MOLECULARPHYLOGENETICSAND

Molecular Phylogenetics and Evolution 32 (2004) 152–163

EVOLUTION

www.elsevier.com/locate/ympev

Molecular phylogeny of the armored catfish familyCallichthyidae (Ostariophysi, Siluriformes)

Cristiane Kioko Shimabukuro-Dias,a Claudio Oliveira,a,* Roberto E. Reis,b

and Fausto Forestia

a Departamento de Morfologia, Instituto de Biociencias, Universidade Estadual Paulista, 18618-000 Botucatu, SP, Brazilb Laborat�orio de Ictiologia, Pontif�ıcia Universidade Cat�olica do Rio Grande do Sul, Av. Ipiranga, 6681, Caixa Postal 1429,

90619-900 Porto Alegre, RS, Brazil

Received 17 June 2003; revised 30 October 2003

Available online 17 January 2004

Abstract

The family Callichthyidae comprises eight genera of fishes widely distributed across the Neotropical region. In the present study,

sequences of the mitochondrial genes 12S rRNA, 16S rRNA, ND4, tRNAHis, and tRNASer were obtained from 28 callichthyid

specimens. The sample included 12 species of Corydoras, three species of Aspidoras, two species of Brochis, Dianema, Lepthoplo-

sternum, and Megalechis, and two local populations of Callichthys and Hoplosternum. Sequences of Nematogenys inermis (Nema-

togenyidae), Trichomycterus areolatus, and Henonemus punctatus (Trichomycteridae), Astroblepus sp. (Astroblepidae), and

Neoplecostomus paranensis, Delturus parahybae, and Hemipsilichthys nimius (Loricariidae) were included as the outgroup. Phylo-

genetic analyses were performed by using the methods of maximum parsimony and maximum likelihood. The results of almost all

analyses were very similar. The family Callichthyidae is monophyletic and comprises two natural groups: the subfamilies Co-

rydoradinae (Aspidoras, Brochis, and Corydoras) and Callichthyinae (Callichthys, Dianema, Hoplosternum, Lepthoplosternum, and

Megalechis), as previously demonstrated by morphological studies. The relationships observed within these subfamilies are in

several ways different from those previously proposed on the basis of morphological data. Molecular results were compared with the

morphologic and cytogenetic data available on the family.

� 2003 Elsevier Inc. All rights reserved.

Keywords: Molecular phylogeny; Callichthyidae; Systematic; Fish; Evolution

1. Introduction

The order Siluriformes is a very large fish group,widely

distributed across the tropical regions of the world (Bur-gess, 1989; Ferraris, 1998; Teugels, 1996). The number of

Siluriformes species known is about 2600, but it may ex-

ceed 3000 (Burgess, 1989; Ferraris, 1998; Nelson, 1994;

Teugels, 1996). Their impressive ecological and evolu-

tionary diversity is reflected in many studies of the group

(Fink and Fink, 1996; de Pinna, 1998).

Phylogenetic studies have shown that the families

Callichthyidae, Nematogenyidae, Trichomycteridae, Sco-loplacidae, Astroblepidae, and Loricariidae comprise the

* Corresponding author. Fax: +55-14-6821-3744.

E-mail address: claudio@ibb.unesp.br (C. Oliveira).

1055-7903/$ - see front matter � 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.ympev.2003.11.010

largest monophyletic group of catfishes in the neotrop-

ics, the superfamily Loricarioidea (de Pinna, 1998). The

family Callichthyidae includes 177 species currently

grouped in eight genera distributed throughout all majorriver basins of cis-Andean South America and trans-

Andean Colombia and Panama (Reis, 1998b, 2003).

They are easily recognized by the bony armor formed by

two longitudinal series of dermal plates that almost

completely protect their bodies. The phylogenetic rela-

tionships among the genera of the family have been re-

cently studied by Reis (1998a), who proposed the

cladogram shown in Fig. 1, in which the family iscomposed of two subfamilies, Callichthyinae and Co-

rydoradinae, as previously suggested by Hoedeman

(1952). Reis (1998a) demonstrated that all genera,

with the exception of Corydoras were monophyletic. The

Fig. 1. Phylogenetic relationships among genera of the Callichthyidae

according to Reis (1998a).

C.K. Shimabukuro-Dias et al. / Molecular Phylogenetics and Evolution 32 (2004) 152–163 153

relationships among Corydoras are more complex be-cause of the large number of species involved (143). The

relationships among corydoradines have been further

detailed by Britto (in press), who corroborated the

monophyletic nature of this subfamily and the genera

Aspidoras and Brochis. The monophyly of Corydoras

was not confirmed because some of its species are more

closely related to Aspidoras or Brochis than to nominal

congeners. In order to accommodate the monophyleticgroups found by him, Britto (in press) suggested divid-

ing the genus Corydoras and synonymizing Brochis to

Corydoras.

The development of molecular techniques has invigo-

rated the study of fish systematics. The methods devel-

oped for molecular systematics offer new sets of

characters for the analysis of the relationships among

fishes and have been effectively employed in studies in-cluding analyses from the level of populations to orders

(Kocher and Stephien, 1997). Studies of Neotropical

freshwater fishes including molecular approaches are fast

growing and their results seem to be very promising (Al-

ves-Gomes, 1998, 1999; Alves-Gomes et al., 1995; Farias

et al., 1998; Lovejoy and Collete, 2001; Martins et al.,

2003; Montoya-Burgos, 2003; Montoya-Burgos et al.,

1998; Ort�ı, 1997; Perdices et al., 2002; Sivasundaret al., 2001; Wimberger et al., 1998). In the present study,

part of the mitochondrial genes 12S rRNA, 16S rRNA,

ND4, tRNAHis, and tRNASer of specimens of the eight

callichthyid genera were sequenced and these sequences

were analyzed both alone and combined with morpho-

logical data (Reis, 1998a) so that an alternative phyloge-

netic hypotheses for the family could be suggested.

2. Material and methods

Twenty-five species (28 specimens) representing all

valid callichthyid genera were analyzed. The sample

group consisted of 12 species of Corydoras (including

two cytotypes of C. nattereri), three species of Aspidoras,

two species of Brochis, Dianema, Lepthoplosternum, and

Megalechis, two local populations of Callichthys

callichthys and two local populations of Hoplosternum

littorale. Sequences of Nematogenys inermis (Nematog-

enyidae), Trichomycterus areolatus and Henonemus

punctatus (Trichomycteridae), Astroblepus sp. (Astrobl-

epidae), and Neoplecostomus paranensis, Delturus para-

hybae, and Hemipsilichthys nimius (Loricariidae) were

used as the outgroup. Species, collecting sites and mu-

seum collection numbers are shown in Appendix A.

Total DNA was extracted from ethanol-preservedliver or muscle tissue with the Wizard Genomic DNA

Purification Kit (Promega). Partial sequences of the

mitochondrial genes 12S rRNA, 16S rRNA, ND4, and

tRNASer and the complete sequence of the mitochon-

drial gene tRNAHis were amplified by the polymerase

chain reaction (PCR) with the following primers: L1091

and H1478 (Kocher et al., 1989) for the gene 12S rRNA,

16Sa-L, and 16Sb-H (Palumbi et al., 1991) for the gene16S rRNA, as well as L11935 (50-CCA AAA GCA CAC

GTA GAA GC-30) and H12857 (50-ACC AAG AGT

TTT GGT TCC TA-30) for the genes ND4, tRNAHis,

and tRNASer. Primer concentrations were 5 lM. Am-

plifications were performed in a total volume of 25 ll for35 cycles (30 s at 95 �C, 60 s at 50–60 �C, and 120 s at

72 �C). PCR products were identified in 1% agarose gel.

The amplified segments were extracted from the gel withthe kit GFX PCR DNA and Gel Purification from

Amersham–Pharmacia Biotech. The sequencing reac-

tions were done with the Thermo Sequenase Cy 5.5 Dye

Terminator Cycle Sequencing Kit from Amersham–

Pharmacia Biotech. The sequences were determined in

an automatic sequencer from Visible Genetics, with the

aid of the software Gene Objects 3.0. All sequences were

read at least twice (forward and reverse).The sequences were aligned with the software Clu-

stalW (Thompson et al., 1994) as implemented in the

program DAMBE (Xia and Xie, 2001), with the follow-

ing multiple alignment options: DNA transitions weight

equal to 0.5, IUB DNA weight matrix, option ‘‘delay

divergent sequences’’ equal to 30, three gap open penal-

ties (5, 10, and 15), and three gap extension penalties (0.1,

1.0, and 2.0). Nucleotide saturation was analyzed byplotting the number of observed transitions (Ti) relative

to that of transversions (Tv) against genetic distance

values. Genetic distance analyses were based on a hier-

archical hypothesis test of alternative models imple-

mentedwithModeltest 3.06 (Posada andCrandall, 1998).

Molecular data were divided into three sections: 12S

and 16S rRNA, ND4, and tRNAs. Additionally, the

154 C.K. Shimabukuro-Dias et al. / Molecular Phylogenetics and Evolution 32 (2004) 152–163

published morphological data generated by Reis (1998a)were used. Since the present study was based on a spe-

cies level and the study by Reis (1998a) was mostly

conducted at a genus level, the morphological data re-

garding the genera with multiple species were replicated

(i.e., terminal taxa from the same genera were coded

identically). A similar procedure was successfully em-

ployed in a broad study of Hymenoptera (Dowton and

Austin, 2001). The incongruence length difference (ILD)test (Farris et al., 1995) was used to estimate any dif-

ference in phylogenetic signal among the different

molecular sections and among molecular and morpho-

logical data. The ILD test was performed using PAUP*

beta version 4.0b10 (Swofford, 2002), with 1000 ran-

domized replicates. For each replicate, heuristic searches

with the closest addition sequence option and tree

bisection–reconnection (TBR) branch swamping wereused. All uninformative characters were removed before

the analysis.

Maximum-parsimony (MP) based phylogenetic

analyses were performed using the software PAUP* beta

version 4.0b10 (Swofford, 2002) with heuristic searches

using random addition of sequences and the TBR al-

gorithm. In all analyses the character-state optimization

method employed was the accelerated transformation(ACCTRAN). Parsimony trees were generated using

1:1, 1:2, 1:3, and 2:3 Ti/Tv ratios, considering gaps as

either missing data or a fifth base. Bootstrap resampling

(Felsenstein, 1985) was applied to assess support for

individual nodes using 1000 replicates with 20 random

additions and TBR branch swapping. Decay indexes

(Bremer, 1988) were calculated with SEPAL (Salisbury,

2001).Maximum-likelihood (ML) based phylogenetic rela-

tionships were estimated using the software PAUP* beta

version 4.0b10 (Swofford, 2002). The GTR model

(Yang, 1994) incorporating rate variation (C) and

PINVAR with four C-distributed rate classes (Swofford

et al., 1996) were utilized for all likelihood analyses

based on a hierarchical hypothesis test of alternative

models implemented with Modeltest 3.06 (Posada andCrandall, 1998). The Ti/Tv ratio, gamma shape pa-

rameter, and proportion of non-variant sites were esti-

mated by maximum likelihood from a maximum

parsimony tree. Gaps were considered as missing data.

Bootstrap resampling was applied to assess support for

individual nodes using 100 replicates with 10 random

additions and TBR branch swapping. Additional ML

analyses were conducted with the software MetaPIGAversion 1.0.2b (Lemmon and Milinkovitch, 2002a). This

software implements a heuristic approach, the meta-

population genetic algorithm, involving several popu-

lations of trees that are forced to cooperate in the search

for the optimal tree (Lemmon and Milinkovitch, 2002b).

Analyses were conducted with the default options (one

population of four individuals) using the Hasegawa–

Kishino–Yano (HKY85) nucleotide substitution model(Hasegawa et al., 1985) and 1000 replicates. Consensus

trees were produced with the software TreeExplorer

implemented in MEGA 2.1 (Kumar et al., 2001).

3. Results

The sequences obtained in this study have been de-posited in GenBank under Accession Nos. AY307216–

AY307320 (Appendix A). All sequences were obtained

for all species, with the exception of Lepthoplosternum

tordilho in which the 30 end of the ND4 and the genes

tRNASer and tRNAHis were not sequenced, resulting in

a DNA fragment of 1230 bp.

The nine alignments obtained combining three gap

open penalties (5, 10, and 15) and three gap extensionpenalties (0.1, 1.0, and 2.0) were very similar. Six of the

alignments resulted in an identical matrix with 1614

positions and three alignments resulted in an identical

matrix with 1630 positions. The alignments with 1614

positions were chosen because they were more frequent

and exhibited fewer gap positions. Almost all positions

were very well aligned. However, two sections in the 16S

rRNA gene (with 27 and 8 bp) were difficult to align inall cases, and were excluded from the phylogenetic

analyses. Additionally, in the six alignments with 1614

positions, one section with 5 bp in 12S, and one section

with 4 bp in 16S were also hard to align and therefore

discarded in the phylogenetic analyses. The final matrix

had 1570 bp, from which 811 were conserved, 182 were

phylogenetically uninformative, and 577 were phyloge-

netically informative. The final alignment is availablefrom C. Oliveira upon request.

The percent base composition for sequenced regions

of the L-strand was determined as follows: adenine (A)

31.4; cytosine (C) 25.5; guanine (G) 18.7; and thymine

(T) 24.4. The analysis of these data clearly shows that

the base composition of the L-strand is somewhat A-

rich, similar to that described for several mitochondrial

genes of fishes (Alves-Gomes et al., 1995), lizards(Reeder, 1995), and snakes (Parkinson, 1999). On the

other hand, the base pair composition shows anti-G

bias, characteristic of mitochondrial genes but not of

nuclear genes (Zhang and Hewitt, 1996), as observed in

other fish species (Hrbek et al., 2002; Murphy and

Collier, 1999).

Molecular data were divided in three sections: 12S

and 16S rRNA, ND4, and tRNAs. The transitions/transversions (Ti/Tv) ratio observed was 1.4 for 12S and

16S rRNA, 0.7 for ND4, 3.5 for tRNAs, and 1.0 for the

total data. Fig. 2 shows the results obtained by plotting

transitions and transversions versus genetic distance

(Tamura and Nei, 1993). Linear relationships (coeffi-

cient of determination> 0.85) were found in almost all

analyses, indicating that these data were not saturated.

Fig. 2. Graphics showing the frequency of observed transitions and transversions versus genetic distance (Tamura and Nei, 1993). (A) 12S and 16S

rRNA genes; (B) ND4 gene; (C) tRNAs genes; (D) total molecular data. Transitions are black squares, transversions are open circles.

C.K. Shimabukuro-Dias et al. / Molecular Phylogenetics and Evolution 32 (2004) 152–163 155

The only two regressions which did not showed linear

relationships (coefficient of determination <0.75) were

those related to Ti in the ND4 gene (Fig. 2B) and Tv in

tRNA genes (Fig. 2C), suggesting that these data could

be possibly saturated.

The genetic distance among sequences was estimated

by the GTR model (Yang, 1994) incorporating rate

variation (C) and PINVAR with four C-distributed rateclasses (Swofford et al., 1996) based on a hierarchical

hypothesis test of alternative models implemented with

Modeltest 3.06 (Posada and Crandall, 1998). The mean

values of genetic distance were 0.149� 0.056 (0.000–

0.251) for callichthyids, 0.170� 0.047 (0.000–0.217) for

callichthyines, and 0.091� 0.024 (0.012–0.141) for

corydoradines. The oldest callichthyid fossil known is

Corydoras revelatus from late Paleocene, about 58.5million years ago (Lundberg et al., 1998; Reis, 1998b).

Considering such time as the minimum age of the origin

of Corydoras, the genetic distance among callichthyid

species may be considered low.

The ILD test detected a significant congruence (at the

P < 0:05 level) between molecular and morphological

data (Table 1). On the other hand, a significant incon-

Table 1

Results of incongruence length difference tests (Farris et al., 1994)

ND4 TRNAs Morphological

12S/16S 0.001 0.004 0.532

ND4 — 0.757 0.108

tRNAs — — 0.001

Molecular — — 0.168

Note. Numbers shown are P values. Numbers in bold indicate ILD

test is significant at the P < 0:05 threshold.

gruence (at the P < 0:05 level) was detected in three

pairwise comparisons of the datasets (Table 1). The 12S/

16S dataset was incongruent with both ND4 and tRNA

datasets, and the tRNA dataset was incongruent with

the morphological dataset. Bull et al. (1993) suggested

that incongruent data should not be combined for

phylogenetic analyses. However, Gatesy et al. (1999),

who advocate a ‘‘total evidence’’ methodology, showedthat the combination of incongruent data can increase

the resolution and the support within phylogenetic trees,

revealing if a ‘‘hidden signal’’ is present in the different

datasets. The approach combining putative incongruent

datasets has been recently used (Dowton and Austin,

2001; Lavou�e et al., 2003) with very good results. In the

phylogenetic analyses conducted in the present study,

the same approach was employed.Molecular data were analyzed under a range of

models, from the simplest (all characters changes

weighted equally) to the most parameter-rich (with

separate three-parameter step-matrices applied to each

molecular partition). Initially, a total of eight MP heu-

ristic searches were conducted, including or excluding

gaps, and considering the 1:1, 1:2, 1:3, and 2:3 Ti/Tv

ratios. Considering the differences in Ti/Tv ratios ob-served in the different molecular regions (12 and 16S

rRNA, ND4, and tRNAs), two additional MP heuristic

searches were conducted, including or excluding gaps,

and considering the Ti/Tv ratios of 2:3 for 12 and 16S

rRNA, 3:2 for ND4, and 2:7 for tRNA. The resultant

phylogenies had all the same topology, with a few dif-

ferences in the bootstrap values observed for some in-

ternal nodes. Fig. 3 exhibits the consensus tree obtainedfrom the analysis of 1000 bootstrap replicates (which

Fig. 3. Consensus MP tree produced when gaps were considered as

missing data and Ti/Tv ratio was 1:1 (TL¼ 2984, CI¼ 0.3934,

HI¼ 0.6066, RI¼ 0.5653). Numbers above branches are bootstrap

values based on 1000 replicates. Values below 50% are not shown.

Numbers below branches represent Bremer support index values. Dip-

loid numbers ð2nÞwere taken from several papers cited in the main text.

156 C.K. Shimabukuro-Dias et al. / Molecular Phylogenetics and Evolution 32 (2004) 152–163

generated 2296 trees), where gaps were considered as

missing data and Ti/Tv ratio was 1:1. The values ob-

tained for this tree were: tree length (TL)¼ 2984, con-

sistency index (CI)¼ 0.3934, homoplasy index

(HI)¼ 0.6066, and retention index (RI)¼ 0.5653.

Fig. 4. (A) Consensus ML tree produced with the software PAUP* ()Ln like

based on 100 replicates. (B) Consensus ML tree produced with the softwar

represent the branch support values found on 1000 replicates. Values below

ML analysis conducted with PAUP* resulted in aconsensus phylogeny similar to those obtained in the

MP analyses ()Ln likelihood¼ 14809.67752). Fig. 4A

shows the values P50% found in the 100 bootstrap

replicates. In this ML analysis, an interesting species

group consisting of the three Aspidoras species (boot-

strap value¼ 71) was found. The ML analyses with

MetaPIGA performed with 1000 independent metaGA

searches (strict consensus pruning among four popula-tions) generated 2000 trees. The majority-rule consensus

tree obtained from them was very similar, but

not identical to that obtained in the MP analyses

(Fig. 4B).

A MP analysis, combining molecular and morpho-

logical data equally weighed, resulted in a consensus

phylogeny very similar to those obtained in the MP

analyses of the molecular data. The consensus tree ob-tained from the analysis of 1000 bootstrap replicates

(which generated 2227 trees), where gaps were consid-

ered as missing data, Ti/Tv ratio was 1:1, and the

morphological data were considered ordered is shown in

Fig. 5A. The values obtained for this tree were:

TL¼ 3308, CI¼ 0.4205, HI¼ 0.5795, and RI¼ 0.6126.

The only significant difference observed in the topology

of the phylogeny shown in Fig. 5A when compared withthat shown in Fig. 3 was the presence of both Leptho-

plosternum species forming a monophyletic group with a

bootstrap value of 59.

Weighing morphological data five times the molecu-

lar data, the consensus phylogeny obtained is different

from those obtained in the analyses of the molecular

lihood¼ 14809.67752). Numbers above branches are bootstrap values

e MetaPIGA using the default parameters. Numbers above branches

50% are not shown.

Fig. 5. (A) Consensus MP tree produced combining molecular and morphological data equally weighed (TL¼ 3308, CI¼ 0.4205, HI¼ 0.5795,

RI¼ 0.6126). (B) Consensus MP tree produced weighing the morphological data five times the molecular data (TL¼ 3714, CI¼ 0.4585, HI¼ 0.5415,

RI¼ 0.7277). (C) Consensus MP tree produced weighing the morphological data ten times the molecular data (TL¼ 4199, CI¼ 0.4985, HI¼ 0.5015,

RI¼ 0.7981). Numbers above branches are bootstrap values based on 1000 replicates. Values below 50% are not shown.

C.K. Shimabukuro-Dias et al. / Molecular Phylogenetics and Evolution 32 (2004) 152–163 157

data. The consensus tree obtained from the analysis of1000 bootstrap replicates (which generated 2171 trees),

where gaps were considered as missing data, Ti/Tv ratio

was 1:1, and the morphological data were considered

ordered is shown in Fig. 5B. The values obtained for this

tree were: TL¼ 3714, CI¼ 0.4585, HI¼ 0.5415, and

RI¼ 0.7277. The difference observed in the topology of

the phylogeny shown in Fig. 5B when compared withthat shown in Fig. 3 was the presence of Aspidoras as

monophyletic (as observed in the ML analysis con-

ducted with PAUP*) and the division of the subfamily

Callichthyinae into two genera groups, one consisting of

Dianema and Hoplosternum and the other formed by

Callichthys, Megalechis, and Lepthoplosternum.

158 C.K. Shimabukuro-Dias et al. / Molecular Phylogenetics and Evolution 32 (2004) 152–163

Weighing morphological data 10 times the moleculardata, the consensus phylogeny obtained was very dif-

ferent from those obtained in the analyses of the mo-

lecular data. The consensus tree obtained from the

analysis of 1000 bootstrap replicates (which generated

2025 trees), where gaps were considered as missing data,

and Ti/Tv ratio was 1:1, as well as the ordered mor-

phological data were considered ordered is shown in

Fig. 5C. The values obtained for this tree were:TL¼ 4199, CI¼ 0.4985, HI¼ 0.5015, and RI¼ 0.7981.

The difference observed in the topology of the phylo-

geny shown in Fig. 5C when compared with that shown

in Fig. 3 was the presence of Aspidoras as monophyletic,

the species Corydoras macropterus isolated as the most

basal species of Corydoras, the subfamily Callichthyinae

composed of two lineages: one formed by the genus

Callichthys and the second by the genera Dianema,Hoplosternum, Megalechis, and Lepthoplosternum.

4. Discussion

The molecular phylogenies obtained by the MP and

ML methods were largely congruent and exhibited high

bootstrap and decay indexes supporting the main nodes,as shown in Figs. 3 and 4. Thus, the molecular data

corroborate the hypothesis that the family Callichthyi-

dae, the subfamily Callichthyinae and the subfamily

Corydoradinae are monophyletic groups. The same was

observed when molecular and morphological datasets

were combined (Fig. 5). These results are in accordance

with the initial hypothesis proposed by Hoedeman

(1952) and corroborated by the morphological phylo-genetic analyses conducted by Reis (1998a).

Regarding callichthyines, the topology obtained with

molecular data is, in some ways, distinct from that

proposed by Reis (1998a). Molecular data suggest that

Dianema is the primitive sister group of all other call-

ichthyines, Hoplosternum is the sister group of Callich-

thys, Lepthoplosternum, and Megalechis, and Callichthys

is the sister group of Lepthoplosternum and Megalechis

(Figs. 3 and 4). In all molecular analyses the two species

of Lepthoplosternum studied did not appear as a

monophyletic group, but this may be related to the fact

that complete sequence for L. tordilho was not obtained.

Disregarding this species level problem with the genus

Lepthoplosternum, the relative positions of the genera

Callichthys, Lepthoplosternum, and Megalechis in mo-

lecular phylogenies (Figs. 3 and 4) is in accordance withthe hypothesis of Reis (1998a). However, Dianema and

Hoplosternum as the primitive sister groups of all other

callichthyines is a curious result because the genus

Callichthys has been considered as the most primitive

callichthyid by several authors (Gosline, 1940; Hoed-

eman, 1952; Ribeiro, 1959) and was found to be the

most primitive clade of the callichthyines in the phylo-

genetic study conducted by Reis (1998a). Dianema spe-cies are, both morphologically and behaviorally, the

most differentiated among callichthyines (Reis, 1998a).

Furthermore, from the biogeographic standpoint, Dia-

nema does not seem to be basal, as it is distributed only

across the central Amazon, a region that is extremely

new considering the estimated age of the callichthyines.

By combining both molecular and morphological

data on callichthyines, some different results were ob-served (Fig. 5). Considering that only 72 morphological

characters of the family Callichthyidae were available

(Reis, 1998a) and that 577 nucleotides were phyloge-

netically informative, experiments with different weights

were conducted in order to avoid attributing too much

importance to molecular data. In the first experiment, all

characters were equally weighed, and the phylogeny

obtained was very similar to those obtained with mo-lecular data alone, but for the fact that the genus Lep-

thoplosternum was demonstrated to be monophyletic

(Fig. 5A). By weighing the morphological data five times

the molecular data, the subfamily Callichthyinae was

shown to consist of two units: one formed by Dianema

and Hoplosternum and the other formed by Callichthys,

Lepthoplosternum, and Megalechis with Callichthys as

the sister group of Lepthoplosternum and Megalechis

(Fig. 5B). When the morphological data were weighted

10 times the molecular data, the subfamily Callichthyi-

nae was demonstrated to include two units: one formed

by the genus Callichthys, and the other formed by Dia-

nema, Hoplosternum, Lepthoplosternum, and Megale-

chis, with Callichthys as the sister group of all other

callichthyines (Fig. 5C).

In contrast to the monophyly of the family andsubfamilies, the sister-group relationships among call-

ichthyines genera, as proposed on morphological

grounds, are not very strongly corroborated (Reis,

1998a). However, the topology suggested by the mo-

lecular analyses is not acceptable unless some morpho-

logical characters are considered to have evolved in a

less parsimonious way, increasing from 9 to 15 steps in

three internal nodes within callichthyines (see tree inFig. 36 of Reis, 1998a). Character 69.1 (color pattern of

young with transverse dark bands) would have to have

evolved in the ancestor of the whole subfamily and re-

versed in Callichthys. Similarly, character 56.2 (corac-

oids large and exposed ventrally) would have to have

evolved at the base of the subfamily, decreased in size in

Lepthoplosternum (state 56.1) and become covered by

skin in Callichthys (state 56.0). Character 60.1 (leadingray of pelvic fin with a skin fold dorsally) would have

increased from one to three steps: either evolving at the

base of the subfamily and disappearing in Callichthys

and Lepthoplosternum, or evolving independently in

Dianema, Hoplosternum, and Megalechis. Similarly,

characters 8.1 (first lateral-line ossicle with lateral ex-

pansions), 9.1 (second lateral-line ossicle with lateral

C.K. Shimabukuro-Dias et al. / Molecular Phylogenetics and Evolution 32 (2004) 152–163 159

expansions), and 54.1 (caudal fin bilobed), would haveto have evolved independently in Dianema and Hoplo-

sternum, or evolved in the ancestor of the subfamily and

subsequently disappeared in the ancestor of Callichthys,

Lepthoplosternum, and Megalechis. On the other hand,

character 5.1 (trigemino-facial foramen separated from

the optic foramen) presents a shorter optimization on

the molecular tree, having evolved in the ancestor of

Callichthys, Lepthoplosternum, and Megalechis. Fur-thermore, by increasing the tree length in six steps, the

molecular topology has no morphological support for

the clade Lepthoplosternum and Megalechis as well as

for the clade including all genera except Dianema, pro-

ducing a quite unresolved tree, if morphology is used to

support the molecular topology.

Some studies have shown that Dianema urostriata has

the lowest value of nuclear DNA content of the sub-family (1.18� 0.07 pg of DNA/nuclei) followed by Ho-

plosternum sp. (1.36� 0.11) and two populations of

Callichthys callichthys, 1.89� 0.24 and 1.94� 0.15

(Oliveira et al., 1993b). These data support the hy-

pothesis of Reis (1998a) and all hypotheses presented in

the present study that suggest that Dianema and Ho-

plosternum are more closely related to each other than to

other callichthyines. Considering that in the subfamilyCorydoradinae the nuclear DNA content tends to in-

crease among genera and species (Oliveira et al., 1993b),

the molecular phylogenies obtained in the present study

also suggest the occurrence of an increase in DNA

content in the evolutionary history of callichthyines. On

the other hand, if Callichthys is the most primitive genus

of Callichthyinae, a reduction in nuclear DNA content

must have occurred in the evolutionary history of thissubfamily.

Cytogenetic studies of callichthyines species have

shown that diploid numbers in this group display a

relatively low variation with values ranging from

2n ¼ 52 to 2n ¼ 66 (Oliveira et al., 1993b; Porto et al.,

1992) and that the karyotypes of Dianema, Hoploster-

num, and Megalechis are very similar with almost all

chromosomes being uniarmed. On the other hand, al-most all Callichthys chromosomes are biarmed (Oliveira

et al., 1993b). These cytogenetic data corroborate the

possible relationship among Dianema, Hoplosternum,

and Megalechis but do not help to identify the most

primitive genera in the subfamily. Considering that the

data available do not satisfactorily resolve the relation-

ships among Callichthyinae genera, further tests, with

additional data, are necessary.The analyses conducted on corydoradines showed

that they consist of at least three groups of species. The

first group is formed by the species of the genus Aspi-

doras and C. macropterus. The second group is formed

by Corydoras prionotos and C. barbatus, and the third

group is formed by the other species of Corydoras and

Brochis (Figs. 3, 4, 5A and B). These results are similar

to those obtained by Reis (1998a), who showed thatAspidoras and Brochis were monophyletic groups and

Corydoras was not. More recently, based on morpho-

logical data, Britto (in press) demonstrated in a phylo-

genetic study of corydoradines that Aspidoras and some

Corydoras species (including C. prionotos, C. barbatus,

and C. macropterus) belong to a natural group (tribe

Aspidoradini), and suggested that these Corydoras spe-

cies should be included in a separate genus. Our data donot entirely support this hypothesis.

All MP analyses with molecular data (under different

weight models) (Fig. 3), the ML analysis with MetaP-

IGA (Fig. 4B), and the analysis in which molecular and

morphological data were combined (weighting mor-

phological data equally to molecular data) (Fig. 5A),

showed that the species C. macropterus was the sister

group of Aspidoras poecilus. On the other hand, the MLanalyses conducted with PAUP* (Fig. 5A), and the MP

analysis combining molecular and morphological data

(weighing morphological data five times molecular data)

(Fig. 5B) showed that the three species of Aspidoras

studied comprise a monophyletic group that is the sister

group of C. macropterus. When the morphological data

were weighed 10 times the molecular data, the species

C. macropterus was found to be the sister group of allother species of Corydoras and Brochis (Fig. 5C). In all

analyses, C. prionotos and C. barbatus, were seen as a

monophyletic group that is the sister group of all other

species of Corydoras (excluding C. macropterus) and

Brochis (Figs. 3–5).

These results were unexpected because the morpho-

logical analyses conducted by Britto (in press) showed

that C. prionotos, C. barbatus, and C. macropterus be-long to a monophyletic group that is the sister group of

Aspidoras. Cytogenetic data showed that C. macropte-

rus, C. prionotos, and C. barbatus exhibit almost the

same karyotypic structure, suggesting that these species

should belong to a natural group (Oliveira et al., 1993a).

Additionally, although the nuclear DNA content found

among the species of Aspidoras is very similar to those

found in the species C. macropterus, C. prionotos, andC. barbatus, the diploid number exhibited by Aspidoras,

2n ¼ 44–46, is quite different from those found among

these Corydoras species, 2n ¼ 64–86 (Oliveira et al.,

1993a,b).

The two Brochis species analyzed were found to

represent a natural group in all analyses conducted

(Figs. 3–5). However, they were never found to be the

sister group of all other Corydoras species. Reis (1998a)suggested that Corydoras is not monophyletic if Brochis

is considered as a recognized genus. Phylogenetic studies

conducted by Britto (in press) also showed that the ge-

nus Corydoras is not monophyletic if Brochis is accepted

as a valid genus, and therefore suggested that Brochis

should be considered a synonym of Corydoras. Our

molecular study supports this view.

160 C.K. Shimabukuro-Dias et al. / Molecular Phylogenetics and Evolution 32 (2004) 152–163

The study conducted by Britto (in press) suggestedthat Corydoras difluviatilis is the primitive sister group

of all other Corydoradini species. In the present phylo-

genetic analyses this hypothesis was not corroborated

since C. difluviatilis was grouped with Corydoras sodalis

(Figs. 3 and 5) or as sister group of Brochis (Fig. 4).

Cytogenetic studies showed that C. difluviatilis has

2n ¼ 78 chromosomes (Shimabukuro-Dias et al., in

press) and C. sodalis has 2n ¼ 74 (our unpublished data)and the karyotypes of both species are very similar with

a high number of uniarmed chromosomes. Unfortu-

nately, C. sodalis was not included in the phylogenetic

study of Britto (in press). The presence of a high diploid

number and a high number of uniarmed chromosomes is

also a characteristic of Brochis species (Oliveira et al.,

1993b), reinforcing the hypothesis that these four species

may belong to a natural group (Fig. 3).Although only a small portion of the large genus

Corydoras has been analyzed, our molecular study

supports the monophyly of some species groups in the

tribe Corydoradini. Thus, the species Corydoras metae

and Corydoras araguaiensis were found to be sister

species in all analyses. These species are characterized by

the presence of a high diploid number, 2n ¼ 92 and

2n ¼ 94, respectively, and very similar karyotypicstructure (Oliveira et al., 1992; Shimabukuro-Dias et al.,

submitted). Additionally, in all analyses the species

Corydoras aeneus was found to be the sister group of

C. metae and C. araguaiensis.

All analyses performed provided a significant statis-

tical support for a second group, which included Cory-

doras flaveolus, Corydoras paleatus, two cytotypes of

C. nattereri and C. ehrhardti (Figs. 3–5). Although thephylogenetic study conducted by Britto (in press) pro-

vided no evidence that the species C. paleatus, C. nat-

tereri, and C. ehrhardti comprise a monophyletic group,

Oliveira et al. (1992, 1993a), based on the fact that these

species have almost the same karyotypic structure and

nuclear DNA content, suggested that they belong to a

natural group. The finding that one cytotype of

C. nattereri ð2n ¼ 42Þ is more closely related to C. ehr-

hardti than to a second cytotype of C. nattereri ð2n ¼ 44Þwas not expected (Figs. 3–5). Cytogenetic studies con-

ducted in three isolated samples identified as C. nattereri,

showed that each had their own diploid number (2n ¼ 40,

42 or 44) and karyotype (Oliveira et al., 1990). This

finding allowed the authors to suggest that these cytotypes

represent different unidentified species. The molecular

data herein presented support this hypothesis. However,additional samples should be analyzed before any final

conclusion is drawn.

The results obtained in the present study show that

the mitochondrial genes employed were able to recover

the high-level relationship patterns among the calli-

chthyid genera. However, considering that the rela-

tionship pattern among genera was not congruent with

that found in morphological studies, and that the rela-tionships among some Corydoras species were not well

resolved, two main points should be focused in future

studies: the use of additional sequences and a larger

number of species. The analysis combining molecular

and morphological data was very useful in demon-

strating the monophyletic nature of the genus Leptho-

plosternum, but as the amount of morphological data

was much reduced when compared to that of moleculardata, significant changes in molecular phylogenies were

observed only when special weight models were em-

ployed. Considering that the use of species weight

models may not be justifiable, further analyses of addi-

tional morphological data may be very helpful in the

understanding of the phylogeny of Callichthyidae.

Acknowledgments

The authors are grateful to Renato Devid�e for his

technical assistance, to Hern�an Ortega, Margarida

Carvalho, C�esar Cuevas, and Miguel Velasquez for their

help during the collection of several specimens, Marcelo

Britto for his help in the identification of some Cory-

doras and Aspidoras , M�ario de Pinna for his help in theidentification of Trichomycteridae species, and two

anonymous reviewers for their valuable comments.

Funds supporting this study were provided by FAPESP,

CAPES, and CNPq.

Appendix A

Museum numbers and collection sites of fish from

which DNA was extracted for this study (LBP¼Labo-

rat�orio de Biologia de Peixes, Instituto de Biociencias ofUniversidade Estadual Paulista, Botucatu, S~ao Paulo,

Brazil; MCP¼Museu de Ciencias e Tecnologia, Pon-

tif�ıcia Universidade Cat�olica do Rio Grande do Sul,

Porto Alegre, Brazil) and GenBank Accession Nos. (12S

rRNA/16S rRNA/ND4+ tRNAHis + tRNASer, in pa-

renthesis and in order).

A.1. Ingroup taxa

Aspidoras poecilus, c�orrego �Aguas Quentes, Araguaia

Riber basin, Barra do Garc�as, MT, Brazil, LBP1272

(AY307224/AY307259/AY307294); Aspidoras fuscogutta-

tus, c�orrego Araponga, Tiete River basin, Pen�apolis, SP,Brazil, LBP1295 (AY307223/AY307258/AY307293); As-

pidoras cf. fuscoguttatus, aquarium, LBP453 (AY307222/

AY307257/AY307292); Brochis britskii, a tributary of therio Pirai, Paraguay River basin (16�25.6800S,56�25.1430W), Pocon�e, MT, Brazil, LBP688 (AY307228/

AY307263/AY307298); Brochis splendens, aquarium,

LBP432 (Y307229/AY307264/AY307299); Callichthys

C.K. Shimabukuro-Dias et al. / Molecular Phylogenetics and Evolution 32 (2004) 152–163 161

callichthys, cytotype 1 ð2n ¼ 58Þ, ribeir~ao Santa Rita,Tiete River basin (23�55.5940S, 46�53.3360W), Embu

Guac�u, SP, Brazil, LBP485 (AY307240/AY307275/

AY307310);Callichthys callichthys, cytotype 2 ð2n ¼ 56Þ,c�orrego do Pombo, Tiete River basin,Mar�ılia, SP, Brazil,LBP485 (AY307241/AY307276/AY307311); Corydoras

aeneus, rio Araqu�a, Tiete River basin (22�47.1350S,48�28.8920W), Botucatu, SP, Brazil, LBP410 (AY307234/

AY307269/AY307304); Corydoras araguaiensis, aquar-ium, LBP435 (AY307232/AY307267/AY307302); Cory-

doras barbatus, rio S~ao Jo~ao (25�58,6490S, 48�52,9930W),

Garuva, PR, Brazil, LBP743 (AY307227/AY307262/

AY307297); Corydoras difluviatilis, c�orrego �Agua Boa,

Mogi-Guac�u River basin (22�23.0040S, 47�25.8190), Ar-

aras, SP, Brazil, LBP382 (AY307230/AY307265/

AY307300); Corydoras ehrhardti, c�orrego Ribeir~ao Ca-

valo, Itapucu River basin (26�28.2500S, 49�10.9580W),Jaragu�a do Sul, SC, Brazil, LBP741 (AY307235/

AY307270/AY307305); Corydoras flaveolus, rio Alam-

bari, Tiete River basin (22�5600800S, 48�1901500W), Bot-

ucatu, SP, Brazil, LBP397 (AY307239/AY307274/

AY307309);Corydoras macropterus, a tributary of the rio

Preto, Itanha�em River basin (24�10.8900S, 46�50.5630W),

Itanha�em, SP, Brazil, LBP1238 (AY307225/AY307260/

AY307295); Corydoras metae, aquarium, LBP428(AY307233/AY307268/AY307303); Corydoras nattereri,

cytotype 1 ð2n ¼ 44Þ, rio Marumbi (25�29.1970S,48�49.9780W), Morretes, PR, Brazil, LBP778

(AY307237/AY307272/AY307307); Corydoras nattereri,

cytotype 2 ð2n ¼ 42Þ, rio Fau, Ribeira de Iguape River

basin (24�12.4410S, 47�28.6160W), Miracatu, SP, Brazil,

LBP1266 (AY307236/AY307271/AY307306); Corydoras

paleatus, rio Gua�iba River basin (30�02.8200S,51�22.3470W), Eldorado do Sul, RS, Brazil, LBP568

(AY307238/AY307273/AY307308);Corydoras prionotos,

rio Fau, Ribeira de Iguape River basin (24�12.4410S,47�28.6160W),Miracatu, SP,Brazil, LBP1267 (AY307226/

AY307261/AY307296); Corydoras sodalis, aquarium,

LBP434 (AY307231/AY307266/AY307301); Dianema

longibarbis, aquarium, Purus River basin, AM, Brazil,

LBP557 (AY307242/AY307277/AY307312); Dianema

urostriata, aquarium, Purus River basin, AM, Brazil,

LBP558 (AY307243/AY307278/AY307313); Hoploster-

num littorale, Represa de Jurumirim, Paranapanema

River basin (19�34.6300S, 57�01.1230W), Angatuba, SP,

Brazil, LBP466 (AY307244/AY307279/AY307314); Ho-

plosternum littorale, rio Gua�ıba (30�02.8200S,51�22.3470W), Eldorado do Sul, RS, Brazil, LBP569

(AY307245/AY307280/AY307315); Lepthoplosternum

altamazonicum, swamp at the Pacaya River, Ucayali

River basin, Peru, MCP uncat. (AY307247/AY307282/

AY307317); Lepthoplosternum tordilho, a tributary of

the rio Gua�ıba (30�0203900S, 51�2600700W), Eldorado do

Sul, RS, Brazil, MCP 29383 (AY307249/AY307284/

AY307319); Megalechis personata, igarap�e do Almoc�o,Acre River basin, Rio Branco, AC, Brazil, LBP239

(AY307246/AY307281/AY307316); Megalechis thora-

cata, aquarium, upper rio Negro, AM, Brazil, LBP526

(AY307248/AY307283/AY307318).

A.2. Outgroup taxa

Nematogenyidae: Nematogenys inermis, estero Aguasde laGloria, Pacific drainage (S36�50.3040,W72�55.6420),Aguas de la Gloria, VIII Region, Chile, LBP1002

(AY307250/AY307285/AY307320). Trichomycteridae:

Trichomycterus areolatus, R�ıo Rehue, R�ıo Biobio

(S38�09.2030, W72�37.0050), Quechereguas, IX Region,

Chile, LBP994 (AY307217/AY307252/AY307287); He-

nonemus punctatus, lago Amap�a, Acre River basin

(S10�03.0380, W67� 50.8740), Rio Branco, AC, Brazil,LBP1125 (AY307216/AY307251/AY307286). Astrobl-

epidae: Astroblepus sp., Rio Santa, Pacific drainage

(S08�57054.60, W77�50007.400), Sucre, Ancash, Peru,

LBP1357 (AY307219/AY307254/AY307289). Loricarii-

dae: Neoplecostomus paranensis, c�orrego Hortel~a,Paranapanema River basin (S22�550, W48�300), Botuc-atu, SP, Brazil, LBP709 (AY307218/AY307253/

AY307288);Delturus parahybae, rio Pombas, Para�ıba doSul River basin (S21�2602800,W42�2601900), Laranjal,MG,

Brazil, MCP31467 (AY307220/AY307255/AY307290);

Hemipsilichthys nimius, rio Pereque-ac�u, Paraty, RJ,

Brazil, MCP31990 (AY307221/AY307256/AY307291).

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