ha

lier ue th as Ad ff aa AG 64 OD

cae Rs cls OO
b* ae
in vi Uh UR on
‘liek \ TIRE) ey

‘\¢¢ GO QR OH AA OA 84 Be Oe Op

23 24 Fase Bal IR: 20 28 30 = 31 32

#6 44 of fs
33 3403538

Figure 2. Hypostomus regani karyotypes from the confluence of the Piumhi and Sao Francisco Rivers a
chromosome stained with Giemsa and b sequentially labeled by C-banding; ¢ box indicates chromosome

pair labeled with silver nitrate locating the nucleolus organizer regions (pair no. 15). Bar = 10 pm.

armored catfish that include microhabitat preferences, territoriality and specialized
reproductive tactics, with consequences for the fixation of chromosomal rearrange-
ments and speciation (Oliveira et al. 2009).

Description of karyotype in Aypostomus regani... 139

Figure 3. Mitotic metaphases in Hypostomus regani from the confluence of the Piumhi and Sao Francisco

Rivers submitted to fluorescent in situ hybridization a showing two 18S rDNA sites (arrows) b nine 5S
rDNA sites (arrows) € chromosomes bearing 18S rDNA sites d chromosomes bearing 5S rDNA sites.
Bar = 10 um.

In a previous study, Artoni and Bertollo (2001) point to evolutionary trends for
the karyotype of the subfamilies of Loricariidae. Among those that exhibit extensive
variation in the diploid number, the genus Hypostomus stands out, with inter-species
variation ranging from 2n = 54 to 84 chromosomes, which demonstrates the strong
action of events of centric fission in the karyotype diversification of this group when
compared to more basal forms found in sister groups of Hypostominae, such as Lipo-
sarcus Gunther, 1864 (2n = 52), Rhinelepsis Agassiz, 1829 (2n = 54) and Pogonopoma
Regan, 1904 (2n=54) (Artoni et al. 1999, Artoni and Bertollo 2001). The data present
here for H. regani, in relation for the most basal Hypostomus species, also support the
Artoni and Bertollo’s hypothesis (op. cit.) regarding the location and distribution of
heterochromatin, especially in relation to the accumulation of equilocal and inter-
stitial heterochromatic blocks preferentially located in subtelocentric and acrocentric
chromosomes. Artoni and Bertollo (1999) propose that this tendency increases among
species of Hypostomus that have higher diploid numbers as a consequence of likely
translocations between non-homologous chromosomes in the interphase nucleus.

Especially regarding the species H. regani, we can highlight the location of nucleolus
organizer regions (NORs) as a variable inter-population character. ‘The results reveal dis-
tinct patterns of chromosome types and location that enable the 1. regani population in
the Piumhi River of the Sao Francisco Basin to be distinguished from the populations

140 Ernani de Oliveira Mendes-Neto et al. / Comparative Cytogenetics 5(2): 133-142 (2011)

in the Mogi-Guacu and Tieté Rivers analyzed by Artoni and Bertollo (1996, 2001)
and Alves et al. (2006), respectively. With these data on the gene activity of the NORs
reinforced by the in situ chromosome location of the 18s rDNA sites (FISH), we may
suggest that the colonizing H. regani individuals that invaded the Sao Francisco River
Basin facilitated by the transposition of the Piumhi River (Moreira-Filho and Buckup
2005) may have originated from a population of this species that inhabits the Grande
River Basin. In its turn, the last population is karyotypically distinct from other popula-
tions of this species that occur in the Upper Parana River Basin (Fig. 4).

The results obtained with the chromosomal location of 5S gene do not currently
allow any evolutionary inferences. However this cytotaxonomical marker may be im-
portant in future studies, especially regarding the number and location of this class of
ribosomal DNA in Hypostomus.

Besides the identification of a population of 1. regani belonging to the native ichthyo-
fauna of the Piumhi River basin, originally founded from the ichthyofauna of the Upper
Parana River, we also verified the introduction of an exotic species in the Sao Francisco
River Basin, with unpredictable consequences for the homeostasis of this environment.

Acknowledgements

The authors are grateful to the Instituto Estadual de Floresta de Minas Gerais (License
number 091/07) and IBAMA (Instituto Brasileiro do Meio Ambiente IBAMA/MMA/
SISBIO license number: 472897). This work was supported by FAPESP (Fundagao de
Amparo a Pesquisa do Estado de Sao Paulo, Processo: 06/54290-6), CNPg (Conselho
Nacional de Desenvolvimento Cientifico e Tecnolégico), CAPES (Coordenagao de
Aperfeicoamento de Pessoal de Nivel Superior).

Figure 4. Idiogram of chromosomes bearing nucleolus organizer regions labeled by silver nitrate, com-
paring Hypostomus regani populations from the Piumhi River in the Upper Sao Francisco Basin a and
Mogi Guacu b and Tieté c Rivers in the Upper Parana Basin. Bar = 1 um.

Description of karyotype in Aypostomus regani... 141

References

Alves AL, Oliveira C, Nirchio M, Granado A, Foresti F (2006) Karyotypic relationships among the
tribes of Hypostominae (Siluriformes: Loricariidae) with description of XO sex chromosome
system in a Neotropical fish species. Genetica 128: 1-9. doi:10.1007/s10709-005-0715-1

Armbruster JW (2004) Phylogenetic relationships of the suckermouth armoured catfishes (Lo-
ricariidae) with emphasis on the Hypostominae and the Ancistrinae. Zoological Journal of
the Linnean Society 141: 1-80. doi:10.1111/j.1096-3642.2004.00109.x

Artoni RE Bertollo LAC (1996) Cytogenetic studies on Hypostominae (Pisces, Siluriformes, Loricari-
idae). Considerations on karyotype evolution in the genus Hypostomus. Caryologia 49: 81-90.

ArtoniRE Bertollo LAC (1999) Nature and distribution of constitutive heterochromatin in fishes,
genus Hypostomus (Loricariidae). Genetica 106: 209-214. doi:10.1023/A:1003957719178

Artoni RF, Bertollo LAC (2001) Trends in the karyotype evolution of Loricariidae fish (Siluri-
formes). Hereditas 134: 201-210. doi:10.1111/j.1601-5223.2001.00201.x

Artoni RF, Molina WE, Bertollo LAC, Galetti Jr. PM (1999) Heterochromatin analysis in the
fish species Liposarcus anisitsi (Siluriformes) and Leporinus elongatus (Characiformes). Ge-
netics and Molecular Biology 22: 1-6. doi:10.1590/S1415-47571999000100009

Artoni RE Shibatta OA, Gross MC, Schneider CH, Almeida MC, Vicari MR, Bertollo LAC
(2006) Astyanax aff. fasciatus Cuvier, 1819 (Teleostei; Characidae): evidences of a species com-
plex in the upper rio Tibagi basin (Parana, Brazil). Neotropical Ichthyology 4(2): 197-202.

Artoni RE, Vicari MR, Almeida MC, Moreira-Filho O, Bertollo LAC (2009) Karyotype diver-
sity and fish conservation of southern field from South Brazil. Review in Fish Biology and
Fisheries 19: 393—401. doi:10.1007/s11160-009-9109-2

Bertollo LAC, Takahasha CS, Moreira-Filho O (1978) Cytotaxonomic considerations on Hop-
lias lacerdae (Pisces, Erythrinidae). Brazilian Journal of Genetics 1(2): 103-120.

Cardoso AR, Silva JFP (2004) Two new species of the genus Hemiancistrus Bleeker (Teleostei: Silu-
riformes: Loricariidae) from the upper Rio Uruguai basin. Neotropical Ichthyology 2(1): 1-8.
doi:10.1590/S1679-62252004000100001

Carvalho M, Bockmann FA (2007) Familia Loricariidae: Hypostominae. In: Buckup PA, Men-
ezes NA, Ghazzi MS (Eds.) Catalogo das espécies de peixes de agua doce do Brasil. Museu
Nacional. Rio de Janeiro, 91-98.

Cereali SS, Pomini E, Rosa R, Zawadzki CH, Froehlich O, Giuliano-Caetano L (2008) Karyo-
type description of two species of Hypostomus (Siluriformes, Loricariidae) of the Planalto
da Bodoquena, Brazil. Genetics and Molecular Research 7(3): 583-591. doi:10.4238/
vol7-3gmr404

Ferraris CJ Jr. (2007) Checklist of catfishes, recent and fossil (Osteichthyes: Siluriformes), and
catalogue of siluriform primary types. Zootaxa 1418: 1-628.

Hatanaka TE, Galetti Jr. PM (2004) Mapping of the 18S and 5S ribosomal RNA genes in the
fish Prochilodus argenteus, Agassiz, 1829 (Characiformes, Prochilodontidae). Genetica 122:
239-244. doi:10.1007/s10709-004-2039-y

Howell WM, Black DA (1980) Controled silver-staining of nucleolus organizer regions with the
protective coloidal developer: a 1-step method. Experientia 36: 1014-1015. doi:10.1007/
BF01953855

142 Ernani de Oliveira Mendes-Neto et al. / Comparative Cytogenetics 5(2): 133-142 (2011)

Isbriicker JH (1980) Classification and catalogue of the mailed Loricariidae (Pisces, Siluri-
formes). Verslagen en Technische Gegevens, Universiteit van Amsterdan 22: 1-181.

Kavalco KF, Pazza R, Bertollo LAC, Moreira-Filho O (2005) Karyotypic diversity and evolution
of Loricariidae (Pisces, Siluriformes). Heredity 94: 180-186. doi:10.1038/sj.hdy.6800595

Levan A, Fredga K, Sandberg AA (1964) Nomenclature for centromeric position of chromo-
somes. Hereditas 52: 201-220. doi:10.1111/j.1601-5223.1964.tb01953.x

Martins C, Galetti Jr. PM (1999) Chromosomal localization of 5S rDNA genes in
Leporinus fish (Anostomidae, Characiformes). Chromosome Research 7: 363-367.
doi:10.1023/A:1009216030316

Milhomem SSR, Castro RR, Nagamachi CY, Souza ACP, Feldeberg E, Pieczarka JC (2010)
Different cytotypes in fishes of the genus Hypostomus Lacépéde, 1803, (Siluriformes: Lori-
cariidae) from Xingu river (Amazon region, Brazil). Comparative Cytogenetics 4: 45—54.
doi:10.3897/compcytogen.v4il.31

Montoya-Burgos JI (2003) Historical biogeography of the catfish genus Hypostomus (Siluri-
formes: Loricariidae), with implications on the diversification of Neotropical ichthyofau-
na. Molecular Ecology 12: 1855-1867. doi:10.1046/j.1365-294X.2003.01857.x

Moreira-Filho O, Buckup PA (2005) A poorly known case of watershed transposition between
the Sao Francisco and upper Parana river basins. Neotropical Ichthyology 3(3): 449-452.

Muramoto JI, Ohno S, Atkin NB (1968) On the diploid state of the fish order Ostariophys.
Chromosoma 24: 59-66. doi:10.1007/BF00329607

Oliveira RR, Feldberg E, Dos Anjos MB, Zuanon J (2009) Mechanisms of chromosomal
evolution and its possible relation to natural history characteristics in Ancistrus catfishes
(Siluriformes: Loricariidae). Journal of Fish Biology 75: 2209-2225. doi:10.1111/j.1095-
8649.2009.02450.x

Paiva S, Renesto E, Zawadzki CH (2005) Genetic variability of Hypostomus (Teleostei, Loricari-
idae) from the Ribeirao Maringa, a stream of the upper Rio Parana basin, Brazil. Genetics
and Molecular Biology 28: 370-375. doi:10.1590/S1415-47572005000300005

Pereira EHL, Oyakawa OT (2003) Lsbrueckerichthys epakmos, a new species of loricariid catfish
from the rio Ribeira de Iguape basin, Brazil (Teleostei: Siluriformes). Neotropical Ichthyol-
ogy 1(1): 3-9. doi:10.1590/S1679-62252003000100001

Pinkel D, Straume T, Gray JW (1986) Cytogenetic analysis using quantitative, high-sensitivity,
fluorescent hybridization. Proceedings of the National Academy of Sciences 83: 2934-2938.
doi:10.1073/pnas.83.9.2934

Reis RE, Kullander SO, Ferraris Jr. CJ (2003) Check list of the freshwater fishes of South and
Central America. EDIPURCS, Porto Alegre, 729 pp.

Reis RE, Pereira EHL, Armbruster JW (2006) Delturinae, a new loricariid catfish subfamily
(Teleostei, Siluriformes), with revision of Delturus and Hemipsilichthys. Zoological Journal
of the Linnean Society 147: 277-299. doi:10.1111/j.1096-3642.2006.00229.x

Sumner AT (1972) A simple technique for demonstrating centromeric heterocromation. Ex-
perimental Cell Research 75: 304-306. doi:10.1016/0014-4827(72)90558-7

Weber C (2003) Subfamily Hypostominae (Armored Cartfishes). In: Reis RE, Kullander SO,
Ferraris Jr. CJ (Eds) Check list of the freshwater fishes of South and Central America. ED-
IPURCS, Porto Alegre, 351-372.