Biochemical evidence of a possible new species of Neoplecostomus (Teleostei: Loricariidae) from the upper Rio Paraná basin, Brazil

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Biochemical Systematics and Ecology 32 (2004) 573–582

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Biochemical evidence of a possible new speciesof Neoplecostomus (Teleostei: Loricariidae)from the upper Rio Parana basin, Brazil

Claudio Henrique Zawadzki a,�, Anderson Luis Alves c,Erasmo Renesto b, Claudio Oliveira c

a Universidade Estadual do Centro-Oeste, CEDETEG, R. Simeao Camargo Varela de Sa 03, 85040-080,

Guarapuava, PR, Brazilb Universidade Estadual de Maringa, Nucleo de Pesquisas em Limnologia, Ictiologia e Aquicultura G-90,

Av. Colombo, 5790, 87020-900, Maringa, PR, Brazilc Universidade Estadual Paulista, Instituto de Biociencias, Departamento de Morfologia, Rubiao Jr., s/n,

18618-000, Botucatu, Sao Paulo, Brazil

Received 1 August 2002; accepted 17 September 2003

Abstract

Neoplecostomus paranensis Langeani, 1990, from the upper Rio Parana, is the only Neo-plecostomus species described in this basin and is distinguished from its congeners by thelack or reduction of the adipose fin. Neoplecostomus specimens with a normal and alwayspresent adipose fin were caught in the Rio Corumba, upper Rio Parana basin. In the presentstudy two samples of populations, one from a tributary of Rio Paranapanema (identified asa typical N. paranensis) and the other from the Rio Corumba were compared through allo-zyme electrophoresis. Six diagnostic loci were found, Acp-A, Adh-A, Est-A, Gpi-A, Ldh-Aand Ldh-B. In addition, the locus Gpi-B showed significant differences between allelicfrequencies for the two samples. Nei’s genetic identity between the populations was 0.731.The expressive genetic divergence together with the presence of an adipose fin show that thesample from the Rio Corumba is distinct from N. paranensis and probably represents a newspecies.# 2003 Published by Elsevier Ltd.

Keywords: Loricariidae; Neoplecostomus; Systematic; Allozymes; Heterozygosity; Genetic diversity;

Taxonomy

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C.H. Zawadzki et al. / Biochemical Systematics and Ecology 32 (2004) 573–582574

1. Introduction

The Neotropical subfamily Neoplecostominae holds a key position in the Lor-

icariidae phylogeny and has been considered a sister group of all other non-Litho-

geninae Loricariidae (de Pinna, 1998). The Neoplecostominae is a monogeneric

subfamily with the genus Neoplecostomus Eigenmann & Eigenmann, 1888, com-

prising seven species (Bizerril, 1995). Among these, Neoplecostomus paranensis Lan-

geani, 1990 is endemic from the upper Rio Parana basin, being diagnosed by the

lack or reduction of the adipose fin. In contrast to the original description of Neo-

plecostomus paranensis, specimens of Neoplecostomus collected in the Rio Corumba

(upper Rio Parana basin) bear a regular adipose fin. They have a similar mor-

phology and, despite the adipose fin evidence (Fig. 1), one could question the val-

idity of this character to separate these populations into distinct species. In such a

situation, genetic techniques, as auxiliary tools to systematic and taxonomy have

been used in several taxa including the Loricariidae. Zawadzki et al. (2000) showed

the usefulness of allozyme electrophoresis in the discrimination of similar species of

the loricariid genus Loricariichthys from southern Brazil. The allozyme electro-

phoresis was also performed by Fisch-Muller et al. (2001) to discriminate two cryp-

tic species of the genus Ancistrus from the upper Rio Tocantins basin and by

Zawadzki (2001) to diagnose 15 Hypostominae species from Itaipu reservoir in the

upper Rio Parana basin. In the present study the allozyme electrophoresis tech-

nique is used to compare the Neoplecostomus population from the Rio Corumba to

N. paranensis from the Hortela stream (upper Rio Parana basin) in order to resolve

the taxonomic status of the Corumba sample. The genetic variability of these two

) Neoplecostomus sp., NUP 2528, 77.1 mm SL; Brazil: Upper Rio Parana basin, Rio C

Fig. 1. (A orumba;

and (B) Neoplecostomus paranensis Langeani, 1990, NUP 2608, 73.0 mm SL; Brazil: Upper Rio Parana

basin, Hortela stream. Arrows indicate the adipose fin.

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575C.H. Zawadzki et al. / Biochemical Systematics and Ecology 32 (2004) 573–582

samples and the tissue-specific enzyme distribution for the genus Neoplecostomusare investigated.

2. Materials and methods

Two samples of populations from tributaries of the upper Rio Parana basin wereobtained: 15 specimens of Neoplecostomus sp. from tributaries of the Rio Cor-umba, Caldas Novas (Goias, Brazil) and 15 specimens of N. paranensis from Hor-tela stream, a tributary of the Rio Paranapanema, Botucatu (Sao Paulo, Brazil)(Figs. 1 and 2). Samples of white skeletal muscle, liver and heart tissues from livingspecimens were collected and stored in liquid nitrogen until the analyses. Electro-phoretic procedures follow Zawadzki et al. (1999). The nomenclature used forenzymes and alleles was proposed by the International Union of Biochemistry andMolecular Biology (1992). Data were analyzed using Biosys 1 software (Swofford

Fig. 2. Southern portion of South America showing the site of collection of Neoplecostomus sp., from

the Corumba reservoir (dot) and from Neoplecostomus paranensis Langeani, 1990 from Hortela stream

(star).

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C.H. Zawadzki et al. / Biochemical Systematics and Ecology 32 (2004) 573–582576

and Selander, 1981). The genetic interpretation of the enzymatic patterns wasbased on the quaternary structure of the enzymes described by Ward et al. (1992).Due to their small size, most individuals were totally destroyed at the excision of

tissues. Voucher specimens are deposited in the collections of Nupelia (NUP) atthe State University of Maringa, Brazil. Data concerning the material was arran-ged in the text as follow: museum acronym and catalog number, number of speci-mens, minimum and maximum standard length in millimeters, locality of collect,town of reference, geographic coordinates, date and collector.

2.1. Voucher specimens

Neoplecostomus paranensis: NUP 2608, 4, 59.2–91.3, Hortela stream, Rio Pardo,upper Rio Parana basin, Botucatu, Sao Paulo, S 25

v1401200 W 54

v1400200,

15.iv.1997, Anderson L. Alves.Neoplecostomus sp.: NUP 2528, 5, 46.9–77.1, corrego Taquari, Rio Corumba,

upper Rio Parana basin, Caldas Novas, Goias, 17 to 18vS/48

vW, iii.1996 to

iii.2000, Nupelia.

3. Results

Fourteen enzymatic systems were analyzed, allowing the detection of 31 allelesin 22 loci. Table 1 shows the enzymatic systems analyzed and Table 2 shows theallele frequencies of the two samples analyzed. All the polymorphic loci analyzed

Table 1

Names, number of enzyme commission (E.C. No.), tissues, buffers, quaternary structure (QS) and num-

ber of locus of each enzyme of Neoplecostomus sp. from Corumba reservoir and Neoplecostomus para-

nensis Langeani, 1990 from Hortela stream. L: liver; M: muscle; H: Heart; TBE: Tris/borate/EDTA

(pH 8.7) (Boyer et al., 1963); TC: Tris/citrate (pH 7.0) (Shaw and Prasad, 1970)

Enzyme (abbreviation)

E.C. No. Tissue Buffer QS L oci

Aspartate transaminase (Aat)

2.6.1.1 L, H, M TBE Dimeric 1

Acid phosphatase (Acp)

3.1.3.2 L TC Dimeric 1

Alcohol dehydrogenase (Adh)

1.1.1.1 L TBE Dimeric 1

Esterase (Est)

L TBE Dimeric 1

Glycerol-3-phosphate dehydrogenase

(G3pdh)

1.1.1.8

L, H, M TC Dimeric 2

Glucose-6-phosphate dehydrogenase

(G6pdh)

1.1.1.49

L TBE Tetrameric 1

Glucose-6-phosphate isomerase (Gpi)

5.3.1.9 L, H, M TC Dimeric 2

Isocitrate dehydrogenase, NADP+ (Icdh)

1.1.1.42 L, H, M TC Dimeric 2

L-Lactate dehydrogenase (Ldh)

1.1.1.27 H, M TC Tetrameric 2

Malate dehydrogenase (Mdh)

1.1.1.37 L, H, M TC Dimeric 3

Malate dehydrogenase-NADP+ (Mdhp)

1.1.1.40 L, H, M TC Tetrameric 2

Peroxidase (Per)

1.11.1..6 L, H TC Tetrameric 2

Phosphoglucomutase (Pgm)

5.4.2.2 L, H, M TC Monomeric 1

Superoxide dismutase (Sod)

1.15.1.1 L, H, M TBE Dimeric 1

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were in Hardy–Weinberg equilibrium. Neoplecostomus paranensis shows no poly-

morphism, whereas for Neoplecostomus sp. polymorphisms were observed at loci

Gpi-B, Ldh-B and Pgm-A. The loci Acp-A, Adh-A, Est-A, Gpi-A and Ldh-A

were fixed for different alleles in both species (Fig. 3). The unbiased genetic

identity of Nei (1978) for the two samples was I ¼ 0:731 and the genetic dis-

tance was D ¼ 0:314. The measures of genetic variability for the two samples

are presented in Table 3.The tissue-specific enzyme distribution for the genus Neoplecostomus was very

similar to the loricariid genus Hypostomus investigated by Zawadzki et al.

(2001).

Table 2

Allele frequencies estimated for Neoplecostomus sp. from Rio Corumba and N. paranensis Langeani,

1990 from Hortela stream

Locus A

llele N eoplecostomus sp. (n ¼ 15) N . paranensis (n ¼ 15)

sAat-A a

1 .000 1 .000

Acp-A a

� ��� 1 .000

b

1 .000 � ��� Adh-A a 1 .000 � ���

b

� ��� 1 .000

Est-A a

1 .000 � ��� b � ��� 1 .000

G3pdh-A a

1 .000 1 .000

G3pdh-B a

1 .000 1 .000

G6pdh-A a

1 .000 1 .000

Gpi-B a

0 .367 1 .000

b

0 .633 � ��� Gpi-A a � ��� 1 .000

b

1 .000 � ��� mIcdh-A a 1 .000 1 .000

sIcdh-A a

1 .000 1 .000

Ldh-A a

1 .000 � ��� b � ��� 1 .000

Ldh-B a

0 .033 � ��� b 0 .967 � ��� c � ��� 1 .000

sMdh-A a

1 .000 1 .000

sMdh-B a

1 .000 1 .000

mMdh-A a

1 .000 1 .000

sMdhp-A a

1 .000 1 .000

mMdhp-B a

1 .000 1 .000

Per-1 a

1 .000 1 .000

Per-2 b

1 .000 1 .000

Pgm-A a

0 .100 � ��� b 0 .900 1 .000

Sod-A a

1 .000 1 .000

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4. Discussion

According to Langeani (1990), Neoplecostomus species are morphometricallyvery similar and few morphologic characters are useful in distinguishing them.Neoplecostomus paranensis, N. microps (Steindachner, 1876), and N. espir-itosantensis Langeani, 1990 comprises a very homogeneous group without clearmorphological differentiation (Langeani, 1990).Cytogenetic techniques have been able to diagnose many non-described fish spe-

cies (e.g. Albert et al., 1999; Jesus and Moreira-Filho, 2000). Otherwise, Alves et al.(2001), found a diploid number of 54 chromosomes and interstitial NOR (nuc-leolar organizer region) in two species analyzed (Neoplecostomus paranensis and N.microps), suggesting that this genus has a stable karyotypic constitution makingdifficult the use of these data to identify species in the Neoplecostominae sub-family.Due to the morphological and karyotypic similarity among Neoplecostomus spe-

cies, other characters are necessary to identify new species in this group. In theNeotropical region, biochemical genetic approaches, as auxiliary tools to system-atic and taxonomy, show their usefulness for defining differences in morphologi-cally similar species.Allozyme electrophoresis has its strength in fixing species boundaries of sympa-

trid species. Among sexual diploid organisms the detection of a single allele fixed indifferent populations reflects a restriction in the gene flow and consequently theexistence of putative biologic species (Richardson et al., 1986; Murphy et al., 1996).In the analysis of allopatrid populations their taxonomic status are ascertained bythe extension of genetic divergence among them.In a previous work on the Rio Corumba basin, Zawadzki (2001) was able to dif-

ferentiate Neoplecostomus sp. (Neoplecostominae) from 10 species of the genusHypostomus (Hypostominae) obtaining 10 diagnostic loci (G3pdh-A, G3pdh-B,sIcdh-A, sMdh-A, sMdhp-A, sMdhp-B, Per-1, Per-2, Per-3 and Sod-A) and from a

Table 3

Genetic variability measures for 22 loci of Neoplecostomus sp. and N. paranensis Langeani, 1990. Num-

bers in parentheses are standard deviations. N: number of specimens; K: mean number of alleles per

locus; P0,99: frequency of polymorphic loci; Ho: mean observed heterozygosity; He: mean expected het-

erozygosity

Population N

K P 0.99 Ho He

Neoplecostomus sp. 1

5 1.1 (0.1) 1 2.5 0.019 (0.069) 0.030 (0.100)

N. paranensis 1

5 1.0 (0.0) 0 0.0 0.000 (0.000) 0.000 (0.000)

Fig. 3. (a) Schematic representation of electrophoretic patterns of eight enzymatic systems of Neocple-

costomus fishes. Ns: Neoplecostomus sp.; Np: Neoplecostomus paranensis Langeani, 1990; H: Heart; L:

Liver; M: Muscle. Only the most frequent allele is shown for each locus. (b) Schematic representation of

electrophoretic patterns of six enzymatic systems of Neocplecostomus fishes. Ns: Neoplecostomus sp.; Np:

Neoplecostomus paranensis Langeani, 1990; H: Heart; L: Liver; M: Muscle. Only the most frequent allele

is shown for each locus.

l

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sympatrid Hisonotus sp. (Hypoptopomatinae) at five loci (mAat-A, Acp-A, Adh-A,Gcdh-A and Pgm-A). The high genetic differences obtained by the authors wereexpected considering that these three genera belong to different subfamilies.The high genetic differences obtained between Neoplecostomus sp. and N. para-

nensis can be better understood when these data are compared to a similar study.Zawadzki et al. (2002) compared two populations of Hypostomus margaritifer(Regan, 1907) through allozyme electrophoresis, one from the Corumba and theother from Itaipu reservoir and did not find any fixed allelic difference between thetwo populations, only one significant allelic frequency difference at locus sAat-A,and an identity value of 0.988. For two conspecific samples inhabiting distinct geo-graphical areas the restriction of gene flow could lead to some independent evolv-ing loci, as could be the case of sAat-A. In the H. margaritifer study, the lowgenetic divergence is expected because the genome had not as yet reached repro-ductive isolation. In the present work the six fixed diagnostic loci on the 22 studiedsuggested the existence of two distinct Neoplecostomus species in the upper RioParana basin.The hypothesis that the populations from the Rio Corumba could represent

another described species of Neoplecostomus from neighboring basins, although nottested here, seems to be very unlikely. Loricariid fish, in general, are not known asgood migrators and besides, the small length of the species of the genus Neoplecos-tomus and their actual distribution restricted to headwater rivers also seems toindicate a low migration range. The Rio Parana basin has been isolated from otherclose related basins (such as the Sao Francisco and the Amazon basins) for severalmillions of years in which the local fauna of these basins evolved independently.The possibility that the Neoplecostomus sp. population from the Rio Corumba is asample of another described species present in the upper Rio Parana by headwaterriver captures is also not excluded here, although, this phenomena is also veryunlikely.The Rio Corumba is considered to present a relative endemism of its ichthyo-

fauna (Julio and Agostinho, in press) due to the presence of some rapids and can-yons at the Rio Paranaıba (Pavanelli and Britski, 1999) which reduces theprobability of this population to represent a sample of some southern describedspecies. From the close northern hydrographic drainage, the Rio Sao Franciscobasin, the population of the Rio Corumba can be separated from N. franciscoensisLangeani, 1990 by an expanded dorsal fin locking mechanism wider than the baseof the first dorsal ray (this mechanism is minor than the width of the first dorsal finray in N. franciscoensis) and from N. variipictus Bizerril, 1995 by a greater numberof teeth in the pre-maxillary and dentary, 15 to 21 and 10 to 14 (counts ofZawadzki, Pavanelli and Langeani, personal comunication) vs. 12 to 14 and 7, res-pectively.The unusual lack of allozyme genetic variability in N. paranensis from Hortela

stream could be explained by high endogamic force and ecological restriction para-meters maintained by efficient geographical barriers to species of Neoplecostomus.Thus, the low levels of genetic variability of N. paranensis from Hortela streamseems to indicate that this population is sedentary and restricted to small areas.

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Since Langeani (1990) cited N. paranensis to be widespread over the upper RioParana basin, our results could lead to the hypothesis that a morphological con-servatism could be hiding a higher diversity with undescribed species of Neoplecos-tomus waiting for new morphological, osteological and molecular investigation.

Acknowledgements

The authors are grateful to Francisco Langeani for reading the manuscript. Wealso thank Samuel Verıssimo and Wladimir M. Domingues for collecting samplesat Corumba reservoir and Maria S. Ribelatto Arita for reference corrections.Nupelia and FURNAS Centrais Eletricas provided us with logistical support. Thisstudy was partially supported by grants from the Coordenacao de Aperfei-coamento de Pessoal de Nıvel Superior (CAPES) and Fundacao de Amparo aEducacao do Estado de Sao Paulo (FAPESP).

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