Molecular phylogeny of the armored catfish family Callichthyidae (Ostariophysi, Siluriformes)

MOLECULAR PHYLOGENETICS AND EVOLUTION Molecular Phylogenetics and Evolution 32 (2004) 152–163 www.elsevier.com/locate/ympev

Molecular phylogeny of the armored catfish family Callichthyidae (Ostariophysi, Siluriformes) Cristiane Kioko Shimabukuro-Dias,a Claudio Oliveira,a,* Roberto E. Reis,b and Fausto Forestia a b

Departamento de Morfologia, Instituto de Bioci^ encias, Universidade Estadual Paulista, 18618-000 Botucatu, SP, Brazil 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, Lepthoplosternum, and Megalechis, and two local populations of Callichthys and Hoplosternum. Sequences of Nematogenys inermis (Nematogenyidae), Trichomycterus areolatus, and Henonemus punctatus (Trichomycteridae), Astroblepus sp. (Astroblepidae), and Neoplecostomus paranensis, Delturus parahybae, and Hemipsilichthys nimius (Loricariidae) were included as the outgroup. Phylogenetic 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 Corydoradinae (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. Ó 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 (Burgess, 1989; Ferraris, 1998; Teugels, 1996). The number of Siluriformes species known is about 2600, but it may exceed 3000 (Burgess, 1989; Ferraris, 1998; Nelson, 1994; Teugels, 1996). Their impressive ecological and evolutionary 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, Scoloplacidae, Astroblepidae, and Loricariidae comprise the * Corresponding author. Fax: +55-14-6821-3744. E-mail address: [email protected] (C. Oliveira).

1055-7903/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2003.11.010

largest monophyletic group of catfishes in the neotropics, the superfamily Loricarioidea (de Pinna, 1998). The family Callichthyidae includes 177 species currently grouped in eight genera distributed throughout all major river basins of cis-Andean South America and transAndean 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 relationships among the genera of the family have been recently studied by Reis (1998a), who proposed the cladogram shown in Fig. 1, in which the family is composed of two subfamilies, Callichthyinae and Corydoradinae, as previously suggested by Hoedeman (1952). Reis (1998a) demonstrated that all genera, with the exception of Corydoras were monophyletic. The

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2. Material and methods

Fig. 1. Phylogenetic relationships among genera of the Callichthyidae according to Reis (1998a).

relationships among Corydoras are more complex because 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 monophyletic groups found by him, Britto (in press) suggested dividing the genus Corydoras and synonymizing Brochis to Corydoras. The development of molecular techniques has invigorated the study of fish systematics. The methods developed for molecular systematics offer new sets of characters for the analysis of the relationships among fishes and have been effectively employed in studies including 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 (Alves-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; Sivasundar et 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 morphological data (Reis, 1998a) so that an alternative phylogenetic hypotheses for the family could be suggested.

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 (Nematogenyidae), Trichomycterus areolatus and Henonemus punctatus (Trichomycteridae), Astroblepus sp. (Astroblepidae), and Neoplecostomus paranensis, Delturus parahybae, and Hemipsilichthys nimius (Loricariidae) were used as the outgroup. Species, collecting sites and museum collection numbers are shown in Appendix A. Total DNA was extracted from ethanol-preserved liver 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 mitochondrial 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 gene 16S 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. Amplifications were performed in a total volume of 25 ll for 35 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 with the kit GFX PCR DNA and Gel Purification from Amersham–Pharmacia Biotech. The sequencing reactions 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 ClustalW (Thompson et al., 1994) as implemented in the program DAMBE (Xia and Xie, 2001), with the following multiple alignment options: DNA transitions weight equal to 0.5, IUB DNA weight matrix, option ‘‘delay divergent sequences’’ equal to 30, three gap open penalties (5, 10, and 15), and three gap extension penalties (0.1, 1.0, and 2.0). Nucleotide saturation was analyzed by plotting the number of observed transitions (Ti) relative to that of transversions (Tv) against genetic distance values. Genetic distance analyses were based on a hierarchical hypothesis test of alternative models implemented with Modeltest 3.06 (Posada and Crandall, 1998). Molecular data were divided into three sections: 12S and 16S rRNA, ND4, and tRNAs. Additionally, the

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published morphological data generated by Reis (1998a) were used. Since the present study was based on a species level and the study by Reis (1998a) was mostly conducted at a genus level, the morphological data regarding the genera with multiple species were replicated (i.e., terminal taxa from the same genera were coded identically). A similar procedure was successfully employed 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 difference in phylogenetic signal among the different molecular sections and among molecular and morphological data. The ILD test was performed using PAUP* beta version 4.0b10 (Swofford, 2002), with 1000 randomized replicates. For each replicate, heuristic searches with the closest addition sequence option and tree bisection–reconnection (TBR) branch swamping were used. 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 algorithm. 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 relationships 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 and Crandall, 1998). The Ti/Tv ratio, gamma shape parameter, and proportion of non-variant sites were estimated 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 MetaPIGA version 1.0.2b (Lemmon and Milinkovitch, 2002a). This software implements a heuristic approach, the metapopulation genetic algorithm, involving several populations 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 deposited 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 extension penalties (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 in all 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 phylogenetically informative. The final alignment is available from 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 Arich, 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 (coefficient of determination > 0.85) were found in almost all analyses, indicating that these data were not saturated.

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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.

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 rate classes (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 58.5 million 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 inconTable 1 Results of incongruence length difference tests (Farris et al., 1994)

12S/16S ND4 tRNAs Molecular

ND4

TRNAs

Morphological

0.001 —

0.004 0.757

—

—

—

—

0.532 0.108 0.001 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, showed that 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 heuristic 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 observed 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 differences in the bootstrap values observed for some internal nodes. Fig. 3 exhibits the consensus tree obtained from the analysis of 1000 bootstrap replicates (which

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Fig. 3. Consensus MP tree produced when gaps were considered as missing data and Ti/Tv ratio was 1:1 (TL ¼ 2984, CI ¼ 0.3934, HI ¼ 0.6066, RI ¼ 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. Diploid numbers ð2nÞ were taken from several papers cited in the main text.

generated 2296 trees), where gaps were considered as missing data and Ti/Tv ratio was 1:1. The values obtained for this tree were: tree length (TL) ¼ 2984, consistency index (CI) ¼ 0.3934, homoplasy index (HI) ¼ 0.6066, and retention index (RI) ¼ 0.5653.

ML analysis conducted with PAUP* resulted in a consensus phylogeny similar to those obtained in the MP analyses ()Ln likelihood ¼ 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 (bootstrap value ¼ 71) was found. The ML analyses with MetaPIGA performed with 1000 independent metaGA searches (strict consensus pruning among four populations) 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 morphological data equally weighed, resulted in a consensus phylogeny very similar to those obtained in the MP analyses of the molecular data. The consensus tree obtained from the analysis of 1000 bootstrap replicates (which generated 2227 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. 5A. The values obtained for this tree were: TL ¼ 3308, CI ¼ 0.4205, HI ¼ 0.5795, and RI ¼ 0.6126. The only significant difference observed in the topology of the phylogeny shown in Fig. 5A when compared with that shown in Fig. 3 was the presence of both Lepthoplosternum species forming a monophyletic group with a bootstrap value of 59. Weighing morphological data five times the molecular data, the consensus phylogeny obtained is different from those obtained in the analyses of the molecular

Fig. 4. (A) Consensus ML tree produced with the software PAUP* ()Ln likelihood ¼ 14809.67752). Numbers above branches are bootstrap values based on 100 replicates. (B) Consensus ML tree produced with the software MetaPIGA using the default parameters. Numbers above branches represent the branch support values found on 1000 replicates. Values below 50% are not shown.

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Fig. 5. (A) Consensus MP tree produced combining molecular and morphological data equally weighed (TL ¼ 3308, CI ¼ 0.4205, HI ¼ 0.5795, RI ¼ 0.6126). (B) Consensus MP tree produced weighing the morphological data five times the molecular data (TL ¼ 3714, CI ¼ 0.4585, HI ¼ 0.5415, RI ¼ 0.7277). (C) Consensus MP tree produced weighing the morphological data ten times the molecular data (TL ¼ 4199, CI ¼ 0.4985, HI ¼ 0.5015, RI ¼ 0.7981). Numbers above branches are bootstrap values based on 1000 replicates. Values below 50% are not shown.

data. The consensus tree obtained from the analysis of 1000 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: TL ¼ 3714, CI ¼ 0.4585, HI ¼ 0.5415, and RI ¼ 0.7277. The difference observed in the topology of

the phylogeny shown in Fig. 5B when compared with that shown in Fig. 3 was the presence of Aspidoras as monophyletic (as observed in the ML analysis conducted 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.

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Weighing morphological data 10 times the molecular data, the consensus phylogeny obtained was very different from those obtained in the analyses of the molecular 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 morphological data were considered ordered is shown in Fig. 5C. The values obtained for this tree were: TL ¼ 4199, CI ¼ 0.4985, HI ¼ 0.5015, and RI ¼ 0.7981. The difference observed in the topology of the phylogeny 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 Callichthyidae, 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 phylogenetic 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 callichthyines, Hoplosternum is the sister group of Callichthys, 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 molecular phylogenies (Figs. 3 and 4) is in accordance with the 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; Hoedeman, 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 species are, both morphologically and behaviorally, the most differentiated among callichthyines (Reis, 1998a). Furthermore, from the biogeographic standpoint, Dianema 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 observed (Fig. 5). Considering that only 72 morphological characters of the family Callichthyidae were available (Reis, 1998a) and that 577 nucleotides were phylogenetically 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 molecular data alone, but for the fact that the genus Lepthoplosternum 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 Callichthyinae was demonstrated to include two units: one formed by the genus Callichthys, and the other formed by Dianema, Hoplosternum, Lepthoplosternum, and Megalechis, with Callichthys as the sister group of all other callichthyines (Fig. 5C). In contrast to the monophyly of the family and subfamilies, the sister-group relationships among callichthyines genera, as proposed on morphological grounds, are not very strongly corroborated (Reis, 1998a). However, the topology suggested by the molecular analyses is not acceptable unless some morphological 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 in Fig. 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 reversed in Callichthys. Similarly, character 56.2 (coracoids 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 (leading ray 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 expansions), 9.1 (second lateral-line ossicle with lateral

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expansions), and 54.1 (caudal fin bilobed), would have to have evolved independently in Dianema and Hoplosternum, 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. Furthermore, 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, producing 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 subfamily (1.18
0.07 pg of DNA/nuclei) followed by Hoplosternum 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 hypothesis of Reis (1998a) and all hypotheses presented in the present study that suggest that Dianema and Hoplosternum are more closely related to each other than to other callichthyines. Considering that in the subfamily Corydoradinae the nuclear DNA content tends to increase 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 this subfamily. 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, Hoplosternum, and Megalechis are very similar with almost all chromosomes being uniarmed. On the other hand, almost 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 relationships 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 Aspidoras 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

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to those obtained by Reis (1998a), who showed that Aspidoras and Brochis were monophyletic groups and Corydoras was not. More recently, based on morphological data, Britto (in press) demonstrated in a phylogenetic 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 species should be included in a separate genus. Our data do not entirely support this hypothesis. All MP analyses with molecular data (under different weight models) (Fig. 3), the ML analysis with MetaPIGA (Fig. 4B), and the analysis in which molecular and morphological data were combined (weighting morphological 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 ML analyses 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 all other 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 morphological analyses conducted by Britto (in press) showed that C. prionotos, C. barbatus, and C. macropterus belong to a monophyletic group that is the sister group of Aspidoras. Cytogenetic data showed that C. macropterus, 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, and C. 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 genus 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.

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The study conducted by Britto (in press) suggested that Corydoras difluviatilis is the primitive sister group of all other Corydoradini species. In the present phylogenetic 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. Unfortunately, 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 karyotypic structure (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 statistical support for a second group, which included Corydoras flaveolus, Corydoras paleatus, two cytotypes of C. nattereri and C. ehrhardti (Figs. 3–5). Although the phylogenetic study conducted by Britto (in press) provided no evidence that the species C. paleatus, C. nattereri, 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. ehrhardti than to a second cytotype of C. nattereri ð2n ¼ 44Þ was not expected (Figs. 3–5). Cytogenetic studies conducted 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 callichthyid genera. However, considering that the relationship pattern among genera was not congruent with

that found in morphological studies, and that the relationships 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 demonstrating the monophyletic nature of the genus Lepthoplosternum, but as the amount of morphological data was much reduced when compared to that of molecular data, significant changes in molecular phylogenies were observed only when special weight models were employed. Considering that the use of species weight models may not be justifiable, further analyses of additional 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 Corydoras and Aspidoras , M
ario de Pinna for his help in the identification 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 (LBP ¼ Laborat
orio de Biologia de Peixes, Instituto de Bioci^encias of Universidade Estadual Paulista, Botucatu, S~ao Paulo, Brazil; MCP ¼ Museu de Ci^encias e Tecnologia, Pontif
ıcia Universidade Cat
olica do Rio Grande do Sul, Porto Alegre, Brazil) and GenBank Accession Nos. (12S rRNA/16S rRNA/ND4 + tRNAHis + tRNASer , in parenthesis and in order). A.1. Ingroup taxa
Aspidoras poecilus, c
orrego Aguas Quentes, Araguaia Riber basin, Barra do Garcßas, MT, Brazil, LBP1272 (AY307224/AY307259/AY307294); Aspidoras fuscoguttatus, c
orrego Araponga, Tiet^e River basin, Pen
apolis, SP, Brazil, LBP1295 (AY307223/AY307258/AY307293); Aspidoras cf. fuscoguttatus, aquarium, LBP453 (AY307222/ AY307257/AY307292); Brochis britskii, a tributary of the rio Pirai, Paraguay River basin (16°25.6800 S, 56°25.1430 W), 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

callichthys, cytotype 1 ð2n ¼ 58Þ, ribeir~ ao Santa Rita, Tiet^e River basin (23°55.5940 S, 46°53.3360 W), Embu Guacßu, SP, Brazil, LBP485 (AY307240/AY307275/ AY307310); Callichthys callichthys, cytotype 2 ð2n ¼ 56Þ, c
orrego do Pombo, Tiet^e River basin, Mar
ılia, SP, Brazil, LBP485 (AY307241/AY307276/AY307311); Corydoras aeneus, rio Araqu
a, Tiet^e River basin (22°47.1350 S, 48°28.8920 W), Botucatu, SP, Brazil, LBP410 (AY307234/ AY307269/AY307304); Corydoras araguaiensis, aquarium, LBP435 (AY307232/AY307267/AY307302); Corydoras barbatus, rio S~ ao Jo~ ao (25°58,6490 S, 48°52,9930 W), Garuva, PR, Brazil, LBP743 (AY307227/AY307262/
AY307297); Corydoras difluviatilis, c
orrego Agua Boa, 0 Mogi-Guacßu River basin (22°23.004 S, 47°25.8190 ), Araras, SP, Brazil, LBP382 (AY307230/AY307265/ AY307300); Corydoras ehrhardti, c
orrego Ribeir~ ao Cavalo, Itapucu River basin (26°28.2500 S, 49°10.9580 W), Jaragu
a do Sul, SC, Brazil, LBP741 (AY307235/ AY307270/AY307305); Corydoras flaveolus, rio Alambari, Tiet^e River basin (22°560 0800 S, 48°190 1500 W), Botucatu, SP, Brazil, LBP397 (AY307239/AY307274/ AY307309); Corydoras macropterus, a tributary of the rio Preto, Itanha
em River basin (24°10.8900 S, 46°50.5630 W), 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°29.1970 S, 48°49.9780 W), Morretes, PR, Brazil, LBP778 (AY307237/AY307272/AY307307); Corydoras nattereri, cytotype 2 ð2n ¼ 42Þ, rio Fau, Ribeira de Iguape River basin (24°12.4410 S, 47°28.6160 W), Miracatu, SP, Brazil, LBP1266 (AY307236/AY307271/AY307306); Corydoras paleatus, rio Gua
iba River basin (30°02.8200 S, 51°22.3470 W), Eldorado do Sul, RS, Brazil, LBP568 (AY307238/AY307273/AY307308); Corydoras prionotos, rio Fau, Ribeira de Iguape River basin (24°12.4410 S, 47°28.6160 W), 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); Hoplosternum littorale, Represa de Jurumirim, Paranapanema River basin (19°34.6300 S, 57°01.1230 W), Angatuba, SP, Brazil, LBP466 (AY307244/AY307279/AY307314); Hoplosternum littorale, rio Gua
ıba (30°02.8200 S, 51°22.3470 W), 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°020 3900 S, 51°260 0700 W), Eldorado do Sul, RS, Brazil, MCP 29383 (AY307249/AY307284/ AY307319); Megalechis personata, igarap
e do Almocßo, Acre River basin, Rio Branco, AC, Brazil, LBP239

161

(AY307246/AY307281/AY307316); Megalechis thoracata, aquarium, upper rio Negro, AM, Brazil, LBP526 (AY307248/AY307283/AY307318). A.2. Outgroup taxa Nematogenyidae: Nematogenys inermis, estero Aguas de la Gloria, 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); Henonemus punctatus, lago Amap
a, Acre River basin (S10°03.0380 , W67° 50.8740 ), Rio Branco, AC, Brazil, LBP1125 (AY307216/AY307251/AY307286). Astroblepidae: Astroblepus sp., Rio Santa, Pacific drainage (S08°570 54.60 , W77°500 07.400 ), Sucre, Ancash, Peru, LBP1357 (AY307219/AY307254/AY307289). Loricariidae: Neoplecostomus paranensis, c
orrego Hortel~ a, Paranapanema River basin (S22°550 , W48°300 ), Botucatu, SP, Brazil, LBP709 (AY307218/AY307253/ AY307288); Delturus parahybae, rio Pombas, Para
ıba do Sul River basin (S21°260 2800 , W42°260 1900 ), Laranjal, MG, Brazil, MCP31467 (AY307220/AY307255/AY307290); Hemipsilichthys nimius, rio Perequ^e-acßu, Paraty, RJ, Brazil, MCP31990 (AY307221/AY307256/AY307291).

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