Syst. Biol. 46(1)75-100, 1997
THE RADIATION OF CHARACIFORM FISHES AND THE LIMITS OF RESOLUTION OF MITOCHONDRIAL RIBOSOMAL DNA SEQUENCES
GUILLERMO ORT11'3 AND AXEL MEYER1'2 ^Department of Ecology and Evolution, State University of New York, Stony Brook, New York 11794-5245, USA 2Program in Genetics, State University of New York, Stony Brook, New York 11794, USA
Abstract.—Phylogenetic relationships among fishes from ostariophysan orders, characiform fami- Downloaded from https://academic.oup.com/sysbio/article/46/1/75/1685535 by guest on 28 September 2021 lies, and serrasalmin genera (e.g., "piranhas") were examined using partial mitochondrial ribo- somal DNA sequences of the 12S and 16S genes. Phylogenetic information content of these se- quences was assessed at three levels of taxonomic inclusiveness by analyzing the patterns of nucleotide substitution using secondary structure models. Conserved and variable regions were identified, mapped onto the structural models, and compared at increasing levels of taxonomic divergence. In general, loop regions (unpaired) exhibited a higher level of variation than did stem regions (paired). A high proportion of compensatory substitutions was observed in stem regions in three data sets, suggesting strong selection to maintain the secondary structure. Saturation due to multiple substitutions was indicated by decreasing transition / transversion ratios and strong structural constraints on variation in comparisons among orders of Ostariophysi but was not obvious among familes of Characiformes and was not detected among serrasalmin genera. Reli- able phylogenetic signal successfully reconstructed relationships among serrasalmin genera. How- ever, aside from a few well-supported clades, relationships could not be reconstructed with con- fidence among characiform families and ostariophysan orders. The reciprocal monophyly of African and Neotropical characiform lineages was rejected (based on maximum likelihood ratio tests), and some support for previous hypotheses based on morphology was provided by the molecular data. The radiation of characiform fishes is discussed in a historical biogeographic context. [Biogeography; Characiformes; mitochondrial DNA; molecular phylogenetics; Ostario- physi; phylogenetic information; secondary structure.]
Characiform fishes provide a prime ex- etfishes") have adaptations for "flight" up ample of the complex evolutionary and to several centimeters above water. Size biogeographical patterns often seen in range among characiforms is also remark- tropical and subtropical faunas. They con- able: the largest species are predatory stitute a group of ecologically and mor- forms known to reach 130 cm in length phologically diverse fish that live in rivers and a weight of 38 kg (Gery, 1977; Sverlij and lakes in Africa and the Neotropics. and Espinach Ros, 1986), whereas the The vast array of trophic specializations smallest, known as miniature species found among characiforms is comparable (Weitzman and Vari, 1988), have adults to that of dchlids and includes detritivory that do not exceed 26 mm in length (e.g., (mud eating), herbivory, planktivory some "tetras," glandulocaudines, lebiasin- (plankton filtering), predation, fin and ids). Only a few groups exhibit parental scale eating, and the notorious group pre- care. dation of piranhas. Some species have pe- The order Characiformes is divided into culiar morphological and physiological ad- 16 families (Greenwood et al., 1966) or 14 aptations for survival in extreme hypoxic families (Gery, 1977) (see the Appendix); 4 conditions commonly found in their flood- of these families are endemic to Africa (ca. plain environments (e.g., air breathing and 200 species), and the rest are endemic to special membranous extensions of the lips South and Central America (more than for gaseous exchange in the surface film of 1,200 species). The confinement of charac- the water column). Other species ("hatch- iforms to exclusively freshwater habitats renders them closely linked to the dynam- 3 Present address: Department of Genetics, Univer- ics of geological history and makes them sity of Georgia, Athens, Georgia 30602, USA. E-mail: an important model group for biogeo- [email protected]. graphic studies (e.g., Myers, 1938). A major 75 76 SYSTEMATIC BIOLOGY VOL. 46 issue in characiform biogeography con- multiple substitutions, and at "deep" lev- cerns the evolutionary consequence of els of divergence, where the more con- drift-vicariant events caused by the sepa- served sites contain useful phylogenetic in- ration of the African and South American formation. continents, approximately 84-106 million A recurring theme in all of these studies, years ago (Parrish, 1993; Pitman et alv and one of the most crucial issues in mo- 1993). The geographic distribution of lecular systematics, is understanding what Characiformes has been interpreted in a makes a molecule a suitable marker to phylogenetic framework by Lundberg trace organismal history. An understand- Downloaded from https://academic.oup.com/sysbio/article/46/1/75/1685535 by guest on 28 September 2021 (1993), using a provisional hypothesis of ing of the evolutionary behavior of molec- relationships. Phylogenetic relationships ular characters provides the basis for reli- among characiform lineages have been able phylogenetic analyses. The nucleotide controversial (e.g., Weitzman and Fink, substitution patterns (e.g., frequency of 1983; Uj, 1990; Buckup, 1991; Lucena, 1993; change, transition/transversion ratios, Vari, 1995), and so far have been addressed base compositional biases) might also be using morphological characters only (Fig. good indicators of the strengths and limi- 1). Because African and Neotropical forms tations of particular data sets. For some do not seem to form reciprocally mono- groups of organisms, highly corroborated phyletic groups, Lundberg (1993) suggest- phylogenies are available. These "known" ed that the origin of most major clades phylogenies, which are derived from con- must have preceded the continental split, gruent hypotheses based on several inde- and as a consequence, their current distri- pendent data sets, may provide good bution can only be explained by either un- benchmarks for empirical tests of candi- likely marine dispersals or a remarkably date molecular markers (e.g., Friedlander disproportionate extinction of African et al., 1992, 1994; Graybeal, 1994). A mol- taxa. To test current hypotheses of rela- ecule shown to harbor information that al- tionships as well as these biogeographic lows researchers to correctly arrive at these scenarios, a molecular phylogeny of the known phylogenies is deemed suitable for major groups of Characiformes is present- the study of other groups of taxa of com- ed. parable age of divergence. But at least two Most phylogenetic research using mito- possible shortcomings of this approach chondrial DNA (mtDNA) sequences of an- have been noted: (1) nucleotide substitu- imals has been most successful at the pop- tion rates may differ among taxa (e.g., Li ulation, species, and genus levels and Tanimura, 1987), and (2) the time of (reviewed by Wilson et al., 1985; Moritz et the radiation of a particular group of in- al., 1987; Avise et al., 1988; Meyer, 1994a, terest might not necessarily be well known 1994b; Simon et al., 1994). MtDNA evolves from the fossil record (Friedlander et al., at a high rate (e.g., Brown et al., 1982), and 1992,1994; Graybeal, 1994). Clearly, a gene phylogenetic information is presumably shown to be a good marker because it pro- lost over evolutionary time. However, Min- vided data for the recovery of the phylog- dell and Honeycutt (1990) and Hillis and eny of a particular group of taxa may not Dixon (1991) suggested that mitochondrial be appropriate for other taxa. ribosomal genes (which are some of the For any molecular marker, the limit of most conserved in the mitochondrial ge- phylogenetic resolution for recent diver- nome) could be used as phylogenetic gences is easily defined by the lack of vari- markers to resolve relationships among able characters among taxa. At the other taxa that had diverged as long as 300 or extreme, the limit of resolution for ancient 65 million years ago, respectively. Simon divergences must be determined by using et al. (1994) concluded that the 12S and 16S known phylogenies and looking for the de- ribosomal RNA (rRNA) genes are likely to gree of support that particular relation- be useful at the population level, where ships receive (e.g., Smith, 1989) and/or by highly variable sites are not saturated with analyzing the pattern of molecular evolu- 1997 ORTI AND MEYER—MOLECULAR PHYLOGENY OF CHARACIFORMES 77
(a) after Lucena, 1993 (b) Uj, 1990 I— Curimatidae fl—l— Brycon I '— Prochilodontidae I— '— Chalceus I I— Anostomidae Serrasalminae '— Chilodidae Triportheus Hemiodidae Astyanax Characidiidae Cheirodon I— Cynodontidae
Pseudocorynopoma j '— Acestrorhynchidae Downloaded from https://academic.oup.com/sysbio/article/46/1/75/1685535 by guest on 28 September 2021 Oligosarcus I I— Cynopotamidae Aphyocharax '— Characidae I— Poptella Aphyocharacidae '— Tetragonopterus Tetragonopteridae Cynopotamus Serrasalmidae Gnathocharax Brycon idae Acestrorhynchus C Rhaphiodon I— Lebiasinidae I '— Erythrinidae Erythrinidae
'— Ctenoluciidae Ctenoluciidae
(C) after Buckup, 1991 i—Ctenoluciidae (d) Vari, 1995
Lebiasinidae * Ctenoluciidae Erythrinidae Acestrorhynchus - Erythrinidae Cynopotamus Tetragonopterus Lebiasinidae Oligosarcus Brycon (e) Fink and Fink, 1981 Hemiodontidae Charaddiinae Curimatidae I Gymnotiformes Prochilodontidae J '— Siluriformes Anostomidae Chilodontidae H ' Charadformes Parodontidae I Cypriniformes HHiMilUWHJI Gonorynchiformes •c Distichodontidae
FIGURE 1. Phylogenetic hypothesis among ostariophysan and characiform taxa, based on morphological evidence. African taxa are enclosed in black boxes, (a) Modified from Lucena (1993). (b) From Uj (1990). (c) Modified from Buckup (1991). (d) From Vari (1995). (e) From Fink and Fink (1981). tion. The latter alternative may constitute DNA sequences for the phylogeny of cha- a complementary approach useful in the raciform fishes. We examined in detail nu- absence of preexisting information. We cleotide substitution patterns for data sets present an empirical study in which we with increasing levels of taxonomic inclu- used this approach to assess the limit of siveness, from closely related species and resolution of mitochondrial ribosomal genera (within the subfamily Serrasalmi- 78 SYSTEMATIC BIOLOGY VOL. 46
nae, which includes piranhas) to families (1989), for direct sequencing from both di- (of Characiformes) and orders (of Ostar- rections (Gyllensten and Erlich, 1988). Sin- iophysi, which includes Characiformes). gle-stranded DNA was concentrated and Assuming that increasing taxonomic di- desalted in Millipore Ultrafree-MC filters vergence translates into increasing diver- and sequenced by the dideoxy chain-ter- gence times, we describe the evolutionary mination method (Sanger et al., 1977) with behavior of the molecular characters, from the limiting primer from the asymmetric the earliest stages of divergence, to deter- PCR amplification, using a commercial kit
mine at what taxonomic level they cease to (Sequenase 2, U.S. Biochemical). Downloaded from https://academic.oup.com/sysbio/article/46/1/75/1685535 by guest on 28 September 2021 contain reliable phylogenetic information. Sequence Alignment and Phylogenetic MATERIALS AND METHODS Analyses With the DNA sequences obtained in Fish Specimens and DNA Sequences this study plus sequences of Eigenmannia, A total of 53 specimens representing all Rhamphichthys, and Apteronotus (Gymnoti- families of Characiformes (15 currently ac- formes), Hypostomus, Cetopsis, Trichomycte- cepted, see the Appendix) and all orders rus, and Malapterurus (Siluriformes), and of ostariophysan fishes were included in cyprinids (see Appendix for GenBank ac- this study. Species identifications, localities cession numbers), three major data sets of origin, museum collection numbers, representing different taxonomic levels GenBank accession numbers, and other were compiled: two were restricted to the relevant information are given in the Ap- characiforms, and the third included taxa pendix. Fish tissues for DNA extraction from all five ostariophysan orders. These were preserved in 70% ethanol. Genomic data sets were collected with the intention DNA was extracted from muscle tissue by of testing five major hypotheses of charac- Proteinase K/SDS dissolution and purified iform relationships (Vari, 1979, 1983, 1995; by phenol-chloroform extraction and eth- Buckup, 1991; Lucena, 1993) and one hy- anol precipitation (Maniatis et al., 1982; pothesis on the relationships among all Kocher et al., 1989). The polymerase chain ostariophysan orders (Fink and Fink, reaction (PCR; Saiki et al., 1988) was used 1981). The first data set contained 27 spe- to amplify segments of the small (12S) and cies, mostly from the family Characidae, large (16S) subunit RNA mitochondrial following Lucena (1993) in taxon selection genes. Double-stranded amplifications (Fig. la). The second characiform data set were performed in 25-|xl volumes contain- is more inclusive, containing 38 taxa rep- ing 67 mM Tris (pH 8.8), 6.7 mM MgCl^ resenting all characiform families. All four 16.6 mM (NH4)2SO4, 10 mM 2-mercapto- families of African characiforms were in- ethanol, 0.5 mM of each dNTP, 1 |xM of cluded to test whether African and Neo- each primer, 10-1,000 ng genomic DNA, tropical groups form separate monophy- and 0.5 units of Taq Polymerase (Perkin/ letic groups. The third data set included 22 Elmer-Cetus). The following primers were ostariophysan taxa. used: for 12S, L1091 and H1478 (Kocher et DNA sequences were aligned using al., 1989); and for 16S, 16Sar-L and CLUSTAL W 1.5, with default settings 16Sbr-H (Palumbi et al., 1991). These prim- (Thompson et al., 1994). For each data set, ers amplify fragments of the 12S and 16S sequences from both ribosomal subunits mitochondrial rRNA genes corresponding were concatenated into a single file, and a to positions 1091-1478 and 2510-3059 in single alignment was obtained. Additional the human mitochondrial genome, respec- alignments with different gap: change cost tively (Anderson et al., 1981). Gel purifi- ratios were also performed to detect align- cation of double-stranded products was ment-ambiguous sites, following Gatesy et followed by generation of single-stranded al. (1994a), where a site is considered am- DNA, obtained by asymmetric PCR using biguous when gap assignments are unsta- the protocol described by Kocher et al. ble, differing among alternative align- 1997 ORTI AND MEYER—MOLECULAR PHYLOGENY OF CHARACIFORMES 79 ments. Default settings for CLUSTAL W consistency index [RC]). Maximum likeli- are opening gap cost = 10, extending gap hood searches were done using empirical cost = 5; the alternative settings used were base frequencies, with the "quick" and opening gap cost = 5, extending gap cost "global" options in effect, and by jumbling = 4, and opening gap cost = 20, extending the input order of taxa until the same best gap cost = 8. Computer-generated align- tree was obtained at least three times. ments were then refined based on pub- Neighbor-joining reconstructions were lished secondary structure models de- based on Kimura's two-parameter distanc- scribed by Guttel et al. (1985), Dams et al. es (Kimura, 1981). Different character Downloaded from https://academic.oup.com/sysbio/article/46/1/75/1685535 by guest on 28 September 2021 (1988), and Neefs et al. (1991) for the small weightings and inclusion schemes were subunit rRNA molecule and by Guttel and used with these programs according to the Fox (1988) and Guttel et al. (1993) for the analysis of the nucleotide substitution pat- large subunit rRNA molecule. From these terns and are discussed with the resulting models, a structure for the piranha rRNA topologies. Bootstrapping (Felsenstein, sequences was previously derived (Orti et 1985) was used to estimate confidence in al., 1996) and was used here to adjust the the results (100 pseudoreplications for par- sequences to a secondary structure format. simony, 500 pseudoreplications for neigh- Alignment gaps were placed by CLUSTAL bor joining). W in stem regions only rarely, and they were either left there and considered as Patterns of Nucleotide Change "bulges" in the paired structure or moved Nucleotide substitution patterns and to contiguous unpaired regions. These compensatory mutations in stems are de- gaps were moved in agreement with our scribed for the 27-taxon characiform data structural model only if the gap assign- set and the 22-taxon ostariophysan data set ment within the stem disrupted several only because they represent both extremes otherwise complementary base-pair inter- of taxonomic divergence sampled in this actions. study. These patterns were compared with Phylogenetic inference was based on those of a previous study (Orti et al., 1996) maximum parsimony using PAUP 3.1.1 of a 33-taxon data set of piranhas and pa- (Swofford, 1993) and MacClade 3.0 (Mad- cus (subfamily Serrasalminae). The com- dison and Maddison, 1992), maximum putation of nucleotide substitutions among likelihood (Felsenstein, 1981) using fast- sequences was done by tracing the substi- DNAml 1.0.8 (Olsen et al., 1994) and tutions on the best tree obtained by the NUCML 2.2 (Adachi and Hasegawa, 1994), above methods rather than by pairwise and neighbor joining (Saitou and Nei, comparisons (Fitch and Markowitz, 1970; 1987) using MEGA 1.0 (Kumar et al., 1993). but see Collins et al., 1994). This approach Heuristic searches using PAUP were done provides an estimate of the changes that by stepwise random addition of taxa, with occurred across the phylogeny under the at least 20 replications and TBR branch assumption of maximum parsimony swapping with the MULPARS option in (Swofford and Maddison, 1992). Average effect, and by collapsing zero-length changes of character state were computed branches. Overall consistency indices (con- using MacClade (Maddison and Maddi- sistency index [CI], Kluge and Farris, 1969; son, 1992). Based on the secondary struc- retention index [RI], Farris, 1989) were cal- ture models, nucleotide composition and culated as a measure of fit between the substitution rates for stem and loop data and the reported topologies. Succes- regions were calculated separately, follow- sive approximations, or a posteriori re- ing the method outlined by Vawter and weighting (Farris, 1969; Carpenter, 1988), Brown (1993). Relative rates of change for was used to test the stability of resulting each structural category and for each gene topologies and also to choose among sev- fragment were calculated with and with- eral equally parsimonious trees (reweight- out corrections for category size. Relative ing was done by maximizing the rescaled rates of each kind of nucleotide substitu- 80 SYSTEMATIC BIOLOGY VOL. 46
TABLE 1. Summary of variable and phylogeneti- For stem regions, a tally of the changes cally informative sites by region based on the second- ary structure models from the 12S and 16S rRNA in paired nucleotides (single changes and genes. Values for the data set containing 22 ostario- double changes) that maintain and disrupt physan (O) taxa and the data set containing 27 charac- the pairing was used for comparison with iform (C) taxa are given. Data sets excluding align- expected values, following the method of ment-ambiguous sites are also shown. Alignment Dixon and Hillis (1993). gaps were treated as missing.
No. No. No. RESULTS
aligned variable informa- Downloaded from https://academic.oup.com/sysbio/article/46/1/75/1685535 by guest on 28 September 2021 nucleo tides sites tive sites DNA sequence fragments of approxi- Data O C O C O C mately 340 bp and 530 bp were obtained for the 12S and 16S genes, respectively. 12S These fragments were aligned to a second- Stems 155 155 51 47 35 31 ary structure format following the piranha Loops 142 130 81 70 62 53 All 325 319 142 123 104 89 12S and 16S models (Orti et al., 1996), and 16S stem and loop regions were identified. A synopsis of the sites assigned to each re- Stems 171 174 49 49 33 31 Loops 258 273 160 157 129 127 gion and the observed variation for the All 545 548 283 270 189 191 27-taxon characiform and 22-taxon ostar- Ambiguous iophysan data sets is shown in Table 1. For excluded 747 729 320 270 200 169 these two data sets, the alignments (12S Total (12S + 16S) 870 867 425 393 293 280 and 16S combined) required a total of 18- 25 and 14-27 alignment gaps per sequence, respectively. Indels (insertion/deletion tion (e.g., A <-» C, A <-» G, A <-> T) for each events) represented between 1.6% and structural category and for each gene frag- 3.1% of the aligned sequence length. Most ment were corrected for base composition. indels were 1 bp in length, and maximum Because loops, for example, are AC rich, indel length was 4 bp and 9 bp for the more changes between A and C than be- characiform and ostariophysan data sets, tween any other pair of nucleotides are ex- respectively. Alignment-ambiguous sites pected to occur there. Correction factors (Gatesy et al., 1994a) were detected in loop were defined by adding the percent com- regions and comprised 122 and 138 sites position for the category of both bases in- for the ostariophysan and characiform data volved in the change and dividing the sum sets, respectively. The aligned sequences by 50%. The observed number of changes (PAUP files) are available electronically at was then divided by this factor, so that if http: / / www.utexas.edu / ftp / depts / syst- there were compositional bias in favor of a biol and were published previously (Orti, pair of nucleotides, division by a number 1995). larger than unity would correct for this Sequence divergence among taxa for the overrepresentation (Vawter and Brown, 12S and 16S fragments combined is sum- 1993). marized in Table 2. Variation between os-
TABLE 2. Percent sequence divergence within and between taxa for the 12S and 16S fragments combined (ranges) for 22 ostariophysan taxa.
Between Taxa Within 2 3 4 5 1. Kneriidae 6.2 21.5-22.5 20.5-21.6 20.3-22.4 19.3-24.1 2. Cypriniformes 12.2 — 15.7-18.5 16.9-21.0 17.8-21.9 3. Siluriformes 11.3-14.0 — 15.1-19.5 11.4-20.0 4. Gymnotiformes 12.3-16.0 — 13.7-20.9 5. Characiformes 9.2-21.3 — 1997 ORTI AND MEYER—MOLECULAR PHYLOGENY OF CHARACIFORMES 81
Freq. of Change No. of Changes tariophysan orders ranged from 11.4% per Category per Site when Distichodus (Characiformes) and Hy- All 16S postomus (Siluriformes) were compared to Sites 24.1% for Kneria (Gonorhynchiformes) and 12S Nannostomus (Characiformes). Mean se- 16S Among genera Stems Zl (Serrasalminae) quence divergence among orders was 12S 1 >21% for comparisons between Gono- 16S Loops J rhynchiformes and the other ostariophysan 12S =r Downloaded from https://academic.oup.com/sysbio/article/46/1/75/1685535 by guest on 28 September 2021 orders and 17-18% among cyprinids, cat- 0 0.2 0.4 0.6 ) 1 2 3 fish, electric fish, and characiforms (oto- ( All 16S physans, sensu Fink and Fink, 1981). With- Sites 12S I in Characiformes, the most divergent = = • sequences (Boulengerella and Aphyocharax) 16S Among families Stems were 21.3% different, but average sequence 12S n (Characiformes) divergence among characiform taxa was 16S Loops I I 15-16%. 12S ZZ3 I Structural Constraints on Variation All 16S Sequence variation was similar among Sites 16S and 12S sequences but was larger in 12S = i loop than in stem regions (Fig. 2). For ex- 16S Among orders Stems ample, mean pairwise sequence diver- 12S z (Ostanophysi) 3-, gence was only 7% in stems but was 22% 16S Loops i in loops for the 27-taxon characiform data 12S =j—' I set. The relative frequency of change among genes and structural categories did not differ among comparisons of data sets FIGURE 2. Distribution of variation among struc- tural categories in the 12S and 16S sequences: changes comprising different levels of taxonomic among genera within the subfamily Serrasalminae (33 rank. The same proportions were observed taxa, from Orti et al., 1996), among families within in comparisons among genera within the the order Characiformes (27 taxa), and among orders subfamily Serrasalminae as among orders within the superorder Ostariophysi (22 taxa). The rel- of ostariophysans (Fig. 2, left panels). In ative frequency of change in the different genes and in stems and loops is the number of changes observed contrast, the actual number of changes per in the category divided by the total number of site increased with increasing level of di- changes occurring at all sites. The amount of change vergence, as expected. On average, the 12S per site in each category is the number of changes in and 16S sequences showed approximately the category divided by category size. All changes were reconstructed on the most-parsimonious trees 0.6 changes per site in comparisons among (Figs. 8, 9) with MacClade. serrasalmins but >1.5 changes per site in comparisons among characiform families and ostariophysan orders. No significant common type of change in loop regions increase in the number of changes per site and overall, but A <-> G transitions were was observed in comparisons among os- more abundant among stem substitutions, tariophysan orders relative to comparisons especially in the 12S sequences. This bias among families within Characiformes (Fig. in favor of A <-» G transitions in 12S stems 2, right panels). decreased with increasing sequence diver- Transition substitutions were the most gence. In comparisons among ostariophy- frequent changes observed in both genes, san orders, the number of both kinds of in all structural categories, and in all data transitions was approximately the same sets (Figs. 3, 4). But among transitions, the (Fig. 3, lower left panel). This decrease in proportion of A <-» G and C <-> T changes A <-> G bias was paralleled by an increase differed between structural classes. In both in double substitutions in stems (in both genes, C <-> T transitions were the most strands) as more distantly related sequenc- 82 SYSTEMATIC BIOLOGY VOL. 46
• All sites 12S • Stems 16S S3 Loops
Among genera (Serrasalminae)
i Downloaded from https://academic.oup.com/sysbio/article/46/1/75/1685535 by guest on 28 September 2021 Among families (Characiformes) o
Serrasalminae Characiformes Ostariophysi FIGURE 4. Transition /transversion ratios for changes reconstructed on the most-parsimonious tree If! ttiln- _ II lit for each of three fish data sets. Values for stem and Among orders (Ostariophysi) loop regions for each gene fragment are shown sep- arately. • = 16S stems; • = 12S stems; O = 12S loops; A = 16S loops.
might reach saturation levels sooner than U nit the 12S molecule. CT AG AT AC CG GT CT AG AT AC CG GT The most remarkable effect of structural Ts Tv Ts Tv constraints on level of variation among se- FIGURE 3. Relative frequencies of the different quences can be seen in Figures 5 and 6. types of base change (corrected for base composition) Regions within the sequences that are able in the 12S and 16S fish sequences. The average num- to accept mutations were well defined for ber of changes was reconstructed on the most-parsi- monious trees using MacClade. For each gene and both genes. In comparisons among closely structural category, the rates are expressed as a pro- related serrasalmin genera, these regions portion of the number of C «-» T changes. Ts = tran- were already well defined, and variation sitions; Tv = transversions. only accumulated within these regions in comparisons involving more distantly re- lated taxa. This effect is more pronounced es are compared. In the 16S stem sequenc- in 16S than in 12S because a few more vari- es, no such bias in favor of A <-» G transi- able regions were "recruited" in 12S, in tions was observed. Overall, transition bias comparisons among more distantly related was more pronounced in stem regions and taxa. Sequence variation seemed to reach in comparisons among recently diverged saturation in comparisons beyond the fam- taxa (Fig. 4). The transition/transversion ily level; no obvious difference in sliding ratio was as high as 8-11 among serrasal- window profiles between the characiform min stem sequences but dropped to 3-5 in and ostariophysan data sets could be de- comparisons among ostariophysans. With tected (Figs. 5, 6, lower two panels). This increasing taxonomic divergence the pro- observation, together with the leveling off portion of transversions increased, es- of the transition /transversion ratio and the pecially in loop regions (Figs. 3, 4), but constancy in number of changes per site, there seemed to be no significant change strongly suggests saturation among se- in this proportion when going from cha- quences in the data set comparing ostar- raciform to ostariophysan comparisons. iophysan taxa and to a lesser extent in the This leveling off of the transition/trans- data set comparing characiform families. version ratio is more pronounced in the Base composition for the different struc- 16S sequences than in the 12S sequences tural categories of the three 12S and 16S (Fig. 4), suggesting that the 16S molecule data sets is shown in Figure 7. Overall, 1997 ORTI AND MEYER—MOLECULAR PHYLOGENY OF CHARACIFORMES 83 Downloaded from https://academic.oup.com/sysbio/article/46/1/75/1685535 by guest on 28 September 2021
(a)
(b)
(c) Position FIGURE 5. Secondary structure model for the piranha (Pygocentrus nattereri) 12S sequence (from Orti et al., 1996). The three panels under the model show a sliding window analysis of variation (window size = 7, overlap = 1) for the serrasalmin (a), charadform (b), and ostariophysan (c) data sets. The number of variable sites in the window is plotted on the position along the sequence. Letters indicate the regions where variation among sequences is greatest. both gene fragments showed very similar There was no variation in average base base composition, with a slight overrepre- composition among the different data sets, sentation of A and C. Stem and loop but the range of variation in base compo- regions differed in their content of A and sition among taxa increased slightly with G but not in their content of T and C. taxonomic divergence. However, no obvi- 84 SYSTEMATIC BIOLOGY VOL. 46 Downloaded from https://academic.oup.com/sysbio/article/46/1/75/1685535 by guest on 28 September 2021
16S rRNA
UGGUG - A\<7 I I I I • • • ' UUAUCGC
(a)
(b)
(c) Position FIGURE 6. Secondary structure model for the piranha (Pygocentrus nattereri) 16S sequence (from Orti et al., 1996). The three panels under the model show a sliding window analysis of variation (window size = 7, overlap = 1) for the serrasalmin (a), characiform (b), and ostariophysan (c) data sets. The number of variable sites in the window is plotted on the position along the sequence. Letters indicate the regions where variation among sequences is greatest. 1997 ORTI AND MEYER—MOLECULAR PHYLOGENY OF CHARACIFORMES 85
TABLE 3. Substitutions observed in stem regions of 12S 16S the 12S and 16S sequences for the 22 ostariophysan (O) taxa and the 27 characiform (C) taxa.
Expected Observed Type of substitution O Single Base pairing to base pairing 11.6 11.1 44 48 Base pairing to non-base pairing 81.4 77.9 49 41 Downloaded from https://academic.oup.com/sysbio/article/46/1/75/1685535 by guest on 28 September 2021 Double Base pairing to base pairing 21.2 19.7 75 72 Base pairing to non-base pairing 61.8 57.3 8 5
realized (if all mutations were compensa- tory substitutions, this value would be 100%). For the ostariophysan data set, 68.3% of all potential compensatory sub- stitutions were observed (194 of 259, of which only 54 were expected by chance). FIGURE 7. Base composition for stem, loops, and For both data sets analyzed, the proportion all sites for the 12S and 16S sequences in three fish of potential compensatory substitutions data sets. Mean values and ranges are shown. was high, suggesting strong selection for maintaining secondary structure. A simi- ous taxonomic bias in base composition lar observation was reported for a compar- was evident in any of the data sets. ison of the same gene fragments among serrasalmin genera (Orti et al, 1996). How- Secondary Structure and Compensatory ever, sequence divergence among serra- Changes salmins (piranhas and pacus) was maxi- The secondary structure models pro- mally 8.9%, compared with the 11-24% posed for the piranha sequences (Orti et divergence reported here. The percentage alv 1996) showed remarkable conservation of all potential compensatory substitutions across the ostariophysan and characiform observed in stems was 73.6% for the ser- taxa examined. The number of observed rasalmin data set (Orti et al., 1996), almost and expected compensatory substitutions identical to the values reported here. (Dixon and Hillis, 1993) for both gene frag- For both data sets most changes in stems ments and for both data sets is shown in occurred as pairs, rather than singly (when Table 3. Most substitutions in stems did a base changed in one strand, there was a not disrupt base-pairing interactions, and corresponding base change in the comple- there were significantly more compensa- mentary strand). Single substitutions (re- tory mutations than expected by chance (x2 gardless of whether or not they caused = 103.2 and 139.9 for single substitutions compensatory changes) comprised only and x2 = 183.1 and 186.5 for double sub- 36% of all changes in stems (same value stitutions for the ostariophysan and cha- for both data sets), in contrast to 60.9% for raciform data sets, respectively; df = 1, P the piranha data set (Orti et al., 1996). Be- < 0.001). For the characiform data set, 192 cause U-G pairing is accepted in stems, of 243 substitutions in stem regions did single transitions (C <-> T and A <^> G) may not disrupt base-pairing interactions (only not disrupt secondary structure. Transition 50.5 nondisrupting mutations were expect- bias is highest in stems for the serrasalmin ed by chance alone). Thus, 73.5% of all po- data set (Fig. 4). As the level of divergence tential compensatory substitutions were among taxa increases, so does the number 86 SYSTEMATIC BIOLOGY VOL. 46
Hypostomus Trichomycterus Malapterurus Cetopsis Siluriformes
Eigenmannia Downloaded from https://academic.oup.com/sysbio/article/46/1/75/1685535 by guest on 28 September 2021 Rhamphichthys Apteronotus Gymnotiformes Hoplias Leporinus Phenacogrammus Metynnis Chalceus 100 Paracheirodon 100 65 Nannostomus 65 Boulengerella Gasteropelecus Crossostoma Cyprinus Cypriniformes - Kneria - Parakneria FIGURE 8. Shortest tree for the 22-taxon ostariophysan data set (all characters equally weighted, gaps treated as missing data). Gonorhynchiforms (Kneria and Parakneria) were treated as the outgroup. L = 1,460, CI = 0.429 (excluding uninformative characters), and RI = 0.384. Bootstrap support is shown only for those branches where at least one value was >50 (neighbor-joining bootstrap values above branches; parsimony values below branches). African characiform taxa are shown in black boxes. of double changes and transversions in physans (Fink and Fink, 1981). Parsimony stems (Fig. 4), but the proportion of com- analysis of the ostariophysan 22-taxon data pensatory mutations remains at about the set resulted in a single most-parsimonious same level. Gatesy et al. (1994b) observed tree (length [L] = 1,460) when all charac- that the time lag for compensatory substi- ters were equally weighted and gaps were tutions in the 12S and 16S genes of ante- coded as missing data (Fig. 8). Monophyly lopes was rather small and suggested an of all orders except Characiformes was increase in the rate of compensatory sub- well supported. The clade formed by Cith- stitutions relative to other changes as a arinus and Distichodus, representing two possible cause. The level of compensatory African characiform families, grouped mutations observed for the mitochondrial with catfishes rather than with the other 12S and 16S genes (close to 70%) was high- characiforms. There were nine suboptimal er than that for the 28S gene (38%) report- trees a single step longer (L ^ 1,461), a ed by Dixon and Hillis (1993). strict consensus of which showed no res- olution among otophysan orders and the Phylogenetic Relationships among citharinid-distichodontid clade. Forcing Ostariophysan Orders characiform monophyly required only Gonorhynchiforms (family Kneridae) three extra-steps (L = 1,463). Bootstrap were used as outgroup taxa for the oto- values <50 for all relationships among or- 1997 ORTI AND MEYER—MOLECULAR PHYLOGENY OF CHARACIFORMES 87 ders indicate that resolution of deep Bryconinae, in part), (7) Pygocentrus + Co- branches in the tree is poor, and low con- lossoma (subfamily Serrasalminae), and (8) sistency indices (CI = 0.429, RI = 0.384) Citharinus + Distichodus. NJ also supported indicate that the level of homoplasy in the clades 1-8 with high bootstrap values, but data set is high. The tree resulting from relationships among clades differed from neighbor-joining (NJ) analysis also those obtained with parsimony and were grouped Distichodus and Citharinus with not supported by bootstrap analysis (Fig. catfishes and placed Gymnotiformes as the 9). Additionally, NJ and parsimony boot- sister group of Characiformes + Silurifor- strap analysis both supported a clade Downloaded from https://academic.oup.com/sysbio/article/46/1/75/1685535 by guest on 28 September 2021 mes. The same result was obtained with formed by Cynopotamus and Cheirodon with maximum likelihood (ML) analyses. values of 99 and 48, respectively (not When the 123 alignment-ambiguous shown in Fig. 9). The best ML tree (In like- sites were excluded from the analysis, lihood = -8829.15) was obtained in 6 of three equally most-parsimonious trees 10 "jumbled" replications using the were obtained (L = 940, CI = 0.369, RI = fastDNAml program. It contained clades 0.391). A strict consensus of these trees 1-8 and had a topology very similar to showed highly unlikely relationships, plac- that shown in Figure 9. ing the cypriniforms nested within the cat- When the 138 alignment-ambiguous fish and the distichodontid-citharinid sites were excluded from the analyses, par- clade as the sister group of all other oto- simony yielded four shortest trees (L = physans. When alignment-ambiguous sites 835, CI = 0.355, RI = 0.391). A strict con- were included in the analysis and only sensus of these trees was mostly unre- transversions were used, six equally par- solved but contained clades 1-8 plus a simonious trees were obtained, leaving the clade formed by Tetragonopterus + Chal- deeper nodes unresolved and characiform ceus. The strict consensus tree also monophyly unsupported. As suggested by grouped serrasalmins (clade 7) with Hep- the substitution pattern analysis, the se- setus + Hoplias (clade 2) as sister groups quences contained in this data set do not and grouped this combined clade with le- provide reliable information to solve this biasinids (clade 1) + Rhaphiodon. A poster- phylogeny with confidence. iori reweighting resulted in a single com- pletely resolved tree with all the Phylogenetic Relationships among previously identified clades, but it was dif- Characiform Families: 27-taxon Data Set ferent in several respects from the tree The citharinid-distichodontid clade is shown in Figure 9. However, only clades the sister group of all characiform lineages 1-8 were supported by bootstrap analysis (Fink and Fink, 1981; Orti and Meyer, with values >50 (similar to those shown 1996) and was used as the outgroup for in Fig. 9). All other relationships received this data set. Parsimony analysis, giving all poor support. Similar results were ob- sites equal weight and treating alignment tained with NJ and ML analyses. For ex- gaps as missing data, resulted in three ample, the best ML tree obtained when shortest trees (L = 1,564, CI = 0.344, RI = alignment-ambiguous sites were excluded 0.361). With a posteriori reweighting, one (see Table 4) was only four steps longer of the shortest trees was obtained (Fig. 9). than the most-parsimonious trees and dif- However, bootstrap analysis only support- fered in several ways from the topology ed eight clades with values >50: (1) Nan- shown in Figure 9. The only relationships nostomus + Pyrrhulina (family Lebiasini- among clades 1-8 suggested by all three dae), (2) Hoplias + Hepsetus, (3) methods, excluding the 138 alignment-am- Boulengerella + Ctenolucius (family Ctenolubiguou- s sites, were ((Hoplias, Hepse- ciidae), (4) Phenacogrammus + Hydrocynus tus)(Colossoma, Pygocentrus)) and ((Nanno- + Alestes (African subfamily Alestiinae) + stomus, Pyrrhulina)Rhaphiodon), both with Acestrorhynchus, (5) Poptella + Oligosarcus +very low bootstrap support. Astyanax, (6) Brycon + Salminus (subfamily The most conservative strategy for ob- 88 SYSTEMATIC BIOLOGY VOL. 46
Nannostomus Downloaded from https://academic.oup.com/sysbio/article/46/1/75/1685535 by guest on 28 September 2021
Raphiodon Aphyocharax Cynopotamus Cheirodon Poptella Oligosarcus Astyanax Tetragonopterus Triportheus Brycon 95 Salminusninus _J • Chalceus — Pygocentrus Colossoma
FIGURE 9. Characiform data set with 27 taxa. Shortest tree resulting from a posteriori character reweighting on the three equally parsimonious trees obtained when all sites were uniformly weighted (all characters used, gaps coded as missing). The reweighted tree had RC = 0.375. Bootstrap values above branches are from neighbor-joining analysis, and those below branches are from parsimony analyses. Branches without numbers had bootstrap values <50 in both analyses. Numbered braces highlight clades supported by bootstrap analyses. African taxa are shown in black boxes. taining reliable phylogenetic information, not supported in this analysis. NJ using according to the substitution pattern anal- only transversions gave a similar result, ysis described above, would be to elimi- except that Serrasalminae was supported, nate saturated transitions and alignment- but Lebiasinidae and Hoplias + Hepsetus ambiguous sites (which are in the most were not. Parsimony and NJ analyses were quickly evolving regions within loops). Us- also congruent in denning a clade formed ing this strategy, parsimony analysis re- by Hoplias and Ctenoluciidae (clade 3). sulted in 302 shortest trees. A posteriori However, bootstrap support for these reweighting of characters based on these groupings was very low (values not trees yielded 15 equally parsimonious shown). trees, a strict consensus of which is shown Although no single set of relationships in Figure 10. Most of clades 1-8 are pres- was firmly supported by this data set (oth- ent in the strict consensus tree. However, er than the components discussed above), Serrasalminae (clade 7), Hoplias + Hepsetus it was used to test alternative topologies (clade 2), and Lebiasinidae (clade 1) were using the likelihood ratio test of Kishino 1997 ORTI AND MEYER—MOLECULAR PHYLOGENY OF CHARACIFORMES 89
Pyrrhulina 1 Hoplias 2 Boulengerella Ctenolucius 2 Acestrorhynchus Downloaded from https://academic.oup.com/sysbio/article/46/1/75/1685535 by guest on 28 September 2021
Poptella Oligosarcus Astyanax Tetragonopterus Cheirodon Aphyocharax Brycon 1 _ Salminus I Gnathocharax Triportheus Chalceus Cynopotamus Raphiodon Colossoma 7 Pygocentrus 7 Nannostomus 1
' I^ FIGURE 10. Characiform data set with 27 taxa. This strict consensus of 15 trees was obtained by a posteriori reweighting, excluding alignment-ambiguous sites and considering only transversions. Numbered braces iden- tify clades also obtained in Figure 9, and dashed lines with numbers are clades shown in Figure 9 but not supported in this strict consensus. African taxa are shown in black boxes. and Hasegawa (1989). Table 4 shows par- These alternative topologies were obtained simony tree lengths (from MacClade) and by optimizing (with parsimony or with log likelihood values (computed with the ML) while enforcing topological con- program NUCML; Adachi and Hasegawa, straints. For example, the African taxa 1994) for alternative topologies, including (Hepsetus and Alestiinae) were forced to a and excluding the alignment-ambiguous basal position, joining the Citharinus + sites. Nine tree topologies were compared Distichodus clade in the "Neotropical with this method (see Table 4) and were monophyly" constraint tree, and the best included in the "trees" block in the (elec- solution for a fully resolved tree was tronically available) data file. In addition to searched for using PAUP or NUCML. Sim- testing different results obtained with dif- ilarly, the topology used to test Lucena's ferent phylogenetic methods and inclusion (1993) hypothesis was the best (ML or par- sets, the main purpose of these compari- simony) tree among the 945 fully resolved sons was to evaluate a previous hypothesis trees that agree with the partially resolved (Lucena, 1993; Fig. la) and the phylogeny topology shown in Figure la. When all implied by a single viacariant event sepa- sites were considered, the best parsimony rating African and Neotropical lineages tree (tree 1, Table 4; also shown in Fig. 9) (i.e., monophyly of the Neotropical taxa). was not significantly different from the TABL E4. Tree lengths for parsimony (PARS) analyses (from MacClade) and log likelihoods for maximum likelihood (ML) analyses (from NUCML) for alternative phylogenetic hypotheses using the characiform 27-taxon data set, including and excluding alignment-ambiguous sites. Tree topologies are as follows: (1) best parsimony "reweighted" tree using all sites (shown in Fig. 9); (2) best ML tree from fastDNAml, using all sites; (3) best ML tree from fastDNAml, excluding alignment-ambiguous sites; (4) best parsimony tree using all data, constraining monophyly of all Neotropical taxa; (5) best ML tree using all data, constraining monophyly of all Neotropical taxa; (6) best parsimony tree (equal to best ML tree) excluding alignment-ambiguous sites, constraining monophyly of all Neotropical taxa; (7) best ML tree using all sites, constraining the topology to satisfy Lucena's (1993) hypothesis (see Fig. la); (8) best parsimony "reweighted" tree, excluding alignment-ambiguous sites and transitions (TS) (the best ML tree of the 15 trees summarized in Fig. 10); (9) best ML tree excluding alignment-ambiguous sites, constraining the topology to satisfy Lucena's (1993) hypothesis (see Fig. la). Extra steps are counted with respect to the shortest tree in this table, for each inclusion set. AL, is the difference in log likelihood between the best tree and tree i (SE = standard error). The best topologies for each method and inclusion set are underlined, and log likelihood values significantly worse than the best estimate have asterisks.
Maximum likelihood Parsimonv (tree lengths 4- extra steps) All sites Ambiguous excluded Ambiguous Log Log Topology All sites excluded likelihood AL, + SE likelihood AL, ± SE 1. PARS all data 1,564 849 ( + 10) -8870.2 -24.4 ± 23.0 -5455.2* -51.3 ± 25.5 2. ML all data 1,568 (4-4) 858 (+19) -8845.9 0.0 ± 0.0 -5462.0* -58.2 ± 23.1 3. ML amb. excluded 1,569 (4-5) 839 -8865.1 -19.3 ± 28.1 -5403.9 0.0 ± 0.0 4. PARS all data, Neo. monophyly 1,590 (+26) 863 (+24) -8931.2* -85.3 ± 30.6 -5480.8* -77.0 ± 25.5 5. ML all data, Neo. monophyly 1,593 (+29) 866 (+27) -8896.9* -51.1 ± 25.2 -5488.2* -84.4 ± 30.9 6. PARS-ML amb. excluded, Neo. monophyly 1,606 (+42) 847 (+8) -8966.8* -120.9 ± 40.5 -5448.1 -44.2 ± 27.0 7. ML all data, Lucena 1,649 (+85) 901 (+62) -9056.8* -210.9 ± 42.9 -5604.8* -200.9 ± 42.3 8. PARS amb. and TS excluded 1,655 (+91) 890 (+51) -9033.1* -187.3 ± 42.1 -5507.8* -103.9 ± 36.2
9. ML amb. excluded, Lucena 1,660 (+96) 903 (+64) -9072.3* -226.5 ± 43.4 -5602.0* -198.1 ± 42.8 Downloaded from https://academic.oup.com/sysbio/article/46/1/75/1685535 by guest on 28 September 2021 September 28 on guest by https://academic.oup.com/sysbio/article/46/1/75/1685535 from Downloaded 1997 ORTI AND MEYER—MOLECULAR PHYLOGENY OF CHARACIFORMES 91 best ML tree (tree 2, Table 4), but all other best tree from ML searches (In likelihood alternative topologies tested (trees 4-9) = -6604.3, using fastDNAml) was the were significantly worse. Following Kishi- same as 1 of the 14 shortest parsimony no and Hasegawa (1989), alternative to- trees; all 14 shortest trees from parsimony pologies were considered significantly had very similar log likelihoods and did worse than the best ML tree when the 95% not differ significantly from each other confidence interval of the log likelihood when tested simultaneously (Kishino and difference between them did not include Hasegawa, 1989). NJ analysis supported zero. Significantly worse alternative topol- the same components as parsimony (see Downloaded from https://academic.oup.com/sysbio/article/46/1/75/1685535 by guest on 28 September 2021 ogies are shown with asterisks in Table 4 NJ bootstrap values in Fig. 11) but resulted for both inclusion sets. Hypotheses of Neo- in a globally different topology overall, tropical monophyly and Lucena's (1993) which required 28 extra steps for parsi- hypothesis were not supported by the 12S mony and had a significantly worse like- and 16S data sets. Of all alternative topol- lihood than the best tree. ogies tested, only tree 6 (Table 4) was not Forcing the monophyly of Neotropical rejected but was close to being significant- taxa resulted in seven equally parsimoni- ly worse than the best ML tree (tree 3) ous trees with 13 extra steps (L = 1,065). when tested with the data set excluding When these seven trees were tested against alignment-ambiguous sites (AL6 = -44.2 the best ML tree with NUCML, they all ± 27). had significantly worse log likelihood val- ues (not shown). Alternative hypotheses Phylogenetic Relationships among (Fig. 1) such as those from Buckup (1991) Characiform Families: 38-taxon Data Set and Uj (1990) were not tested with this Parsimony analysis of the 38-taxon cha- data set because they did not include all raciform data set, weighting all sites equal- taxa used in this analysis. ly and treating alignment gaps as missing, resulted in eight shortest trees (L = 2,059, DISCUSSION CI = 0.283, RI = 0.371), but their strict con- sensus is highly unresolved. Clades 1-8 Sequence Variation and the Limits of (see Figs. 9,10) are also present in the con- Phylogenetic Resolution sensus, plus a few others shown in Figure Most phylogenetic relationships among 11. For simplicity, only results obtained taxa were not resolved with confidence in when excluding alignment-ambiguous this study using ribosomal mtDNA se- sites will be presented because all major quences. Lack of resolution might be at- differences between inclusion sets are not tributed to saturation of nucleotide strongly supported. changes and/or to the mode of evolution- Parsimony analysis excluding 140 align- ary diversification of the taxa involved. ment-ambiguous sites resulted in 14 short- Comparisons of 12S and 16S sequences est trees (L = 1,052, CI = 0.290, RI = among characiform families showed a 0.392), and with a posteriori reweighting a slightly lower level of mean sequence di- single completely resolved tree was ob- vergence (14.9%) than did comparisons tained (Fig. 11). The same pattern as that among orders of otophysans (17.3%). As- for the 27-taxon data set emerges, but in suming rate constancy across all lineages, addition to the previously described eight this observation could be taken as evidence clades, four more clades are supported. for dating the origination of the major lin- Clade 9 (family Gasteropelecidae), clade 10 eages of Characiformes very close to the (Prochilodus + family Curimatidae), and origin of the otophysan orders (cyprini- clade 11 (subfamily Glandulocaudinae, forms, catfishes, electric fishes). Alterna- family Characidae) are strongly supported tively, similar values of sequence diver- by bootstrap analysis (both NJ and parsi- gence among lineages may reflect mony), and each constitutes a well-sup- saturation at the DNA level, given the ported unit in terms of morphology. The structural constraints on sequence varia- 92 SYSTEMATIC BIOLOGY VOL. 46
66 Nannostomus} Lebiasinidae 54 Pyrrhulina I 98 Pygocentrus t Serrasalminae 98 Colossoma 62 Hoplias Erythrinidae 51 AFR.I Raphiodon
Apareiodon Parodontidae Downloaded from https://academic.oup.com/sysbio/article/46/1/75/1685535 by guest on 28 September 2021 82 • Carnegiella 9 96 ' G,asteropet^] G^steropelecidae Abramites "i Leporinus j Anostom.dae Chilodus Chilodontidae Characidium Crenuchidae Hemiodus Hemiodontidae \ 1 92 Prochilodus Prochilodontidae Cyphocharax 10 60 100 Curimatidae 95 Steindachnerinafma j 91 Boulengerella Ctenoluciidae 84 Ctenolucius Gnathocharax
Chalceus j* Cynopotamus Cheirodon Corynopoma Glandulo- Gephyrocharax caudinae Poptella Oligosarcus Astyanax Aphyocharax Tetragonopterus
98 Cithanmdae AFR. 89 Distichodontidae FIGURE 11. Characiform data set with 38 taxa. Shortest tree obtained with a posteriori reweighting on 14 equally parsimonious trees, when alignment-ambiguous sites were excluded. Numbers above and below branch- es are bootstrap values from neighbor joining and parsimony analyses, respectively (only values >50 are shown). Numbered braces (9-11) identify clades, other than those (1-8) shown in Figure 9, that are supported by bootstrap analyses. Family names are given next to each taxon, except for the family Characidae. For the Characidae, only subfamilial groupings supported by bootstrap analyses are indicated (Serrasalminae and Glandulocaudinae). Numbered arrows mark putative drift-vicariant events between African and Neotropical groups; t = clades postulated to have gone extinct in Africa. African taxa are shown in black boxes. 1997 ORTI AND MEYER—MOLECULAR PHYLOGENY OF CHARACIFORMES 93 tion. The higher average divergence ob- 50- served in comparisons between gonorhyn- Ependymin chiforms and otophysans (21.1%) suggests 40- that the divergence values among otophy- o 30- sans might be close to but not yet at com- o plete saturation. However, maximum di- o 20J vergence values among characiform families, otophysan orders, and ostario- physan orders were essentially all the o Downloaded from https://academic.oup.com/sysbio/article/46/1/75/1685535 by guest on 28 September 2021 o same (21.3%, 21.9%, and 24%, respectively; C Fig. 12), indicating that saturation is a
1993). However, no one has proposed a Figure 11 is taken at face value, and the close relationship between alestins and nodes separating Hepsetus and alesines Acestrorhynchus. from their Neotropical sister taxa are as- Mean percentages of sequence diver- sumed to reflect drift-vicariant events, the gence (uncorrected, 12S and 16S genes) be- extinction of at least six major lineages of tween the African taxa and their corre- charadforms in Africa is implied (see Fig. sponding Neotropical sister groups were 11). 16.2% for Distichodus + Citharinus, 11.2% Although the fossil record of Charad- for Hepsetus, and 15.1% for Alestiinae. Di- formes is not very useful for testing the Downloaded from https://academic.oup.com/sysbio/article/46/1/75/1685535 by guest on 28 September 2021 vergence between Hepsetus and ctenolucids above scenario, intriguing fossils have (putative sister groups according to mor- been described by Greenwood and Howes phological studies) was 16.6%. These val- (1975) and Stewart (1994). Teeth and skulls ues are within the same range of diver- of Miocene to Lower Pleistocene age were gence values recorded among the other assigned to now-extinct charadform fishes families of Characiformes, below the 21- (Sindacharax lepersonnei and S. deserti), ap- 24% saturation value (Fig. 12), suggesting parently widespread in northern and east- that most lineages (families) of characi- ern Africa. These teeth are more similar to form fishes had originated before the vi- the teeth of modern serrasalmins such as cariant event separating African and Neo- Colossoma and Piaractus than to those of tropical taxa, approximately 100 million any African charadform fish (Greenwood years ago. If Characiformes experienced a and Howes, 1975; Stewart, 1994). Serrasal- rapid evolutionary radiation comparable to mins form a well-supported monophyletic that of cichlid fishes in East African lakes taxon endemic to South America (Macha- (e.g., Greenwood, 1984; Meyer et al., 1990; do-Allison, 1982; clade 7, Figs. 9, 11) and Meyer, 1993), but 100 million years ago, include herbivorous forms such as Colos- resolution of phylogenetic relationships soma and Piaractus, considered the primi- among the major lineages is not expected tive sister group to the more derived pred- to be easily obtained. Poor resolution of re- atory piranhas (e.g., Pygocentrus; Orti et al., lationships among charadform taxa using 1996). The systematic position of serrasal- phylogenetic analyses of ependymin DNA mins within Charadformes could not be sequences (Orti and Meyer, 1996) and con- resolved with confidence in the present flicting phylogenetic hypotheses from study (Figs. 9-11), and no close relation- morphological data seem to agree with ship of serrasalmins with other Neotropi- this prediction. cal charadds is suggested. South American Analyzing Buckup's (1991) phylogenetic serrasalmin fossils indicate that forms sim- hypothesis in a biogeographic context, ilar to Cobssoma had differentiated by at Lundberg (1993) also arrived at the conclu- least 13 million years ago (Lundberg et al., sion that the major groups of charadforms 1986; Lundberg, 1996). Considering that had originated before the African-South serrasalmins are exclusively freshwater American vicariant event (although the fishes, if Sindacharax really belongs to the proposed African-South American sister- serrasalmin clade, the origin of serrasal- group relationships differed). He also mins would have to be unequivocally raised the important question of why more placed before the African-South American charadform subgroups now endemic to continental split (>84 million years ago), the Neotropics do not have close relatives in agreement with conclusions from DNA in the African fauna. Assuming a strict vi- sequence divergences. Sindacharax would cariant view and no dispersal of charad- also provide an example of extirpation in forms across the widening Atlantic ocean, Africa of one trans-South Atlantic clade the present biogeographic distribution im- (Lundberg, 1993). Fossil fishes from the plies a remarkably uneven rate of extinc- Miocene fauna of the Magdalena Basin in tion among African charadforms (Lund- Colombia provide a good example for ex- berg, 1993). If the topology shown in tirpation of serrasalmins from a formerly 96 SYSTEMATIC BIOLOGY VOL. 46 diverse Amazon-Orinoco fauna (Lund- earlier versions of the manuscript. This paper was pre- pared in partial fulfillment of requirements for the berg et alv 1986). The depauperate fauna Ph.D. in Ecology and Evolution by G.O. This is con- of the present Magdalena River does not tribution 958 from the Graduate Program in Ecology include Colossoma and piranha species, and and Evolution at SUNY-Stony Brook. local extinction due to tectonism and cli- matic changes during the Cenozoic has REFERENCES been suggested to explain the loss of di- versity (Lundberg et al., 1986; Lundberg ADACHI, J., AND M. HASEGAWA. 1994. MOLPHY: A program package for molecular phylogenetics, ver- and Chernoff, 1992). Similar geological sion 2.2. Institute of Statistical Mathematics, Tokyo. Downloaded from https://academic.oup.com/sysbio/article/46/1/75/1685535 by guest on 28 September 2021 and climatic processes might have affected ALVES-GOMES, J. A., G. ORTI, M. HAYGOOD, W. HEI- a previously characiform-rich African fau- LIGENBERG, AND A. MEYER. 1995. Phylogenetic na and may be invoked to explain why analysis of the South American electric fishes (order Gymnotiformes) and the evolution of their electro- only three lineages of characiforms are genie system: A synthesis based on morphology, found there at present. Paleocene tectonic electrophysiology, and mitochondrial sequence data. movements of the African plate and post- Mol. Biol. Evol. 12:298-318. Miocene aridification affected the African ANDERSON, S., A. T. BANKIER, B. G. BARRELL, M. H. L. continent more severely than they did DE BRUIJN, A. R. COULSON, J. DROUIN, I. C. EPERON, D. P. NIERLICH, B. A. ROE, F. SANGER, P. H. 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with a revisionary study. Smithson. Contrib. Zool. Subfamily Characinae 564:1-97. Tribe Charatini VAWTER, L., AND W. M. BROWN. 1993. Rates and pat- Cynopotamus sp. (R. Uruguay, Salto Grande, Ar- terns of base change in the small subunit ribosomal gentina; USNM 325689). GB: U33961, RNA gene. Genetics 134:597-608. U33998. WEITZMAN, S. H., AND W. L. FINK. 1983. Relationships Gnathocharax steindachneri (GO 123). GB: of the neon tetras, a group of South American fresh- U33589, U33624. water fishes (Teleostei, Charatidae), with comments Tribe Acestrorhynchini on the phylogeny of New World Characiformes. Acestrorhynchus sp. (GO 76). GB: U33962, Bull. Mus. Comp. Zool. 150:339-395. U33999. Downloaded from https://academic.oup.com/sysbio/article/46/1/75/1685535 by guest on 28 September 2021 WEITZMAN, S. HV AND R. P. VARI. 1988. Miniaturi- Oligosarcus sp. (R. Uruguay, Salto Grande, Ar- zation in South American freshwater fishes: An gentina; USNM 235690). GB: U33963, overview and discussion. Proc. Biol. Soc. Wash. 101: U34000. 444-465. Subfamily Raphiodontinae WILSON, A. C, R. L. CANN, S. M. CARR, M. GEORGE, Rhaphiodon vulpinus (R. Uruguay, Salto Grande, U. B. GYLLENSTEN, K. M. HELM-BYCHOWSKI, R. G. Argentina; GO 124). GB: U33964, U34001. HIGUCHI, S. R. PALUMBI, E. M. PRAGER, R. D. SAGE, Subfamily Bryconinae AND M. STONEKING. 1985. Mitochondrial DNA and Tribe Salminini two perspectives on evolutionary genetics. Biol. J. Salminus sp. (R. de la Plata, Buenos Aires, Ar- Linn. Soc. 26:375-400. gentina; GO IB). GB: U33965, U34002. Received 25 July 1995; accepted 25 May 1996 Tribe Bryconini Associate Editor: David Cannatella Brycon sp. (R. Parana, Bella Vista, Argentina; USNM 326005). GB: U33966, U34003. Chalceus macrolepidotus (GO 40). GB: U33587, APPENDIX U33622. SPECIMENS EXAMINED Tribe Triportheini Information provided includes locality of origin Triportheus paranensis (R. Parana, Bella Vista, (when known, otherwise fish are from commercial Argentina; GO 109). GB: U33588, U33623. sources; AFR = African taxa), G.O. (GO) collection Subfamily Aphyocharacinae numbers, GenBank (GB) accession numbers for the Aphyocharax sp. (GO 93). GB: U33968, U34005. 12S and 16S sequences, and Museum catalog numbers Subfamily Glandulocaudinae for voucher specimens deposited in museum collec- Corynopoma riisei (cultured stock from R. Man- tions: USNM = U.S. Museum of Natural History; zanares, Venezuela; GO 73). GB: U33969, INPA = Instituto Nacional de Pesquisas da Amazonia. U34006. Characiform family-level classification follows that of Gephyrocharax sp. (GO 122). GB: U33970, Greenwood et al. (1966), with a few exceptions: the U34007. family Cynodontidae is included as a tribe in the fam- Subfamily Stethaprioninae ily Characidae, as suggested by Howes (1976); the Poptella sp. (GO 174). GB: U33971, U34008. ichthyborids are included within the family Disticho- Subfamily Tetragonopterinae dontidae, following Vari (1979); and the characid sub- Astyanax fasciatus (GO 65). GB: U33972, families Characidiinae and Crenuchinae are grouped U34009. in the new family Crenuchidae (Buckup, 1991). As a Tetragonopterus sp. (R. Parana, Bella Vista, Ar- consequence, 15 characiform families are recognized. gentina; GO 108). GB: U33973, U34010. Subfamily Cheirodontinae Order Characiformes Cheirodon sp. (R. Parana, Bella Vista, Argentina, 1. Family Hepsetidae (AFR) USNM 325676). GB: U33974, U34011. Hepsetus odoe (GO 126). GB: U33852, U33992. Paracheirodon innesi (GO 43). GB: U33975, 2. Family Citharinidae (AFR) U34012. Citharinus congicus (R. Malagarasi, Tanzania; Subfamily Serrasalminae GO 195). GB: U33826, U33993. Pygocentrus nattereri (R. Solimoes, Ilha da Mar- 3. Family Distichodontidae (AFR) chantaria, AM, Brazil; INPA 10143). GB: Distichodus sp. (GO 72). GB: U33827, U33994. U33558, U33590. 4. Family Crenuchidae Colossoma macropomum (R. Solimoes, Ilha da Characidium sp. (NE Brazil, USNM 318101). GB: Marchantaria, AM, Brazil; INPA 10149). U33828, U34030. GB: U33581, U33616. 5. Family Characidae 6. Family Erythrinidae Subfamily Alestiinae (AFR) Hoplias malabaricus (R. Uruguay, Salto Grande, Alestes sp. (R. Malagarasi, Tanzania; GO 147). Argentina; GO 113). GB: U33976, U34013. GB: U33829, U33995. 7. Family Ctenoluciidae Phenacogrammus sp. (GO 49). GB: U33830, Ctenolucius sp. (GO 67). GB: U33977, U34014. U33996. Boulengerella maculata (GO 66). GB: U33978, Hydrocyon sp. (R. Malagarasi, Tanzania; GO U34015. 127). GB: U33960, U33997. 8. Family Lebiasinidae 100 SYSTEMATIC BIOLOGY VOL. 46
Nannostomus sp. (GO 120). GB: U33979, Family Rhamphichthyidae U34016. Rhamphichthys sp. GB: U15257, U15233 (from Alves- Pyrrhulina sp. (R. Parana, Bella Vista, Argenti- Gomes et al., 1995). na, USNM 325675). GB: U33980, U34017. Family Apteronotidae 9. Family Hemiodontidae Apteronotus albifrons. GB: U15275, U15226 (from Al- Hemiodus sp. (GO 191). GB: U33981, U34018. ves-Gomes et al., 1995). 10. Family Parodontidae Apareiodon affinis (R. Parana, Bella Vista, Argen- Order Siluriformes tina; GO 156). GB: U33982, U34019. Family Loricariidae 11. Family Gasteropelecidae Hypostomus sp. GB: U15263, U15239 (from Alves- Carnegiella sp. (GO 95). GB: U33983, U34020. Gomes et al., 1995). Downloaded from https://academic.oup.com/sysbio/article/46/1/75/1685535 by guest on 28 September 2021 Gasteropelecus sp. (GO 44). GB: U33984, U34021. Family Cetopsidae 12. Family Curimatidae Cetopsis sp. GB: U15272, U15248 (from Alves-Go- Cyphocharax gilberti (NE Brazil; USNM 318079). mes et al., 1995). GB: U33985, U34022. Family Trichomycteridae Steindachnerina sp. (R. Uruguay, Salto Grande, Trichomycterus sp. GB: U15251, U15227 (from Alves- Argentina; USNM 325691). GB: U33986, Gomes et al., 1995). U34023. Family Malapteruridae 13. Family Prochilodontidae Malapterurus sp. GB: U15261, U15237 (from Alves- Pwchilodus lineatus (R. de la Plata, Buenos Ai- Gomes et al., 1995). res, Argentina; GO Bl). GB: U33987, U34034. Order Cypriniformes 14. Family Anostomidae Family Cyprinidae Abramites sp. (GO 77). GB: U33988, U34025. Cyprinus carpio. GB: X61010. Leporinus obtusidens (R. Paraguay, Asuncion, Family Gastromyzontidae Paraguay; GO 133). GB: U34031, U34026. Crossostoma lacustre. GB: M91245. 15. Family Chilodontidae Chilodus sp. (GO 172). GB: 33989, U34027. Order Gonorhynchiformes Family Kneridae Order Gymnotiformes Kneria sp. (R. Malagarasi, Tanzania; GO 194). GB: Family Eigenmaniidae U33990, U34028. Eigenmannia sp. GB: U15269, U15245 (from Alves- Parakneria sp. (R. Malagarasi, Tanzania; GO 193). Gomes et al., 1995). GB: U33991, U34029.